WO2025160307A1

WO2025160307A1 - Treating cancer using biomarkers and gene signatures

Info

Publication number: WO2025160307A1
Application number: PCT/US2025/012809
Authority: WO
Inventors: Rui Wang
Original assignee: University of Texas System; University of Texas at Austin
Current assignee: University of Texas System; University of Texas at Austin
Priority date: 2024-01-25
Filing date: 2025-01-23
Publication date: 2025-07-31
Anticipated expiration: 2026-07-25

Abstract

Provided herein are methods for treating and evaluating cancer in an individual. The methods may include administering one or more treatments or management strategies to the individual after measuring expression levels of one or more genes from a sample from the individual. The methods may also include generating a gene signature score based on the expression levels of the one or more genes, generating a clinically important threshold value for the gene signature score, comparing the gene signature score to a threshold value, and determining risk level of the individual based on the comparison.

Description

TREATING CANCER USING BIOMARKERS AND GENE SIGNATURES

[0001] This application claims priority of U.S. Provisional Patent Application No. 63/624,898, filed January 25, 2024, which is hereby incorporated by reference in its entirety.

BACKGROUND

I. Technical Field

[0002] Embodiments of the disclosure include at least the fields of molecular biology, cancer biology, and medicine, including oncology, endocrinology, otolaryngology, and head and neck surgery.

II. Background

[0003] A cancer patient may benefit from guided treatments based on clinically actionable information of the cancer, such as a cancer biomarker indicating how the cancer may behave, progress over time, or how the cancer may respond to a particular treatment. However, it has been challenging to obtain a reliable biomarker or gene signature for a cancer with consistent clinical utility across patient cohorts, such as for papillary thyroid carcinoma (PTC).

[0004] PTC tumors that lose differentiation are known to be a biologically aggressive subset. Clinical characteristics alone cannot accurately identify high-risk patients with PTC associated with morbidity and mortality. Also, different criteria are used amongst clinicians and researchers to identify dedifferentiated or high-risk PTCs (z.e., based on histopathology, radioactive iodine uptake, and/or clinical behavior), yielding inconsistent clinical associations and limiting the utility of differentiation status as a biomarker.

[0005] Therefore, there is an unmet need for better tools to use at the time of cancer diagnosis to guide treatment selection and risk stratification. For example, since PTC can recur many years after diagnosis, studies that examine performance of a test must take place within cohorts of adequate duration of follow-up. Currently available genomic tests were developed in small cohorts of patients with limited follow-up and were not developed based on adequate time-to event methodology.

[0006] The present disclosure provides a long-felt need in the art of providing better diagnosis, prognosis, and/or treatment for cancer (e.g., PTC). SUMMARY

[0007] Discoveries have been made that provide a solution to at least one or more of the aforementioned problems associated with lacking reliable biomarkers for cancer, such as PTC. In one aspect, the solution can include exemplary expression-based gene signatures to measure differentiation status in PTC, such as by using single-cell and/or bulk RNAseq and/or expression microarray data obtained from human PTC tumors. In certain embodiments, the gene-expression signatures measure the activity of one or more groups of genes that affect how PTC behaves and/or responds to treatment. In particular embodiments, the gene-expression signature(s) is significantly associated with survival outcomes in an example of a cohort of PTC patients. This association is further validated in independent cohorts of PTC patients with appropriate follow-up. The gene-expression signature(s) predicts survival outcomes better than clinical variables alone. In some embodiments, using the gene expression signature, individuals can be stratified as high risk groups and low risk groups, respectively, for developing disease progression, transformation into dedifferentiated histologic subtypes, recurrence, and/or death due to PTC.

[0008] In particular embodiments, the present disclosure is directed to systems, methods, and compositions for identifying and using biomarkers (e.g., gene expression levels) for predicting risk and/or progression of cancer (e.g., thyroid cancer) and/or to guide treatment and clinical management of cancer (e.g., thyroid cancer).

[0009] In one aspect, encompassed are methods of treating thyroid cancer in an individual. In some aspects, the methods may include administering one or more treatments to the individual after measuring expression levels of one or more genes from a sample from the individual. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the methods further include generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; and determining risk level of the individual based on the comparison.

[0010] In some aspects, the one or more treatments to the individual in any of the methods encompassed herein may be administered when the gene signature score is respectively increased compared to the preset threshold value. In some aspects, the one or more treatments to the individual in any of the methods encompassed herein may be administered when the gene signature score is respectively decreased compared to the preset threshold value.

[0011] In some embodiments, a score can guide pretreatment work up of an individual, i.e., the extent and/or type of imaging studies needed, including a follow-up surveillance schedule after the treatment(s). In specific embodiments, a score can determine staging workup. For example, in some embodiments, individuals found to have high-risk disease based on the score may potentially require more extensive work up before treatment initiation to rule out the spread of cancer. Whereas some scores will determine that an individual would receive a neck ultrasound only, other scores indicating high-risk patients may benefit from further workup, such as with CT scans of the neck and chest, PET scans, etc.

[0012] In some aspects, the expression levels of the one or more genes in any of the methods encompassed herein may be measured by a RNA-based assay and/or by a proteinbased assay. In some aspects, the RNA-based assay comprises RNA sequencing (RNA-Seq), single cell RNA-Seq, spatial transcriptomics, polymerase chain reaction (PCR), northern blot, and/or microarray. Protein-based assays may include immunohistochemistry, western blot, and/or mass spectrometry, for example.

[0013] The sample from which the RNA or protein is obtained may be from any tissue and/or fluid from the individual. In some aspects, the sample from the individual in any of the methods encompassed herein includes a thyroid biopsy tissue, as one example.

[0014] In particular embodiments, the methods encompassed herein include analysis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 of the genes encompassed herein. In some aspects, the one or more genes in any of the methods encompassed herein include 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, or 80 or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

[0015] In some aspects, the one or more genes in any of the methods encompassed herein include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof.

[0016] In some aspects, risk level of the individual in any of the methods encompassed herein may be determined further based on one or more clinical variables of the individual. In some aspects, the one or more clinical variables of the individual comprise histopathology, age, biological sex, ethnicity, stage, radioactive iodine uptake, and/or clinical behavior.

[0017] In some aspects, the one or more treatments in any of the methods encompassed herein may include one or more of active surveillance and/or one or more cancer treatments. In some aspects, the one or more cancer treatments may include a surgery. In some aspects, the surgery may include partial thyroidectomy or total thyroidectomy with or without neck dissection/removal of neck lymph nodes. In some aspects, the one or more cancer treatments may include one or more of adjuvant therapy, radiation therapy, proton therapy, hormone therapy, chemotherapy, immunotherapy, bisphosphate therapy, cryotherapy, ultrasound, and/or palliative care. In some aspects, the adjuvant therapy may include radioactive iodine. In some aspects, the active surveillance may include a determination of frequency, imaging modality, timing, and/or duration of one or more follow-up visits of the individual. In some aspects, the one or more cancer treatments may include radioactive iodine.

[0018] In some aspects, the thyroid cancer in any of the methods encompassed herein includes thyroid cancers derived from thyroid follicular cells. In some aspects, the thyroid cancers derived from thyroid follicular cells include follicular thyroid cancer, papillary thyroid cancer, poorly differentiated thyroid cancers, oncocytic thyroid cancer, medullary thyroid cancer, and/or anaplastic thyroid cancers.

[0019] In some aspects, the thyroid cancer in any of the methods encompassed herein may be papillary thyroid cancer. [0020] In one aspect, encompassed are methods of administering a thyroid cancer therapy to an individual having modulation of expression levels of one or more genes. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0021] In one aspect, encompassed are methods of administering one or more thyroid cancer therapies to an individual having measured modulation of expression levels of one or more genes. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4- AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0022] In one aspect, encompassed are methods of treating an individual for thyroid cancer including measuring expression levels of one or more genes from a sample from the individual and administering one or more treatments to the individual based on the levels of expression of the one or more genes. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison.

[0023] In one aspect, encompassed are methods of determining prognosis of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining prognosis of thyroid cancer by analyzing a sample from an individual.

[0024] In one aspect, encompassed are methods of determining recurrence and/or progression of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining the risk of recurrence, progression, and/or transformation of thyroid cancer by analyzing a sample from an individual.

[0025] In one aspect, encompassed are methods of determining grade and/or aggressiveness of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, any method of the present disclosure further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining grade and/or aggressiveness of thyroid cancer by analyzing a sample from an individual.

[0026] In one aspect, encompassed are methods of determining stage, spread, and/or extent of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, any method encompassed herein further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining stage, spread, and/or extent of thyroid cancer by analyzing a sample from an individual. [0027] In one aspect, encompassed are methods of treating an individual in need thereof including measuring expression levels of one or more genes and treating the individual in need thereof with at least one thyroid cancer therapy. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0028] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including measuring expression levels of one or more genes from a sample of the individual; predicting thyroid cancer status of the individual; and determining treatment for the individual. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4- AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0029] In one aspect, encompassed are methods of monitoring efficacy of one or more thyroid cancer therapies in an individual comprising measuring expression levels of one or more genes before and after the one or more thyroid cancer therapies are administered one or more times to the individual. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the measuring may occur after the one or more thyroid cancer therapies are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.

[0030] In one aspect, encompassed are methods of predicting or measuring thyroid cancer treatment response of an individual to one or more treatments including measuring expression levels of one or more genes from a sample of the individual and predicting or measuring whether or not the individual is responsive to the one or more treatments. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0031] In one aspect, encompassed are methods of treating an individual for thyroid cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SEC26A7, MTRNR2E12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SEC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PECG2, SEC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2E8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2E1, GEUE, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPE17, CEIC3, NDUFB1, C16orf89, IPCEF1, SEEENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof; b) generating a gene signature score based on the expression levels measured in step a); c) comparing the gene signature score to a preset threshold value; d) determining the individual’s risk level based on the comparison in step c); e) administering one or more treatments to the individual based on the individual’s risk level determined in step d). [0032] In one aspect, encompassed are methods of determining prognosis of an individual for thyroid cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof; b) generating a gene signature score based on the expression levels measured in step a); c) comparing the gene signature score to a preset threshold value(s); d) determining the individual’s risk level based on the comparison in step c).

[0033] In one aspect, encompassed are methods of determining recurrence and/or progression of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0034] In one aspect, encompassed are methods of determining grade, behavior, and/or aggressiveness of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0035] In one aspect, encompassed are methods of determining stage, extent, and/or spread of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0036] In one aspect, encompassed are methods of treating an individual in need thereof including the steps of detecting a change in expression levels of one or more genes and treating the individual in need thereof with at least one cancer therapy, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0037] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including the steps of detecting a change in expression levels of one or more genes from a sample of the individual, predicting cancer status of the individual, and determining treatment for the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DI01, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SEEENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

[0038] In one aspect, encompassed are methods of determining stage, spread, and/or extent of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include IYD, FHE1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2E1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SEC26A7, ADIRF, PEA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, AEDH1A1, CRABP1, CEIC3, MT1E, KRT8, ID4, NUPR1, CAER, PCP4, NDUFB1, METTE7A, MT1X, SEC25A29, PECG2, MT1F, MAFB, MT1H, GPX3, SEC26A4-AS1, CHCHD10, RPE17, or a combination thereof. In some aspects, any method encompassed herein further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining stage, spread, and/or extent of thyroid cancer by analyzing a sample from an individual.

[0039] In one aspect, encompassed are methods of treating an individual in need thereof including measuring expression levels of one or more genes and treating the individual in need thereof with at least one thyroid cancer therapy. In some aspects, the one or more genes include IYD, FHE1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2E1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SEC26A7, ADIRF, PEA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, AEDH1A1, CRABP1, CEIC3, MT1E, KRT8, ID4, NUPR1, CAER, PCP4, NDUFB1, METTE7A, MT1X, SEC25A29, PECG2, MT1F, MAFB, MT1H, GPX3, SEC26A4-AS1, CHCHD10, RPE17, or a combination thereof.

[0040] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including the steps of detecting a change in expression levels of one or more genes from a sample of the individual, predicting cancer status of the individual, and determining treatment for the individual, wherein the one or more genes comprise IYD, FHE1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2E1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SEC26A7, ADIRF, PEA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0041] In one aspect, encompassed are methods of treating an individual in need thereof including the steps of detecting a change in expression levels of one or more genes and treating the individual in need thereof with at least one cancer therapy, wherein the one or more genes comprise IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0042] In one aspect, encompassed are methods of determining stage, extent, and/or spread of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise IYD, FHL1, IPCEF1, RAP 1 GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0043] In one aspect, encompassed are methods of determining grade, behavior, and/or aggressiveness of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0044] In one aspect, encompassed are methods of determining recurrence and/or progression of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0045] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including measuring expression levels of one or more genes from a sample of the individual; predicting thyroid cancer status of the individual; and determining treatment for the individual. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0046] In one aspect, encompassed are methods of determining prognosis of an individual for thyroid cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0047] In one aspect, encompassed are methods of treating an individual for thyroid cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise IYD, FHL1, IPCEF1, RAP 1 GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof; b) generating a gene signature score based on the expression levels measured in step a); c) comparing the gene signature score to a preset threshold value; d) determining the individual’s risk level based on the comparison in step c); e) administering one or more treatments to the individual based on the individual’s risk level determined in step d).

[0048] In one aspect, encompassed are methods of monitoring efficacy of one or more thyroid cancer therapies in an individual comprising measuring expression levels of one or more genes before and after the one or more thyroid cancer therapies are administered one or more times to the individual. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof. In some aspects, the measuring may occur after the one or more thyroid cancer therapies are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.

[0049] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including measuring expression levels of one or more genes from a sample of the individual; predicting thyroid cancer status of the individual; and determining treatment for the individual. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0050] In one aspect, encompassed are methods of administering a thyroid cancer therapy to an individual having modulation of expression levels of one or more genes. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof. [0051] In one aspect, encompassed are methods of administering one or more thyroid cancer therapies to an individual having measured modulation of expression levels of one or more genes. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0052] In one aspect, encompassed are methods of treating an individual for thyroid cancer including measuring expression levels of one or more genes from a sample from the individual and administering one or more treatments to the individual based on the levels of expression of the one or more genes. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison.

[0053] In one aspect, encompassed are methods of determining prognosis of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining prognosis of thyroid cancer by analyzing a sample from an individual.

[0054] In one aspect, encompassed are methods of determining recurrence and/or progression of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining the risk of recurrence, progression, and/or transformation of thyroid cancer by analyzing a sample from an individual.

[0055] In one aspect, encompassed are methods of determining grade and/or aggressiveness of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof. In some aspects, any method of the present disclosure further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining grade and/or aggressiveness of thyroid cancer by analyzing a sample from an individual.

[0056] In one aspect, encompassed are methods of determining stage, spread, and/or extent of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof. In some aspects, any method encompassed herein further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining stage, spread, and/or extent of thyroid cancer by analyzing a sample from an individual.

[0057] In one aspect, encompassed are methods of treating an individual in need thereof including measuring expression levels of one or more genes and treating the individual in need thereof with at least one thyroid cancer therapy. In some aspects, the one or more genes include IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GADD45B, CPQ, SOD3, CRYAB, TFCP2L1, AAK1, GBP1, ID3, PRDX1, EGR1, EGR2, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, ADIRF, PLA2R1, SORBS2, TFF3, IER2, ATP1A1, CITED2, TNFRSF11B, ALDH1A1, CRABP1, CLIC3, MT1E, KRT8, ID4, NUPR1, CALR, PCP4, NDUFB1, METTL7A, MT1X, SLC25A29, PLCG2, MT1F, MAFB, MT1H, GPX3, SLC26A4-AS1, CHCHD10, RPL17, or a combination thereof.

[0058] In one aspect, encompassed are methods of determining stage, spread, and/or extent of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. In some aspects, any method encompassed herein further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining stage, spread, and/or extent of thyroid cancer by analyzing a sample from an individual.

[0059] In one aspect, encompassed are methods of treating an individual in need thereof including measuring expression levels of one or more genes and treating the individual in need thereof with at least one thyroid cancer therapy. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, S0RBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0060] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including the steps of detecting a change in expression levels of one or more genes from a sample of the individual, predicting cancer status of the individual, and determining treatment for the individual, wherein the one or more genes comprise FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0061] In one aspect, encompassed are methods of treating an individual in need thereof including the steps of detecting a change in expression levels of one or more genes and treating the individual in need thereof with at least one cancer therapy, wherein the one or more genes comprise FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0062] In one aspect, encompassed are methods of determining stage, extent, and/or spread of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0063] In one aspect, encompassed are methods of determining grade, behavior, and/or aggressiveness of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRY AB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0064] In one aspect, encompassed are methods of determining recurrence and/or progression of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRY AB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. [0065] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including measuring expression levels of one or more genes from a sample of the individual; predicting thyroid cancer status of the individual; and determining treatment for the individual. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0066] In one aspect, encompassed are methods of determining prognosis of an individual for thyroid cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0067] In one aspect, encompassed are methods of treating an individual for thyroid cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof; b) generating a gene signature score based on the expression levels measured in step a); c) comparing the gene signature score to a preset threshold value; d) determining the individual’s risk level based on the comparison in step c); e) administering one or more treatments to the individual based on the individual’s risk level determined in step d).

[0068] In one aspect, encompassed are methods of monitoring efficacy of one or more thyroid cancer therapies in an individual comprising measuring expression levels of one or more genes before and after the one or more thyroid cancer therapies are administered one or more times to the individual. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. In some aspects, the measuring may occur after the one or more thyroid cancer therapies are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.

[0069] In one aspect, encompassed are methods of identifying an individual for treatment of thyroid cancer including measuring expression levels of one or more genes from a sample of the individual; predicting thyroid cancer status of the individual; and determining treatment for the individual. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0070] In one aspect, encompassed are methods of administering a thyroid cancer therapy to an individual having modulation of expression levels of one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0071] In one aspect, encompassed are methods of administering one or more thyroid cancer therapies to an individual having measured modulation of expression levels of one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0072] In one aspect, encompassed are methods of treating an individual for thyroid cancer including measuring expression levels of one or more genes from a sample from the individual and administering one or more treatments to the individual based on the levels of expression of the one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison.

[0073] In one aspect, encompassed are methods of determining prognosis of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining prognosis of thyroid cancer by analyzing a sample from an individual.

[0074] In one aspect, encompassed are methods of determining recurrence and/or progression of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining the risk of recurrence, progression, and/or transformation of thyroid cancer by analyzing a sample from an individual.

[0075] In one aspect, encompassed are methods of determining grade and/or aggressiveness of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRY AB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. In some aspects, any method of the present disclosure further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining grade and/or aggressiveness of thyroid cancer by analyzing a sample from an individual.

[0076] In one aspect, encompassed are methods of determining stage, spread, and/or extent of thyroid cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes include FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof. In some aspects, any method encompassed herein further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value(s); and determining risk level(s) of the individual based on the comparison. In some aspects, the method further includes determining stage, spread, and/or extent of thyroid cancer by analyzing a sample from an individual.

[0077] In one aspect, encompassed are methods of treating an individual in need thereof including measuring expression levels of one or more genes and treating the individual in need thereof with at least one thyroid cancer therapy. In some aspects, the one or more genes FHL1, IPCEF1, PEBP1, CPQ, SOD3, CRYAB, TFCP2L1, PRDX1, EGR1, EGR2, FOSB, ZFP36, SNRPN, GCSH, CYR61, PLA2R1, SORBS2, TFF3, IER2, ALDH1A1, CRABP1, ID4, NDUFB1, METTL7A, SLC26A4-AS1, or a combination thereof.

[0078] Any gene set herein may comprise, consist of, or consist essentially of one or more genes in the gene set.

[0079] In particular embodiments, one or more genes encompassed herein decrease or change in expression level in relation to increased risk of having thyroid cancer, presence of thyroid cancer, increased risk of metastasis of thyroid cancer, decreased sensitivity to therapy for thyroid cancer, resistance to therapy for thyroid cancer, and so forth. In particular embodiments, one or more genes encompassed herein decrease in expression level in relation to increased risk of having thyroid cancer, presence of thyroid cancer, increased risk of metastasis of thyroid cancer, sensitivity to therapy for thyroid cancer, resistance to therapy for thyroid cancer, and so forth.

[0080] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0081] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0082] The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or.

[0083] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0084] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0085] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may originate in the thyroid, lymph glands, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some embodiments, the cancer is recurrent cancer and/or second primary cancer. In some embodiments, the cancer is Stage I cancer. In some embodiments, the cancer is Stage II cancer. In some embodiments, the cancer is Stage III cancer. In some embodiments, the cancer is Stage IV cancer.

[0086] In particular embodiments, the cancer is papillary thyroid carcinoma, differentiated thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, oncocytic thyroid carcinoma, poorly differentiated thyroid carcinoma, anaplastic thyroid carcinoma, undifferentiated thyroid carcinoma, tall-cell thyroid carcinoma, or hurthle cell thyroid carcinoma.

[0087] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0088] The term “sample,” as used herein, generally refers to a biological sample, including from any region in the body, such as the thyroid. The sample may be taken from tissue or cells or from the thyroid environment. In some examples, the sample may comprise, or be derived from, any part of the body tissues, including a tissue biopsy, stool, blood, thyroid tissue, tumors, or a combination thereof. The sample may have been isolated from the source prior to collection. In some examples, the sample is isolated from its primary source (such as cells, tissue, bodily fluids such as blood, environmental samples) during sample preparation. The sample may or may not be purified or otherwise enriched from its primary source. In some embodiments, the primary source is homogenized prior to further processing. The sample may be filtered or centrifuged to remove undesired material. The sample may also be purified, dissected, or enriched for particular compositions therein. The sample may contain tissues or cells that are intact, fragmented, or partially degraded.

[0089] “Subject” and “patient” and “individual” may be interchangeable and may refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human. The subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject can be a patient, e.g., have or be suspected of having a disease (that may be referred to as a medical condition), such as one or more infectious diseases, one or more genetic disorders, one or more cancers, or any combination thereof. The “subject” or "individual", as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. An individual may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (e.g., children) and infants and includes in utero individuals. A subject may or may not have a need for medical treatment; an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.

[0090] “Treating” or treatment of a disease or condition refers to utilizing a regimen or executing a protocol, which may include administering one or more therapies (such as radioactive iodine) or cellular therapy products to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease recurrence or progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0091] The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, complete eradication of the tumor, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0092] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0093] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the subject matter of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the subject matter of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0095] FIG. 1 is a Kaplan-Meier analysis showing that in the discovery cohort, a dichotomized gene signature score can separate patients who have high risk of recurrence, progression, or death due to papillary thyroid cancer. A low score corresponds to a more dedifferentiated tumor, which is higher risk for recurrence, progression, or death. A high score corresponds to a tumor with better prognosis.

[0096] FIG. 2 is a Kaplan-Meier analysis showing that in an independent validation cohort, the dichotomized gene signature score generated from FIG. 1 can separate patients who have high risk of recurrence, progression, or death due to papillary thyroid cancer. A low score corresponds to a more dedifferentiated tumor, which is higher risk for recurrence, progression, or death. A high score corresponds to a tumor with better prognosis.

[0097] FIGs. 3A-3B show differential expression between high-risk (TFC_L) and low-risk (TFC_H) groups illustrated using a heatmap plot (3A) and principal components analysis (3B) in the discovery cohort.

[0098] FIG. 4 shows Prognostic RNA Expression Cell-specific Integrated SignaturE (PRECISE) score across patient cohorts.

[0099] FIG. 5 shows PRECISE score predicts response to radioactive iodine (RAI).

[0100] FIG. 6 shows PRECISE score is associated with survival outcomes in PTC.

DETAILED DESCRIPTION

I. General Embodiments

[0101] In embodiments of the disclosure, as examples only any method of the disclosure may have one or more of the following steps, and the one or more steps may occur in any suitable order: analyzing a sample; administering one or more treatments to an individual; administering one or more treatments to an individual in need thereof; administering one or more treatments to an individual after measuring expression levels of one or more genes from a sample from the individual; administering one or more thyroid cancer therapies to an individual; administering one or more thyroid cancer therapies to an individual having modulation of expression levels of one or more genes; administering one or more thyroid cancer therapies to an individual having measured modulation of expression levels of one or more genes; treating an individual for thyroid cancer comprising: measuring expression levels of one or more genes from a sample from the individual, and administering one or more treatments to the individual based on the levels of expression of the one or more genes; determining prognosis of thyroid cancer comprising measuring expression levels of one or more genes; determining prognosis of thyroid cancer by analyzing one or more samples from an individual; determining risk of recurrence and/or progression of thyroid cancer; determining risk of recurrence and/or progression of thyroid cancer comprising measuring expression levels of one or more genes; determining risk of recurrence and/or progression of thyroid cancer by analyzing a sample from an individual; determining risk of transformation of thyroid cancer to an more aggressive subtypel determining behavior, grade and/or aggressiveness of thyroid cancer; determining behavior, grade and/or aggressiveness of thyroid cancer comprising measuring expression levels of one or more genes; determining behavior, grade and/or aggressiveness of thyroid cancer by analyzing a sample from an individual; determining stage and/or spread of thyroid cancer; determining stage and/or spread of thyroid cancer comprising measuring expression levels of one or more genes; determining stage and/or spread of thyroid cancer by analyzing a sample from an individual; treating an individual in need thereof; treating an individual in need thereof comprising measuring expression levels of one or more genes and treating the individual in need thereof with at least one thyroid cancer therapy; identifying an individual for treatment of thyroid cancer; identifying an individual for treatment of thyroid cancer comprising: measuring expression levels of one or more genes from a sample of the individual, predicting thyroid cancer status of the individual, and determining treatment for the individual; monitoring efficacy of one or more thyroid cancer therapies in an individual; monitoring efficacy of one or more thyroid cancer therapies in an individual comprising measuring expression levels of one or more genes before and after the one or more thyroid cancer therapies are administered one or more times to the individual; obtaining a sample from an individual; preparing a sample from an individual; measuring expression levels of one or more genes from a sample from an individual; detecting expression levels of one or more genes from a sample from an individual; identifying expression levels of one or more genes from a sample from an individual; assaying expression levels of one or more genes from a sample from an individual; quantifying expression levels of one or more genes from a sample from an individual; generating a gene signature score; generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; determining risk level of the individual based on the comparison; administering one or more treatments to an individual based on the risk level of the individual; guiding an extent of diagnostic/staging workup for patient to guide treatment; and so forth.

[0102] In embodiments of the disclosure, one or more of the steps in any method of the disclosure may occur at the same time or at different times. For example, in one embodiment an individual may be diagnosed and/or prognosed at the same time as the efficacy of therapy or one or more treatments is determined. In one embodiment an individual is diagnosed at a time different from determination of efficacy of a therapy for the individual.

[0103] In embodiments of the disclosure, one or more genes in any method of the disclosure may include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof. Alternatively and/or additionally, in embodiments of the disclosure, one or more genes in any method of the disclosure may include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof.

[0104] As used herein, a gene signature or biomarker may be based on one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. For example, an expression-based gene signature or biomarker may be based on expressive levels on one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof.

II. Diagnosing, Prognosing, and Treating Cancer

[0105] In certain embodiments there are methods of treating cancer in an individual. In some aspects, the method includes administering one or more treatments to the individual, such as after measuring expression levels of one or more genes from a sample from the individual. In some aspects, the one or more genes may include one or more of all of the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include at least, or no more than, TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include at least, or no more than, TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP 1 GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a threshold value; and determining risk level of the individual based on the comparison.

[0106] In some aspects, the one or more treatments to the individual in any of the methods encompassed herein may be administered when the gene signature score is respectively increased compared to the threshold value. In some aspects, the one or more treatments to the individual in any of the methods encompassed herein may be administered when the gene signature score is respectively decreased compared to the threshold value.

[0107] In some aspects, the threshold value in any of the methods encompassed herein may be preset. In some aspects, the threshold value in any of the methods encompassed herein may not be preset, such as being dynamically adjusted.

[0108] In some aspects, lower expression levels of the one or more genes (e.g., loss of expression) may be associated with higher risk level of the individual (e.g., more aggressive behavior, less responsive to therapy, and/or worse prognosis, such as worse survival probability, higher probability of recurrence/progression, and/or higher probability of death). [0109] In some aspects, higher expression levels of the one or more genes (e.g., increased expression) may be associated with higher risk level of the individual (e.g., more aggressive behavior, less responsive to therapy, and/or worse prognosis, such as worse survival probability, higher probability of recurrence/progression, and/or higher probability of death). [0110] In some aspects, the gene signature score may be positively correlated with expression levels of the one or more genes (e.g., a higher gene signature score may be associated with higher expression levels of the one or more genes). Alternatively, the gene signature score may be negatively correlated with expression levels of the one or more genes (e.g., a higher gene signature score may be associated with lower expression levels of the one or more genes).

[0111] In some aspects, the higher gene signature score may be associated with increased risk level of the individual (e.g., more aggressive behavior, less responsive to therapy, and/or worse prognosis, such as worse survival probability, higher probability of recurrence/progression, and/or higher probability of death). Alternatively, the lower gene signature score may be associated with increased risk level of the individual (e.g., more aggressive behavior, less responsive to therapy, and/or worse prognosis, such as worse survival probability, higher probability of recurrence/progression, and/or higher probability of death). [0112] In some aspects, the expression levels of the one or more genes in any of the methods encompassed herein may be measured by any of the compositions, methods, and/or systems encompassed herein, such as in the parts of Detecting a Gene Signature, Gene and ncRNA Expression Levels, and Assay Methods herein. In some aspects, the expression levels of the one or more genes may be measured by levels of any expression products of the one or more genes, including but not limited to any polynucleotides (e.g., mRNAs and corresponding cDNAs), polypeptides, proteins, and/or any fragments thereof. In some aspects, the expression levels of the one or more genes in any of the methods encompassed herein may be measured by a RNA-based assay. In some aspects, the RNA-based assay comprises RNA sequencing (RNA-Seq), single cell RNA-Seq, PCR, northern blot, and/or microarray.

[0113] In some aspects, the sample from the individual in any of the methods encompassed herein may include any of the compositions, methods, and/or systems encompassed herein, such as in the part of Sample Preparation herein. In some aspects, the sample from the individual in any of the methods encompassed herein includes a thyroid biopsy tissue and/or cells.

[0114] In some aspects, the one or more genes in any of the methods encompassed herein include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 or more, 61 or more, 62 or more, 63 or more, 64 or more, 65 or more, 66 or more, 67 or more, 68 or more, 69 or more, 70 or more, 71 or more, 72 or more, 73 or more, 74 or more, 75 or more, 76 or more, 77 or more, 78 or more, 79 or more, 80 or more genes from all the genes encompassed herein, including but not limited to the genes in the Figures and Examples, such as in Tables 1 to 8. [0115] In some aspects, the one or more genes in any of the methods encompassed herein include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 or more, 61 or more, 62 or more, 63 or more, 64 or more, 65 or more, 66 or more, 67 or more, 68 or more, 69 or more, 70 or more, 71 or more, 72 or more, 73 or more, 74 or more, 75 or more, 76 or more, 77 or more, 78 or more, 79 or more, 80 or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

[0116] In particular embodiments, the risk level for an individual to develop thyroid cancer is determined based on expression levels of one or more genes encompassed herein. In some aspects, risk level of the individual in any of the methods encompassed herein may be determined further based on one or more clinical variables of the individual. In some aspects, the one or more clinical variables of the individual comprise age, biological sex, ethnicity, stage, histopathology, radioactive iodine uptake, and/or clinical behavior.

[0117] In some aspects, the one or more treatments in any of the methods encompassed herein may include any of the compositions, methods, and/or systems encompassed herein, such as in the parts of Cancer Therapy, Therapeutic Methods, Immunotherapy, Administration of Therapeutic Compositions, and Kits. In some aspects, the one or more treatments in any of the methods encompassed herein may include one or more of active surveillance and/or one or more cancer treatments. In some aspects, the one or more cancer treatments include a surgery, such as any surgery encompassed herein. In some aspects, the surgery includes partial thyroidectomy or total thyroidectomy with or without removal of neck lymph nodes. In some aspects, the one or more cancer treatments include one or more of radioactive iodine, adjuvant therapy, radiation therapy, proton therapy, hormone therapy, chemotherapy, immunotherapy, bisphosphate therapy, cryotherapy, ultrasound, and/or palliative care. In some aspects, an adjuvant therapy includes radioactive iodine. In some aspects, the active surveillance includes a determination of frequency, timing, and/or duration of follow-up visits of the individual.

[0118] In some aspects, the cancer in any of the methods encompassed herein includes any types of cancer, including all types of the cancers encompassed herein.

[0119] In some aspects, the cancer is thyroid cancer. In some aspects, the thyroid cancer in any of the methods encompassed herein includes thyroid cancers derived from thyroid follicular cells. In some aspects, the thyroid cancers derived from thyroid follicular cells include follicular thyroid cancer, papillary thyroid cancer, poorly differentiated thyroid cancers, oncocytic thyroid cancer, medullary thyroid cancer, and/or anaplastic thyroid cancers. [0120] In some aspects, the thyroid cancer in any of the methods encompassed herein may be papillary thyroid cancer.

[0121] In one aspect, encompassed are methods of administering one or more cancer therapies to an individual having modulation of expression levels of one or more genes. In some aspects, the one or more genes may include one or more of all of the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT IF, MAFB, CHCHD10, RPL17, and/or a combination thereof.

[0122] In one aspect, encompassed are methods of administering a cancer therapy to an individual having measured modulation of expression levels of one or more genes. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT IF, MAFB, CHCHD10, RPL17, and/or a combination thereof.

[0123] In one aspect, encompassed are methods of treating an individual for cancer including measuring expression levels of one or more genes from a sample from the individual and administering one or more treatments to the individual based on the levels of expression of the one or more genes. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a threshold value; and determining risk level of the individual based on the comparison. [0124] In one aspect, encompassed are methods of determining prognosis of cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a threshold value; and determining risk level of the individual based on the comparison. In some aspects, the method further includes determining prognosis of cancer by analyzing a sample from an individual.

[0125] In one aspect, encompassed are methods of determining recurrence and/or progression of cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a threshold value; and determining risk level of the individual based on the comparison. In some aspects, the method further includes determining recurrence and/or progression of cancer by analyzing a sample from an individual.

[0126] In one aspect, encompassed are methods of determining grade and/or aggressiveness of cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a threshold value; and determining risk level of the individual based on the comparison. In some aspects, the method further includes determining grade and/or aggressiveness of cancer by analyzing a sample from an individual.

[0127] In one aspect, encompassed are methods of determining stage, extent, and/or spread of cancer including measuring expression levels of one or more genes. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4- AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB 16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the method further includes generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a threshold value; and determining risk level of the individual based on the comparison. In some aspects, the method further includes determining stage and/or spread of cancer by analyzing a sample from an individual.

[0128] In one aspect, encompassed are methods of treating an individual in need thereof including measuring expression levels of one or more genes and treating the individual in need thereof with at least one cancer therapy. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof.

[0129] In one aspect, encompassed are methods of identifying an individual for treatment of cancer including measuring expression levels of one or more genes from a sample of the individual; predicting cancer status of the individual; and determining treatment for the individual. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT IF, MAFB, CHCHD10, RPL17, and/or a combination thereof.

[0130] In one aspect, encompassed are methods of monitoring efficacy of one or more cancer therapies in an individual including measuring expression levels of one or more genes before and after the one or more cancer therapies are administered one or more times to the individual. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8. In some aspects, the one or more genes include TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the measuring may occur after the one or more cancer therapies are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.

[0131] In one aspect, encompassed are methods of treating an individual for cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP 1 GAP, ZFP36, ZBTB16, and/or a combination thereof; b) generating a gene signature score based on the expression levels measured in step a); c) comparing the gene signature score to a threshold value, including a clinically informative threshold value (e.g., depending on the gene set used, such as smaller vs larger gene set, a cut off from -0.30 to -0.50 based on a singlesample GSEA derived gene signature score will allow identification of low vs. high risk patients for disease recurrence, progression, or death. The cut off can be further refined within subsets of patients if needed.); d) determining the individual’s risk level based on the comparison in step c); e) administering one or more treatments to the individual based on the individual’s risk level determined in step d). In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0132] In one aspect, encompassed are methods of determining prognosis of an individual for cancer including the steps of: a) measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise TFF3, MT1G, TPO,

MTIF, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof; b) generating a gene signature score based on the expression levels measured in step a); c) comparing the gene signature score to a threshold value; d) determining the individual’s risk level based on the comparison in step c). In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1,

MTIG, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0133] In one aspect, encompassed are methods of determining recurrence and/or progression of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DI02, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHE1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SEC26A7, PEA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, AEDH1A1, CEIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PECG2, MT1F, MAFB, CHCHD10, RPE17, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0134] In one aspect, encompassed are methods of determining grade and/or aggressiveness of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SEC26A7, MTRNR2E12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SEC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PECG2, SEC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2E8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2E1, GEUE, CITED2, FHE1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SEC25A29, AC007952.4, KRT7, PEA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPE17, CEIC3, NDUFB1, C16orf89, IPCEF1, SEEENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHE1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SEC26A7, PEA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, AEDH1A1, CEIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PECG2, MT1F, MAFB, CHCHD10, RPE17, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0135] In one aspect, encompassed are methods of determining stage, extent, and/or spread of a cancer, including identifying a change in expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0136] In one aspect, encompassed are methods of treating an individual in need thereof including the steps of detecting a change in expression levels of one or more genes and treating the individual in need thereof with at least one cancer therapy, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0137] In one aspect, encompassed are methods of identifying an individual for treatment of cancer including the steps of detecting a change in expression levels of one or more genes from a sample of the individual, predicting cancer status of the individual, and determining treatment for the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes include TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0138] In one aspect, encompassed are methods of predicting or measuring thyroid cancer treatment response of an individual to one or more treatments including the steps of measuring expression levels of one or more genes from a sample of the individual and predicting or measuring whether or not the individual is responsive to the one or more treatments, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof. In some aspects, the one or more genes may include one or more of all the genes encompassed herein, including but not limited to one or more of the genes encompassed in the Figures and Examples, such as in Tables 1 to 8.

[0139] In some aspects, the one or more genes comprise TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT IF, MAFB, CHCHD10, RPL17, and/or a combination thereof.

[0140] In some aspects, the one or more treatments may include one or more of active surveillance and/or one or more cancer treatments. In some aspects, the one or more cancer treatments include a surgery, such as any surgery encompassed herein. In some aspects, the surgery includes partial thyroidectomy or total thyroidectomy with or without removal of neck lymph nodes. In some aspects, the one or more cancer treatments include one or more of radioactive iodine, adjuvant therapy, radiation therapy, proton therapy, hormone therapy, chemotherapy, immunotherapy, bisphosphate therapy, cryotherapy, ultrasound, and/or palliative care. In some aspects, an adjuvant therapy includes radioactive iodine. In some aspects, the active surveillance includes a determination of frequency, timing, and/or duration of follow-up visits of the individual. In some aspects, the one or more cancer treatments include radioactive iodine.

III. Detecting a Signature

[0141] Particular embodiments concern the methods of detecting a genetic signature in an individual. In some embodiments, the method for detecting the genetic signature may include selective oligonucleotide probes, arrays, allele- specific hybridization, molecular beacons, restriction fragment length polymorphism analysis, enzymatic chain reaction, flap endonuclease analysis, primer extension, 5’-nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high-resolution melting, DNA mismatch binding protein analysis, surveyor nuclease assay, sequencing, or a combination thereof, for example. The method for detecting the genetic signature may include fluorescent in situ hybridization, comparative genomic hybridization, arrays, polymerase chain reaction, sequencing, or a combination thereof, for example. The detection of the genetic signature may involve using a particular method to detect one feature of the genetic signature and additionally use the same method or a different method to detect a different feature of the genetic signature. Multiple different methods independently or in combination may be used to detect the same feature or a plurality of features.

A. Single Nucleotide Polymorphism (SNP) Detection

[0142] Particular embodiments of the disclosure concern methods of detecting a SNP in an individual. One may employ any of the known general methods for detecting SNPs for detecting the particular SNP in this disclosure, for example. Such methods include, but are not limited to, selective oligonucleotide probes, arrays, allele- specific hybridization, molecular beacons, restriction fragment length polymorphism analysis, enzymatic chain reaction, flap endonuclease analysis, primer extension, 5’-nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high-resolution melting, DNA mismatch binding protein analysis, surveyor nuclease assay, sequencing, or a combination thereof.

[0143] In some embodiments of the disclosure, the method used to detect the SNP comprises sequencing nucleic acid material from the individual and/or using selective oligonucleotide probes. Sequencing the nucleic acid material from the individual may involve obtaining the nucleic acid material from the individual in the form of genomic DNA, complementary DNA that is reverse transcribed from RNA, or RNA, for example. Any standard sequencing technique may be employed, including Sanger sequencing, chain extension sequencing, Maxam-Gilbert sequencing, shotgun sequencing, bridge PCR sequencing, high-throughput methods for sequencing, next generation sequencing, RNA sequencing, or a combination thereof. After sequencing the nucleic acid from the individual, one may utilize any data processing software or technique to determine which particular nucleotide is present in the individual at the particular SNP.

[0144] In some embodiments, the nucleotide at the particular SNP is detected by selective oligonucleotide probes. The probes may be used on nucleic acid material from the individual, including genomic DNA, complementary DNA that is reverse transcribed from RNA, or RNA, for example. Selective oligonucleotide probes preferentially bind to a complementary strand based on the particular nucleotide present at the SNP. For example, one selective oligonucleotide probe binds to a complementary strand that has an A nucleotide at the SNP on the coding strand but not a G nucleotide at the SNP on the coding strand, while a different selective oligonucleotide probe binds to a complementary strand that has a G nucleotide at the SNP on the coding strand but not an A nucleotide at the SNP on the coding strand. Similar methods could be used to design a probe that selectively binds to the coding strand that has a C or a T nucleotide, but not both, at the SNP. Thus, any method to determine binding of one selective oligonucleotide probe over another selective oligonucleotide probe could be used to determine the nucleotide present at the SNP.

[0145] One method for detecting SNPs using oligonucleotide probes comprises the steps of analyzing the quality and measuring quantity of the nucleic acid material by a spectrophotometer and/or a gel electrophoresis assay; processing the nucleic acid material into a reaction mixture with at least one selective oligonucleotide probe, PCR primers, and a mixture with components needed to perform a quantitative PCR (qPCR), which could comprise a polymerase, deoxynucleotides, and a suitable buffer for the reaction; and cycling the processed reaction mixture while monitoring the reaction. In one embodiment of the method, the polymerase used for the qPCR will encounter the selective oligonucleotide probe binding to the strand being amplified and, using endonuclease activity, degrade the selective oligonucleotide probe. The detection of the degraded probe determines if the probe was binding to the amplified strand.

[0146] Another method for determining binding of the selective oligonucleotide probe to a particular nucleotide comprises using the selective oligonucleotide probe as a PCR primer, wherein the selective oligonucleotide probe binds preferentially to a particular nucleotide at the SNP position. In some embodiments, the probe is generally designed so the 3’ end of the probe pairs with the SNP. Thus, if the probe has the correct complementary base to pair with the particular nucleotide at the SNP, the probe will be extended during the amplification step of the PCR. For example, if there is a T nucleotide at the 3’ position of the probe and there is an A nucleotide at the SNP position, the probe will bind to the SNP and be extended during the amplification step of the PCR. However, if the same probe is used (with a T at the 3’ end) and there is a G nucleotide at the SNP position, the probe will not fully bind and will not be extended during the amplification step of the PCR.

[0147] In some embodiments, the SNP position is not at the terminal end of the PCR primer, but rather located within the PCR primer. The PCR primer should be of sufficient length and homology in that the PCR primer can selectively bind to one variant, for example the SNP having an A nucleotide, but not bind to another variant, for example the SNP having a G nucleotide. The PCR primer may also be designed to selectively bind particularly to the SNP having a G nucleotide but not bind to a variant with an A, C, or T nucleotide. Similarly, PCR primers could be designed to bind to the SNP having a C or a T nucleotide, but not both, which then does not bind to a variant with a G, A, or T nucleotide or G, A, or C nucleotide respectively. In particular embodiments, the PCR primer is at least or no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,3 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or more nucleotides in length with 100% homology to the template sequence, with the potential exception of non-homology the SNP location. After several rounds of amplifications, if the PCR primers generate the expected band size, the SNP can be determined to have the A nucleotide and not the G nucleotide.

B. Copy Number Detection

[0148] Particular embodiments of the disclosure concern methods of detecting a copy number variation (CNV) or copy number alteration (CAN), and this may be done by SNP array, bulk DNA sequencing (whole exome sequencing, whole genome sequencing), or single-cell DNA sequencing. One can utilize any known method for detecting CNV and CNV to detect copy number changes. Such methods include fluorescent in situ hybridization, comparative genomic hybridization, arrays, polymerase chain reaction, sequencing, or a combination thereof, for example. Computational software may be used to estimate the CNA/CNV within a sample.

C. DNA Sequencing

[0149] In some embodiments, DNA may be analyzed by sequencing. The DNA may be prepared for sequencing by any method known in the art, such as library preparation, hybrid capture, sample quality control, product-utilized ligation-based library preparation, or a combination thereof. The DNA may be prepared for any sequencing technique. In some embodiments, a unique genetic readout for each sample may be generated by genotyping one or more polymorphic SNPs. In specific embodiments, whole exome sequencing or whole genome sequencing may be used. In some embodiments, sequencing, such as 76 base pair, paired-end sequencing, may be performed to cover approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater percentage of targets at more than 20x, 25x, 30x, 35x, 40x, 45x, 50x, or greater than 50x coverage. In certain embodiments, mutations, SNPS, INDELS, copy number alterations (somatic and/or germline), or other genetic differences may be identified from the sequencing using at least one bioinformatics tool. In some embodiments, one or more bioinformatics tools may be used to infer tumor purity and/or ploidy.

D. RNA Sequencing

[0150] In some embodiments, RNA may be analyzed by sequencing. The RNA may be prepared for sequencing by any method known in the art, such as poly-A selection, cDNA synthesis, stranded or nonstranded library preparation, or a combination thereof. The RNA may be prepared for any type of RNA sequencing technique, including stranded specific RNA sequencing. In some embodiments, sequencing may be performed to generate approximately 10M, 15M, 20M, 25M, 30M, 35M, 40M or more reads, including paired reads. The sequencing may be performed at a read length of approximately 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 105 bp, 110 bp, or longer. In some embodiments, raw sequencing data may be converted to estimated read counts (RS EM), fragments per kilobase of transcript per million mapped reads (FPKM), and/or reads per kilobase of transcript per million mapped reads (RPKM). In some embodiments, one or more bioinformatics tools may be used to infer tumor proportion, stroma content, immune infiltration, and/or tumor immune cell profiles.

E. Proteomics

[0151] In some embodiments, protein may be analyzed by mass spectrometry. The protein may be prepared for mass spectrometry using any method known in the art. Protein, including any isolated protein encompassed herein, may be treated with DTT followed by iodoacetamide. The protein may be incubated with at least one peptidase, including an endopeptidase, proteinase, protease, or any enzyme that cleaves proteins. In some embodiments, protein is incubated with the endopeptidase, LysC and/or trypsin. The protein may be incubated with one or more protein cleaving enzymes at any ratio, including a ratio of pg of enzyme to pg protein at approximately 1:1000, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, or any range between. In some embodiments, the cleaved proteins may be purified, such as by column purification. In certain embodiments, purified peptides may be snap-frozen and/or dried, such as dried under vacuum. In some embodiments, the purified peptides may be fractionated, such as by reverse phase chromatography or basic reverse phase chromatography. Fractions may be combined for practice of the methods of the disclosure. In some embodiments, one or more fractions, including the combined fractions, are subject to phosphopeptide enrichment, including phospho-enrichment by affinity chromatography and/or binding, ion exchange chromatography, chemical derivatization, immunoprecipitation, co-precipitation, or a combination thereof. The entirety or a portion of one or more fractions, including the combined fractions and/or phospho -enriched fractions, may be subject to mass spectrometry. In some embodiments, the raw mass spectrometry data may be processed and normalized using at least one relevant bioinformatics tool.

F. Detection Kits and Systems

[0152] One can recognize that based on the methods described herein, detection reagents, kits, and/or systems can be utilized to detect the gene expression levels related to the genetic signature for diagnosing an individual (the detection either individually or in combination). The reagents can be combined into at least one of the established formats for kits and/or systems as known in the art. As used herein, the terms “kits” and “systems” refer to embodiments such as combinations of at least one gene expression detection reagent, for example at least one PCR primer. The kits could also contain other reagents, chemicals, buffers, enzymes, packages, containers, electronic hardware components, etc. The kits/systems could also contain packaged sets of PCR primers, oligonucleotides, arrays, beads, or other detection reagents. Any number of probes could be implemented for a detection array. In some embodiments, the detection reagents and/or the kits/systems are paired with chemiluminescent or fluorescent detection reagents. Particular embodiments of kits/systems include the use of electronic hardware components, such as DNA chips or arrays, or microfluidic systems, for example. In specific embodiments, the kit also comprises one or more therapeutic or prophylactic interventions in the event the individual is determined to be in need of.

IV. Gene and ncRNA Expression Levels

[0153] Methods encompassed herein include measuring expression of genes and/or noncoding RNAs (ncRNAs). Measurement of expression can be done by a number of processes known in the art. The process of measuring expression may begin by extracting RNA from a tissue sample and/or cells. Extracted mRNA and/or ncRNA can be detected by hybridization (for example by means of Northern blot analysis or DNA or RNA arrays (microarrays) after converting RNA into labeled cDNA) and/or amplification by means of a enzymatic chain reaction. Quantitative or semi-quantitative enzymatic amplification methods such as polymerase chain reaction (PCR) or quantitative real-time RT-PCR or semi- quantitative RT-PCR techniques can be used. Suitable primers for amplification methods encompassed herein can be readily designed by a person skilled in the art. Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA), isothermal amplification of nucleic acids, and nucleic acid sequence based amplification (NASBA). Expression levels of mRNAs and/or ncRNAs may also be measured by RNA sequencing methods known in the art. RNA sequencing methods may include mRNA-seq, total RNA-seq, targeted RNA-seq, small RNA- seq, single-cell RNA-seq, ultra-low-input RNA-seq, RNA exome capture sequencing, ribosome profiling, and spatial transcriptomics. Sequencing data may be processed an aligned using methods known in the art.

[0154] To normalize the expression values of one gene among different samples, comparing the mRNA and/or ncRNA level of interest in the samples from the subject of study with a control RNA level is possible. As it is used herein, a "control RNA" is an RNA of a gene for which the expression level does not differ among different cancer subtypes, for example a gene that is constitutively expressed in all types of cells. A control RNA is preferably an mRNA derived from a housekeeping gene encoding a protein that is constitutively expressed and carrying out essential cell functions. A known amount of a control RNA may be added to the sample(s) and the value measured for the level of the RNA of interest may be normalized to the value measured for the known amount of the control RNA. Normalization for some methods, such as for sequencing, may comprise calculating the reads per kilobase of transcript per million mapped reads (RPKM) for a gene of interest, or may comprise transforming the read count matrix to allow for meaningful comparisons across samples. Any available normalization approaches can be used which accounts for variation due to sequencing depth, gene length, library size, RNA composition, and/or other parameters.

[0155] Methods encompassed herein may include comparing a measured expression level to a reference expression level. The term "reference expression level" refers to a value used as a reference for the values/data obtained from samples obtained from patients. The reference level can be an absolute value, a relative value, a value which has an upper and/or lower limit, a series of values, an average value, a median, a mean value, or a value expressed by reference to a control or reference value. A reference level can be based on the value obtained from an individual sample, such as, for example, a value obtained from a sample from the subject of study but obtained at a previous point in time. The reference level can be based on a number of samples, such as the levels obtained in a cohort of subjects having a particular characteristic. A reference level can be based on the expression levels of the markers to be compared obtained from samples from subjects who do not have a disease state or a particular phenotype. A reference level can be based on expression levels of the markers obtained from normal thyroid tissue and/or cells without cancer. The person skilled in the art will see that the particular reference expression level can vary depending on the specific method to be performed.

[0156] Some embodiments include determining that a measured expression level is higher than, lower than, increased relative to, decreased relative to, equal to, or within a predetermined amount of a reference expression level. In some embodiments, a higher, lower, increased, or decreased expression level is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 50, 100, 150, 200, 250, 500, or 1000 fold (or any derivable range therein) or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900% different than the reference level, or any derivable range therein. These values may represent a predetermined threshold level, and some embodiments include determining that the measured expression level is higher by a predetermined amount or lower by a predetermined amount than a reference level. In some embodiments, a level of expression may be qualified as “low” or “high,” which indicates the patient expresses a certain gene, ncRNA, r gene signature at a level relative to a reference level or a level with a range of reference levels that are determined from multiple samples meeting criteria threshold level. The level or range of levels in multiple control samples is an example of this.

[0157] In particular embodiments, reference levels or values may be determined using normal thyroid tissues from either patients without thyroid cancer or adjacent normal thyroids. In contrast, a threshold level or value may be trained using tumor RNAseq data from patients with cancer.

[0158] In some embodiments, that certain level or a predetermined threshold value is at, below, or above 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,

75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,

100 percentile, or any range derivable therein. Moreover, a threshold level may be derived from a cohort of individuals meeting a particular criteria. The number in the cohort may be, be at least, or be at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more (or any range derivable therein). A measured expression level can be considered equal to a reference expression level if it is within a certain amount of the reference expression level, and such amount may be an amount that is predetermined. This can be the case, for example, when a classifier is used to identify the molecular subtype of a cancer. The predetermined amount may be within 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50% of the reference level, or any range derivable therein.

[0159] For any comparison of gene and/or ncRNA expression levels to a mean expression levels or a reference expression levels, the comparison may be made on a gene-by-gene and ncRNA-by-ncRNA basis. For example, if the expression levels of gene A, gene B, and miRNA X in a patient’s cancer are measured, a comparison to mean expression levels in cancers of a cohort of patients would involve: comparing the expression level of gene A in the patient’s cancer with the mean expression level of gene A in cancers of the cohort of patients, comparing the expression level of gene B in the patient’s cancer with the mean expression level of gene B in cancers of the cohort of patients, and comparing the expression level of ncRNA X in the patient’s cancer with the mean expression level of ncRNA X in cancers of the cohort of patients. Comparisons that involve determining whether the expression level measured in a patient’s cancer is within a predetermined amount of a mean expression level or reference expression level are similarly done on a gene-by-gene and ncRNA-by-ncRNA basis, as applicable.

[0160] A threshold level may be determined by any suitable means. One can utilize a webpaged interface/online tool for clinicians to calculate a gene signature score for their patient(s). Clinicians may upload their patient(s) gene expression data (z.e., from RNAseq) into the web interface. In specific embodiments, the tool extracts gene expression levels for genes included in the gene signature score. Single-gene set GSEA may be applied to generate the score. The tool then applies the cutoff for the gene signature score generated by a machine learning algorithm to specific sample(s) to allow the clinician to estimate disease risk for their patient(s). In specific embodiments, output from the tool may estimate risk category and approximate 2-, 5-, and 10-year risk for disease recurrence, progression, or death due to thyroid cancer.

[0161] A machine learning approach based on recursive partitioning and decision-tree techniques was used to identify score cut-off thresholds for separating patients into low vs. high-risk groups. This in turn generates a dichotomized gene signature score where a high score represents low-risk disease while a low score represents high-risk disease. In specific embodiments, a web interface will calculate the score for each patient based on their tumor expression data and compare it to the threshold to determine risk. V. Assay Methods

A. Detection of methylated DNA

[0162] Aspects of the methods include assaying nucleic acids to determine expression levels and/or methylation levels of nucleic acids. Embodiments of the disclosure include the detection of one or more CpG islands, such as at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands (or any range derivable therein). Each biomarker may comprise or consist of at least or at most or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands (or any range derivable therein). Assays for the detection of methylated DNA are known in the art. Exemplary methods are described herein.

1. HPLC-UV

[0163] The technique of HPLC-UV (high performance liquid chromatography-ultraviolet), developed by Kuo and colleagues in 1980 (described further in Kuo K.C. et al., Nucleic Acids Res. 1980;8:4763-4776, which is herein incorporated by reference) can be used to quantify the amount of deoxycytidine (dC) and methylated cytosines (5 mC) present in a hydrolysed DNA sample. The method includes hydrolyzing the DNA into its constituent nucleoside bases, the 5 mC and dC bases are separated chromatographically and, then, the fractions are measured. Then, the 5 mC/dC ratio can be calculated for each sample, and this can be compared between the experimental and control samples.

2. LC-MS/MS

[0164] Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is an high-sensitivity approach to HPLC-UV, which requires much smaller quantities of the hydrolysed DNA sample. In the case of mammalian DNA, of which ~2%-5% of all cytosine residues are methylated, LC-MS/MS has been validated for detecting levels of methylation levels ranging from 0.05%-10%, and it can confidently detect differences between samples as small as -0.25% of the total cytosine residues, which corresponds to -5% differences in global DNA methylation. The procedure routinely requires 50-100 ng of DNA sample, although much smaller amounts (as low as 5 ng) have been successfully profiled. Another major benefit of this method is that it is not adversely affected by poor-quality DNA (e.g., DNA derived from LEPE samples). 3. ELISA-Based Methods

[0165] There are several commercially available kits, all enzyme-linked immunosorbent assay (ELISA) based, that enable the quick assessment of DNA methylation status. These assays include Global DNA Methylation ELISA, available from Cell Biolabs; Imprint Methylated DNA Quantification kit (sandwich ELISA), available from Sigma-Aldrich; EpiSeeker methylated DNA Quantification Kit, available from abeam; Global DNA Methylation Assay — LINE-1, available from Active Motif; 5-mC DNA ELISA Kit, available from Zymo Research; MethylFlash Methylated DNA5-mC Quantification Kit and MethylFlash Methylated DNA5-mC Quantification Kit, available from Epigentek.

[0166] Briefly, the DNA sample is captured on an ELISA plate, and the methylated cytosines are detected through sequential incubations steps with: (1) a primary antibody raised against 5 Me; (2) a labelled secondary antibody; and then (3) colorimetric/fluorometric detection reagents.

[0167] The Global DNA Methylation Assay — LINE-1 specifically determines the methylation levels of LINE-1 (long interspersed nuclear elements- 1) retrotransposons, of which -17% of the human genome is composed. These are well established as a surrogate for global DNA methylation. Briefly, fragmented DNA is hybridized to biotinylated LINE-1 probes, which are then subsequently immobilized to a streptavidin-coated plate. Following washing and blocking steps, methylated cytosines are quantified using an anti-5 mC antibody, HRP-conjugated secondary antibody and chemiluminescent detection reagents. Samples are quantified against a standard curve generated from standards with known LINE- 1 methylation levels. The manufacturers claim the assay can detect DNA methylation levels as low as 0.5%. Thus, by analysing a fraction of the genome, it is possible to achieve better accuracy in quantification.

4. LINE-1 Pyrosequencing

[0168] Levels of LINE- 1 methylation can alternatively be assessed by another method that involves the bisulfite conversion of DNA, followed by the PCR amplification of LINE-1 conservative sequences. The methylation status of the amplified fragments is then quantified by pyrosequencing, which is able to resolve differences between DNA samples as small as -5%. Even though the technique assesses LINE-1 elements and therefore relatively few CpG sites, this has been shown to reflect global DNA methylation changes very well. The method is particularly well suited for high throughput analysis of cancer samples, where hypomethylation is very often associated with poor prognosis. This method is particularly suitable for human DNA, but there are also versions adapted to rat and mouse genomes.

5. AFLP and RFLP

[0169] Detection of fragments that are differentially methylated could be achieved by traditional PCR-based amplification fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) or protocols that employ a combination of both.

6. LUMA

[0170] The LUMA (luminometric methylation assay) technique utilizes a combination of two DNA restriction digest reactions performed in parallel and subsequent pyrosequencing reactions to fill-in the protruding ends of the digested DNA strands. One digestion reaction is performed with the CpG methylation- sensitive enzyme Hpall; while the parallel reaction uses the methylation-insensitive enzyme MspI, which will cut at all CCGG sites. The enzyme EcoRI is included in both reactions as an internal control. Both MspI and Hpall generate 5'-CG overhangs after DNA cleavage, whereas EcoRI produces 5'-AATT overhangs, which are then filled in with the subsequent pyrosequencing-based extension assay. Essentially, the measured light signal calculated as the Hpall/MspI ratio is proportional to the amount of unmethylated DNA present in the sample. As the sequence of nucleotides that are added in pyro sequencing reaction is known, the specificity of the method is very high and the variability is low, which is essential for the detection of small changes in global methylation. LUMA requires only a relatively small amount of DNA (250-500 ng), demonstrates little variability and has the benefit of an internal control to account for variability in the amount of DNA input.

7. Bisulfite Sequencing

[0171] The bisulfite treatment of DNA mediates the deamination of cytosine into uracil, and these converted residues will be read as thymine, as determined by PCR-amplification and subsequent Sanger sequencing analysis. However, 5 mC residues are resistant to this conversion and, so, will remain read as cytosine. Thus, comparing the Sanger sequencing read from an untreated DNA sample to the same sample following bisulfite treatment enables the detection of the methylated cytosines. With the advent of next-generation sequencing (NGS) technology, this approach can be extended to DNA methylation analysis across an entire genome. To ensure complete conversion of non-methylated cytosines, controls may be incorporated for bisulfite reactions.

[0172] Whole genome bisulfite sequencing (WGBS) is similar to whole genome sequencing, except for the additional step of bisulfite conversion. Sequencing of the 5 mC- enriched fraction of the genome is not only a less expensive approach, but it also allows one to increase the sequencing coverage and, therefore, precision in revealing differentially- methylated regions. Sequencing could be done using any existing NGS platform; Illumina and Life Technologies both offer kits for such analysis.

[0173] Bisulfite sequencing methods include reduced representation bisulfite sequencing (RRBS), where only a fraction of the genome is sequenced. In RRBS, enrichment of CpG-rich regions is achieved by isolation of short fragments after MspI digestion that recognizes CCGG sites (and it cut both methylated and unmethylated sites). It ensures isolation of -85% of CpG islands in the human genome. Then, the same bisulfite conversion and library preparation is performed as for WGBS. The RRBS procedure normally requires -100 ng - 1 pg of DNA.

8. Methods that exclude bisulfite conversion

[0174] In some aspects, direct detection of modified bases without bisulfite conversion may be used to detect methylation. Pacific Biosciences company has developed a way to detect methylated bases directly by monitoring the kinetics of polymerase during single molecule sequencing and offers a commercial product for such sequencing (further described in Flusberg B.A., et al., Nat. Methods. 2010;7:461-465, which is herein incorporated by reference). Other methods include nanopore-based single-molecule real-time sequencing technology (SMRT), which is able to detect modified bases directly (described in Laszlo A.H. et al., Proc. Natl. Acad. Sci. USA. 2013 and Schreiber J., et al., Proc. Natl. Acad. Sci. USA. 2013, which are herein incorporated by reference).

9. Array or Bead Hybridization

[0175] Methylated DNA fractions of the genome, usually obtained by immunoprecipitation, could be used for hybridization with microarrays. Currently available examples of such arrays include: the Human CpG Island Microarray Kit (Agilent), the GeneChip Human Promoter LOR Array and the GeneChip Human Tiling 2. OR Array Set (Affymetrix). [0176] The search for differentially-methylated regions using bisulfite-converted DNA could be done with the use of different techniques. Some of them are easier to perform and analyse than others, because only a fraction of the genome is used. The most pronounced functional effect of DNA methylation occurs within gene promoter regions, enhancer regulatory elements and 3' untranslated regions (3'UTRs). Assays that focus on these specific regions, such as the Infinium HumanMethylation450 Bead Chip array by Illumina, can be used. The arrays can be used to detect methylation status of genes, including miRNA promoters, 5' UTR, 3' UTR, coding regions (~17 CpG per gene) and island shores (regions ~2 kb upstream of the CpG islands).

[0177] Briefly, bisulfite-treated genomic DNA is mixed with assay oligos, one of which is complimentary to uracil (converted from original unmethylated cytosine), and another is complimentary to the cytosine of the methylated (and therefore protected from conversion) site. Following hybridization, primers are extended and ligated to locus- specific oligos to create a template for universal PCR. Finally, labelled PCR primers are used to create detectable products that are immobilized to bar-coded beads, and the signal is measured. The ratio between two types of beads for each locus (individual CpG) is an indicator of its methylation level.

[0178] It is possible to purchase kits that utilize the extension of methylation- specific primers for validation studies. In the VeraCode Methylation assay from Illumina, 96 or 384 user-specified CpG loci are analysed with the GoldenGate Assay for Methylation. Differently from the BeadChip assay, the VeraCode assay requires the BeadXpress Reader for scanning.

10. Methyl- Sensitive Cut Counting: Endonuclease Digestion Followed by Sequencing

[0179] As an alternative to sequencing a substantial amount of methylated (or unmethylated) DNA, one could generate snippets from these regions and map them back to the genome after sequencing. Moreover, coverage in NGS could be good enough to quantify the methylation level for particular loci. The technique of serial analysis of gene expression (SAGE) has been adapted for this purpose and is known as methylation- specific digital karyotyping, as well as a similar technique, called methyl- sensitive cut counting (MSCC).

[0180] In summary, in all of these methods, methylation-sensitive endonuclease(s), e.g., Hpall is used for initial digestion of genomic DNA in unmethylated sites followed by adaptor ligation that contains the site for another digestion enzyme that is cut outside of its recognized site, e.g., EcoP15I or Mmel. These ways, small fragments are generated that are located in close proximity to the original Hpall site. Then, NGS and mapping to the genome are performed. The number of reads for each Hpall site correlates with its methylation level.

[0181] Recently, a number of restriction enzymes have been discovered that use methylated DNA as a substrate (methylation-dependent endonucleases). Most of them were discovered and are sold by SibEnzyme: BisI, BlsI, Glal. Glul, Krol, Mtel, Pcsl, PkrI. The unique ability of these enzymes to cut only methylated sites has been utilized in the method that achieved selective amplification of methylated DNA. Three methylation-dependent endonucleases that are available from New England Biolabs (FspEI, MspJI and LpnPI) are type IIS enzymes that cut outside of the recognition site and, therefore, are able to generate snippets of 32bp around the fully-methylated recognition site that contains CpG. These short fragments could be sequences and aligned to the reference genome. The number of reads obtained for each specific 32-bp fragment could be an indicator of its methylation level. Similarly, short fragments could be generated from methylated CpG islands with Escherichia coli’s methylspecific endonuclease McrBC, which cuts DNA between two half-sites of (G/A) mC that are lying within 50 bp-3000 bp from each other. This is a very useful tool for isolation of methylated CpG islands that again can be combined with NGS. Being bisulfite-free, these three approaches have a great potential for quick whole genome methylome profiling.

B. Sequencing

[0182] DNA, including bisulfite-converted DNA could be used for the amplification of the region of interest followed by sequencing. Primers are designed around the CpG island and used for PCR amplification of bisulfite-converted DNA. The resulting PCR products could be cloned and sequenced. Accordingly, aspects of the disclosure may include sequencing nucleic acids to detect methylation of nucleic acids and/or biomarkers. In some embodiments, the methods of the disclosure include a sequencing method. Exemplary sequencing methods include those described below.

1. Massively parallel signature sequencing (MPSS).

[0183] The first of the next-generation sequencing technologies, massively parallel signature sequencing (or MPSS), was developed in the 1990s at Lynx Therapeutics. MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides. This method made it susceptible to sequence- specific bias or loss of specific sequences. Because the technology was so complex, MPSS was only performed 'in-house' by Lynx Therapeutics and no DNA sequencing machines were sold to independent laboratories. Lynx Therapeutics merged with Solexa (later acquired by Illumina) in 2004, leading to the development of sequencing-by- synthesis, a simpler approach acquired from Manteia Predictive Medicine, which rendered MPSS obsolete. However, the essential properties of the MPSS output were typical of later "next-generation" data types, including hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels. Indeed, the powerful Illumina HiSeq2000, HiSeq2500 and MiSeq systems are based on MPSS.

2. Polony sequencing.

[0184] The Polony sequencing method, developed in the laboratory of George M. Church at Harvard, was among the first next-generation sequencing systems and was used to sequence a full genome in 2005. It combined an in vitro paired- tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of >99.9999% and a cost approximately 1/9 that of Sanger sequencing. The technology was licensed to Agencourt Biosciences, subsequently spun out into Agencourt Personal Genomics, and eventually incorporated into the Applied Biosystems SOLiD platform, which is now owned by Life Technologies.

3. 454 pyrosequencing.

[0185] A parallelized version of pyro sequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picoliter-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other. 4. Illumina (Solexa) sequencing.

[0186] Solexa, now part of Illumina, developed a sequencing method based on reversible dye-terminators technology, and engineered polymerases, that it developed internally. The terminated chemistry was developed internally at Solexa and the concept of the Solexa system was invented by Balasubramanian and Klennerman from Cambridge University's chemistry department. In 2004, Solexa acquired the company Manteia Predictive Medicine in order to gain a massivelly parallel sequencing technology based on "DNA Clusters", which involves the clonal amplification of DNA on a surface. The cluster technology was co-acquired with Lynx Therapeutics of California. Solexa Ltd. later merged with Lynx to form Solexa Inc.

[0187] In this method, DNA molecules and primers are first attached on a slide and amplified with polymerase so that local clonal DNA colonies, later coined "DNA clusters", are formed. To determine the sequence, four types of reversible terminator bases (RT-bases) are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides, then the dye, along with the terminal 3' blocker, is chemically removed from the DNA, allowing for the next cycle to begin. Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera.

[0188] Decoupling the enzymatic reaction and the image capture allows for optimal throughput and theoretically unlimited sequencing capacity. With an optimal configuration, the ultimately reachable instrument throughput is thus dictated solely by the analog-to-digital conversion rate of the camera, multiplied by the number of cameras and divided by the number of pixels per DNA colony required for visualizing them optimally (approximately 10 pixels/colony). In 2012, with cameras operating at more than 10 MHz A/D conversion rates and available optics, fluidics and enzymatics, throughput can be multiples of 1 million nucleotides/second, corresponding roughly to one human genome equivalent at lx coverage per hour per instrument, and one human genome re- sequenced (at approx. 30x) per day per instrument (equipped with a single camera).

5. SOLiD sequencing.

[0189] Applied Biosystems' (now a Thermo Fisher Scientific brand) SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing single copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing. This sequencing by ligation method has been reported to have some issue sequencing palindromic sequences.

6. Ion Torrent semiconductor sequencing.

[0190] Ion Torrent Systems Inc. (now owned by Thermo Fisher Scientific) developed a system based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerization of DNA, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

7. DNA nanoball sequencing.

[0191] DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. The company Complete Genomics uses this technology to sequence samples submitted by independent researchers. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run and at low reagent costs compared to other next generation sequencing platforms. However, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult. This technology has been used for multiple genome sequencing projects. 8. Heliscope single molecule sequencing.

[0192] Heliscope sequencing is a method of single-molecule sequencing developed by Helicos Biosciences. It uses DNA fragments with added poly-A tail adapters which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Heliscope sequencer. The reads are short, up to 55 bases per run, but recent improvements allow for more accurate reads of stretches of one type of nucleotides. This sequencing method and equipment were used to sequence the genome of the M13 bacteriophage.

9. Single molecule real time (SMRT) sequencing.

[0193] SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences, the SMRT technology developer, this methodology allows detection of nucleotide modifications (such as cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of 20,000 nucleotides or more, with average read lengths of 5 kilobases.

C. Additional Assay Methods

[0194] In some embodiments, methods involve amplifying and/or sequencing one or more target genomic regions using at least one pair of primers specific to the target genomic regions. In certain embodiments, the primers are heptamers. In other embodiments, enzymes are added such as primases or primase/polymerase combination enzyme to the amplification step to synthesize primers.

[0195] In some embodiments, arrays can be used to detect nucleic acids of the disclosure. An array comprises a solid support with nucleic acid probes attached to the support. Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodoret al., 1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is used in certain aspects, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated in their entirety for all purposes.

[0196] In addition to the use of arrays and microarrays, it is contemplated that a number of difference assays could be employed to analyze nucleic acids. Such assays include, but are not limited to, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, digital PCR, dd PCR (digital droplet PCR), nCounter (nanoString), BEAMing (Beads, Emulsions, Amplifications, and Magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (Tagged-Amplicon deep sequencing), PAP (Pyrophosphorolysis-activation polymerization), next generation RNA sequencing, northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Bridge Litigation Assay (Genaco).

[0197] Amplification primers or hybridization probes can be prepared to be complementary to a genomic region, biomarker, probe, or oligo described herein. The term "primer" or “probe” as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process and/or pairing with a single strand of an oligo of the disclosure, or portion thereof. Typically, primers are oligonucleotides from ten to twenty and/or thirty nucleic acids in length, but longer sequences can be employed. Primers may be provided in double- stranded and/or single- stranded form, although the single- stranded form is preferred.

[0198] The use of a probe or primer of between 13 and 100 nucleotides, particularly between 17 and 100 nucleotides in length, or in some aspects up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length may be used to increase stability and/or selectivity of the hybrid molecules obtained. One may design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

[0199] In one embodiment, each probe/primer comprises at least 15 nucleotides. For instance, each probe can comprise at least or at most 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein). They may have these lengths and have a sequence that is identical or complementary to a gene described herein. Particularly, each probe/primer has relatively high sequence complexity and does not have any ambiguous residue (undetermined "n" residues). The probes/primers can hybridize to the target gene, including its RNA transcripts, under stringent or highly stringent conditions. It is contemplated that probes or primers may have inosine or other design implementations that accommodate recognition of more than one human sequence for a particular biomarker.

[0200] For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0201] In one embodiment, quantitative RT-PCR (such as TaqMan, ABI) is used for detecting and comparing the levels or abundance of nucleic acids in samples. The concentration of the target DNA in the linear portion of the PCR process is proportional to the starting concentration of the target before the PCR was begun. By determining the concentration of the PCR products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. This direct proportionality between the concentration of the PCR products and the relative abundances in the starting material is true in the linear range portion of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the sampling and quantifying of the amplified PCR products may be carried out when the PCR reactions are in the linear portion of their curves. In addition, relative concentrations of the amplifiable DNAs may be normalized to some independent standard/control, which may be based on either internally existing DNA species or externally introduced DNA species. The abundance of a particular DNA species may also be determined relative to the average abundance of all DNA species in the sample.

[0202] In one embodiment, the PCR amplification utilizes one or more internal PCR standards. The internal standard may be an abundant housekeeping gene in the cell or it can specifically be GAPDH, GUSB and P-2 microglobulin. These standards may be used to normalize expression levels so that the expression levels of different gene products can be compared directly. A person of ordinary skill in the art would know how to use an internal standard to normalize expression levels.

[0203] A problem inherent in some samples is that they are of variable quantity and/or quality. This problem can be overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable DNA fragment that is similar or larger than the target DNA fragment and in which the abundance of the DNA representing the internal standard is roughly 5-100 fold higher than the DNA representing the target nucleic acid region.

[0204] In another embodiment, the relative quantitative RT-PCR uses an external standard protocol. Under this protocol, the PCR products are sampled in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling can be empirically determined for each target DNA fragment. In addition, the nucleic acids isolated from the various samples can be normalized for equal concentrations of amplifiable DNAs.

[0205] A nucleic acid array can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more different polynucleotide probes, which may hybridize to different and/or the same biomarkers. Multiple probes for the same gene can be used on a single nucleic acid array. Probes for other disease genes can also be included in the nucleic acid array. The probe density on the array can be in any range. In some embodiments, the density may be or may be at least 50, 100, 200, 300, 400, 500 or more probes/cm2 (or any range derivable therein).

[0206] Specifically contemplated are chip-based nucleic acid technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization (see also, Pease et al., 1994; and Fodor et al, 1991). It is contemplated that this technology may be used in conjunction with evaluating the expression level of one or more cancer biomarkers with respect to diagnostic, prognostic, and treatment methods.

[0207] Certain embodiments may involve the use of arrays or data generated from an array. Data may be readily available. Moreover, an array may be prepared in order to generate data that may then be used in correlation studies.

VI. Sample Preparation

[0208] In certain aspects, methods involve obtaining a sample from a subject. The methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, endoscopic biopsy, shave biopsy or skin biopsy. In certain embodiments the sample is obtained from a biopsy from thyroid tissue by any of the biopsy methods previously mentioned. In other embodiments the sample may be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, neck, lymph node, or thyroid tissue. Alternatively, the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[0209] A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.

[0210] The sample may be obtained by methods known in the art. In certain embodiments the samples are obtained by biopsy. In other embodiments the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples, such as multiple thyroid samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (for example thyroid) and one or more samples from another specimen (for example lymph node) may be obtained for diagnosis by the methods. In some cases, multiple samples such as one or more samples from one tissue type (e.g. thyroid) and one or more samples from another specimen (e.g. serum) may be obtained at the same or different times. Samples may be obtained at different times are stored and/or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other histopathological analysis methods.

[0211] In some embodiments the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, surgeon, otolaryngologist, or a pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business may consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0212] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy. The method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some embodiments, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0213] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In one embodiment, the sample is a fine needle aspirate of a thyroid or a suspected thyroid tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.

[0214] In some embodiments of the present methods, the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.

[0215] In some embodiments of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.

[0216] In some embodiments, the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample.

VII. Cancer Therapy

[0217] In some embodiments, the method further comprises administering a cancer therapy to the patient. The cancer therapy may be chosen based on the expression level measurements, alone or in combination with the clinicopathologic characteristics of the patient. In some embodiments, the cancer therapy comprises a local cancer therapy and/or a systemic cancer therapy. In some embodiments, the cancer therapy excludes a systemic cancer therapy. In some embodiments, the cancer therapy excludes a local therapy. In some embodiments, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some embodiments, the cancer therapy comprises an immunotherapy, which may be an immune checkpoint therapy. In some embodiments, the treatment possibilities comprise 1. Active surveillance; 2. Surgery; 3. Surgery+radioactive iodine; 4. Radioactive iodine (if recurrent or residual disease); 4. Systemic therapy (targeted therapy, immunotherapy, chemotherapy or combination thereof) with or without surgery +/- radiation. In some embodiments, the cancer therapy may include one or more of Vemurafenib, Encorafenib, Binimetinib, Cabozantinib-S-Malate, Vandetanib, Cabozantinib-S-Malate, Dabrafenib Mesylate, Doxorubicin Hydrochloride, Pralsetinib, Lenvatinib Mesylate, Lenvatinib Mesylate, Trametinib Dimethyl Sulfoxide, Sorafenib Tosylate, Pralsetinib, Selpercatinib, Selpercatinib, Sorafenib Tosylate, Dabrafenib Mesylate, Trametinib Dimethyl Sulfoxide, and/or Vandetanib. In some embodiments, cytotoxic chemotherapy (such as taxanes and/or platinum-based chemotherapy (such as cisplatin) is utilized. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.

VIII. Therapeutic Methods

[0218] In certain embodiments of the disclosure, a therapy is provided based on the outcome of methods encompassed herein. In certain embodiments, therapeutic compositions may be used for in vivo, in vitro, or ex vivo administration. The route of administration of the composition may be, for example, intracutaneous, subcutaneous, intravenous, local (including radio frequency ablation and ethanol ablation), topical, and intraperitoneal administrations.

A. Surgery

[0219] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electro surgery, and microscopically-controlled surgery (Mohs’ surgery).

[0220] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well. B. Chemotherapies

[0221] In some embodiments, the additional therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon- a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0222] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg/m2 to about 20 mg/m2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments. In some embodiments, the amount of cisplatin delivered to the cell and/or subject in conjunction with the construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.

[0223] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter/TNFa construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and/or TNF-a, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a. [0224] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain embodiments, appropriate intravenous doses for an adult include about 60 mg/m2 to about 75 mg/m2 at about 21 -day intervals or about 25 mg/m2 to about 30 mg/m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg/m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone- marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.

[0225] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and/or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria, is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day, intravenous doses include, for example, initially about 40 mg/kg to about 50 mg/kg in divided doses over a period of about 2 days to about 5 days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10 days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5 mg/kg/day to about 3 mg/kg/day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0226] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode- oxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg/m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0227] Gemcitabine diphosphate (GEMZAR®, Eli Filly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.

[0228] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.

C. Radiotherapy

[0229] In some embodiments, the therapy comprises radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art. In specific embodiments, radioactive iodine is provided one or more times to the patient. The radiation can be given with curative or palliative intent with varied dosages and durations, known to those of skill in the art.

D. Checkpoint Inhibitors and Combination Treatment

[0230] Embodiments of the disclosure may include administration of immune checkpoint inhibitors, which are further described below.

1. PD-1, PDL1, and PDL2 inhibitors

[0231] PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and/or PDL1 activity. [0232] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0233] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US2014/0022021, and US2011/0008369, all incorporated herein by reference.

[0234] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2) fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009/ 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0235] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0236] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

2. CTLA-4, B7-1, and B7-2

[0237] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co- stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0238] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0239] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in any of the methods encompassed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001/014424, W02000/037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0240] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1/14424).

[0241] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

E. Polysaccharides

[0242] In some embodiments, the additional therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.

F. Oncolytic virus

[0243] In some embodiments, the additional therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumour. Oncolytic viruses are thought not only to cause direct destruction of the tumour cells, but also to stimulate host anti-tumour immune responses for long-term immunotherapy.

G. Neoantigens

[0244] In some embodiments, the additional therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.

H. Pharmaceutical Compositions

[0245] In certain aspects, the compositions or agents for use in the methods, such as treatments for cancer, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as thyroid or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like. [0246] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0247] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0248] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0249] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0250] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0251] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0252] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. [0253] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0254] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0255] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

I. Other Agents

[0256] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that treat cancer.

J. Additional Agents

1. Immunostimulators

[0257] In some embodiments, the method further comprises administration of an additional agent. In some embodiments, the additional agent is an immuno stimulator. The term “immuno stimulator” as used herein refers to a compound that can stimulate an immune response in a subject, and may include an adjuvant. In some embodiments, an immuno stimulator is an agent that does not constitute a specific antigen, but can boost the strength and longevity of an immune response to an antigen. Such immunostimulators may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL (ASO4), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.), liposomes and liposomal formulations such as AS01, synthesized or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic block co-polymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins, such as bacterial toxoids or toxin fragments.

[0258] In some embodiments, the additional agent comprises an agonist for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinations thereof. In some embodiments, additional agents comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited immunostimulators comprise imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381, U.S. Published Patent Application 2010/0075995, or WO 2010/018132; immuno stimulatory DNA; or immunostimulatory RNA. In some embodiments, the additional agents also may comprise immunostimulatory RNA molecules, such as but not limited to dsRNA, poly I:C or poly Lpoly C12U (available as Ampligen.RTM., both poly I:C and poly I:polyC12U being known as TLR3 stimulants), and/or those disclosed in F. Heil et al., "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway" WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity" U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al., "Immuno stimulatory viral RNA oligonucleotides and use for treating cancer and infections" WO 2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods" WO 2003086280 A2. In some embodiments, an additional agent may be a TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G, and/or HMGB-1. In some embodiments, additional agents may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725.

[0259] In some embodiments, additional agents may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals). In some embodiments, additional agents may be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some embodiments, additional agents may be activated components of immune complexes. Additional agents also include complement receptor agonists, such as a molecule that binds to CD21 or CD35. In some embodiments, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some embodiments, immuno stimulators are cytokines, which are small proteins or biological factors (in the range of 5 kD-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. In some embodiments, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.

2. Immunotherapies

[0260] In some embodiments, the additional therapy comprises a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour- associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immumotherapies are known in the art, and some are described below. a. Activation of co-stimulatory molecules

[0261] In some embodiments, the immunotherapy comprises an activation (i.e., agonist) of a co-stimulatory molecule. In some embodiments, the activator comprises an activator of B7- 1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Activator include agonistic antibodies, polypeptides, compounds, and nucleic acids. b. Dendritic cell therapy

[0262] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0263] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).

[0264] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0265] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0266] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets. c. CAR-T cell therapy

[0267] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0268] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signalling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0269] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19. d. Cytokine therapy

[0270] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0271] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFN ).

[0272] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy. e. Adoptive T-cell therapy

[0273] Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumour death.

[0274] Multiple ways of producing and obtaining tumour targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens. f. Checkpoint Inhibitors and Combination Treatment

[0275] In some embodiments, the additional therapy comprises immune checkpoint inhibitors. Certain embodiments are further described below.

(1) PD-1, PDL1, and PDL2 inhibitors

[0276] PD -1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and/or PDL1 activity.

[0277] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0278] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US2014/0022021, and US2011/0008369, all incorporated herein by reference.

[0279] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2) fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009/ 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0280] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0281] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. (2) CTLA-4, B7-1, and B7-2

[0282] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co- stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0283] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0284] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in any of the methods encompassed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001/014424, W02000/037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0285] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1/14424). [0286] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

K. Polypeptide Expression

[0287] In some aspects, there are nucleic acid molecule encoding polypeptides or peptides of the disclosure (e.g TCR genes). These may be generated by methods known in the art, e.g., isolated from B cells of mice that have been immunized and isolated, phage display, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules or by recombinant methods.

1. Expression

[0288] The nucleic acid molecules may be used to express large quantities of polypeptides. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for humanization of the TCR genes.

2. Vectors

[0289] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, alpha chain, beta chain, or the antigen-binding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well. [0290] To express the polypeptides or peptides of the disclosure, DNAs encoding the polypeptides or peptides are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally complete human CH or CL immunoglobulin or TCR sequence with appropriate restriction sites engineered so that any variable region sequences can be easily inserted and expressed. In some aspects, a vector that encodes a functionally complete human TCR alpha or TCR beta sequence with appropriate restriction sites engineered so that any variable sequence or CDR1, CDR2, and/or CDR3 can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a poly linker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art.

3. Expression Systems

[0291] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system. L. Methods of Gene Transfer

[0292] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

1. Host Cells

[0293] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. Antibodies can be expressed in a variety of cell types. An expression construct encoding an antibody can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the antibody expression construct can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NF AT- 1 or NF-KB, both of which are transcription factors that can be activated upon T-cell activation. Control of antibody expression allows T cells, such as tumor- targeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0294] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.

M. Isolation

[0295] The nucleic acid molecule encoding either or both of the entire heavy, light, alpha, and beta chains of an antibody or TCR, or the variable regions thereof may be obtained from any source that produces antibodies. Methods of isolating mRNA encoding an antibody are well known in the art. See e.g., Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. See, e.g., Kabat et al., 1991, supra. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed in a cell into which they have been introduced and the antibody isolated.

IX. Immunotherapy

[0296] In some embodiments, the methods comprise administration of a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immumotherapies are known in the art, and some are described below.

A. Inhibition of co-stimulatory molecules

[0297] In some embodiments, the immunotherapy comprises an inhibitor of a costimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

B. Dendritic cell therapy

[0298] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0299] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).

[0300] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0301] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0302] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

C. CAR-T cell therapy

[0303] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0304] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signalling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0305] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19.

D. Cytokine therapy

[0306] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0307] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFN ). [0308] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

E. Adoptive T-cell therapy

[0309] Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumour death.

[0310] Multiple ways of producing and obtaining tumour targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.

[0311] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, embodiments of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some embodiments, the patient is one that has been determined to be resistant to a therapy described herein. In some embodiments, the patient is one that has been determined to be sensitive to a therapy described herein.

X. Administration of Therapeutic Compositions

[0312] The therapy provided herein may comprise administration of one or a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second cancer treatments are administered in a separate composition. In some embodiments, the first and second cancer treatments are in the same composition.

[0313] In some embodiments, the first cancer therapy and the second cancer therapy are administered substantially simultaneously. In some embodiments, the first cancer therapy and the second cancer therapy are administered sequentially. In some embodiments, the first cancer therapy, the second cancer therapy, and a third therapy are administered sequentially. In some embodiments, the first cancer therapy is administered before administering the second cancer therapy. In some embodiments, the first cancer therapy is administered after administering the second cancer therapy.

[0314] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0315] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0316] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0317] In some embodiments, the first cancer therapy comprises a first cancer protein, a nucleic acid encoding for the first cancer protein, a vector comprising the nucleic acid encoding for the first cancer protein, or a cell comprising the first cancer protein, a nucleic acid encoding for the first cancer protein, or a vector comprising the nucleic acid encoding for the first cancer protein. In some embodiments, a single dose of the first cancer protein therapy is administered. In some embodiments, multiple doses of the first cancer protein are administered. In some embodiments, the first cancer protein is administered at a dose of between 1 mg/kg and 5000 mg/kg. In some embodiments, the first cancer protein is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,

119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,

138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,

157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,

176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,

195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,

214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,

233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,

252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,

271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,

290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,

309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,

328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,

347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,

366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,

385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,

404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,

423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,

442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,

461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,

480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,

499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517,

518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536,

537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555,

556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700,

800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg/kg.

[0318] In some embodiments, a single dose of the second cancer therapy is administered. In some embodiments, multiple doses of the second cancer therapy are administered. In some embodiments, the second cancer therapy is administered at a dose of between 1 mg/kg and 100 mg/kg. In some embodiments, the second cancer therapy is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,

99, or 100 mg/kg.

[0319] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.

[0320] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0321] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0322] It will be understood by those skilled in the art and made aware that dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0323] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0324] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0325] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0326] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0327] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0328] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum mono stearate and gelatin.

[0329] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0330] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0331] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactic ally effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

XI. Kits

[0332] Certain aspects of the present disclosure also concern kits containing compositions of the disclosure or compositions to implement methods encompassed herein. In some embodiments, kits can be used to evaluate one or more biomarkers. In certain embodiments, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more genes, polynucleotides, probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein. In some embodiments, there are kits for evaluating biomarker activity in a cell.

[0333] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0334] Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.

[0335] Kits for using probes, synthetic nucleic acids, nonsynthetic nucleic acids, and/or inhibitors of the disclosure for prognostic or diagnostic applications are included as part of the disclosure. Specifically contemplated are any such molecules corresponding to any biomarker identified herein, which includes nucleic acid primers/primer sets and probes that are identical to or complementary to all or part of a biomarker, which may include noncoding sequences of the biomarker, as well as coding sequences of the biomarker.

[0336] In certain aspects, negative and/or positive control nucleic acids, probes, and inhibitors are included in some kit embodiments. In addition, a kit may include a sample that is a negative or positive control for methylation of one or more biomarkers.

[0337] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.

[0338] Any embodiment of the disclosure involving specific biomarker by name is contemplated also to cover embodiments involving biomarkers whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified nucleic acid.

[0339] Embodiments of the disclosure include kits for analysis of a cytological or pathological sample by assessing biomarker profile for a sample comprising, in suitable container means, two or more. The kit can further comprise reagents for labeling nucleic acids in the sample. The kit may also include labeling reagents, including at least one of amine- modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an amine-reactive dye.

[0340] In specific embodiments, the biomarker also has an accompanying computational/web tool that will allow individuals/clinicians to input their specific genomic alterations and the tool will determine their risk category using our methods.

XII. Examples

[0341] The following examples are included to demonstrate exemplary embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute exemplary modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. A. Example 1 - Exemplary Genes

[0342] Table 1 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 1.

Table 1

[0343] Table 2 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 2.

Table 2

[0344] Table 3 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 3.

Table 3

[0345] Table 4 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 4.

Table 4

[0346] Table 5 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 5.

Table 5

[0347] Table 6 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 6.

Table 6

[0348] Table 7 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 7.

Table 7

[0349] Table 8 provides an exemplary list of candidate genes for a gene signature or biomarker. For example, an expression-based gene signature or biomarker may be based on expressive levels of one or more of the genes in Table 8.

Table 8

B. Example 2 - Gene Signature Score in a Discovery Cohort

[0350] Baseline characteristics of patients in the discovery cohort are shown in Table 9 below.

Table 9

[0351] As shown in FIG. 1, a Kaplan-Meier analysis was performed to show that in the discovery cohort, a dichotomized gene signature score can separate patients who have high risk of recurrence, progression, or death due to papillary thyroid cancer from those with low risk of recurrence, progression, or death due to papillary thyroid cancer. This cohort contained 111 patients diagnosed with PTC. FIG. 1 shows that, using the gene signature score, 70 patients were categorized as high-risk (low score, the bottom curve in FIG. 1) with significantly decreased progression-free interval values in comparison to the low-risk (high score, the top curve in FIG. 1) group. The gene signature score was derived based on the genes in Table 1 using RNAseq data obtained from patient tumors. This gene signature was generated based on comparing gene expression differences between normal thyroid tissue, papillary thyroid tumors, and the most aggressive type of thyroid cancer, undifferentiated (anaplastic) thyroid cancer, using single-cell RNAseq. Differential gene expression analysis was used to identify candidate genes associated with thyroid cancer dedifferentiation. Dedifferentiation refers to a process whereby thyroid cancer cells lose specialized properties associated with their cell of origin, the thyroid follicular cell. These properties include those related to iodine metabolism and thyroid hormone regulation. De-differentiation has long been recognized as a biological phenomenon in highly lethal histologic subtypes of thyroid cancers including poorly differentiated and anaplastic thyroid carcinomas. In these tumors, dedifferentiation is identifiable based on the presence of canonical histopathologic features such as multiple mitoses, necrosis, and pleomorphic tumor cells. While it is known that papillary thyroid cancer can transform into poorly differentiated and anaplastic thyroid carcinomas through terminal dedifferentiation in rare cases, accumulating evidence suggests that milder degrees of dedifferentiation occurs in a larger subset of papillary thyroid carcinomas. As a result, these thyroid cancers behave aggressively with high risks of disease recurrence and/or progression. Despite a current conceptualization of dedifferentiation in thyroid cancer as a continuum, these earlier states of dedifferentiation are difficult to recognize based on histopathologic features alone.

[0352] The candidate genes in Table 1 were then evaluated in bulk RNAseq data, and a computational method was developed based on single-gene set gene set enrichment analysis (GSEA) to allow the score to be compared across patient cohorts. This approach overcomes one of the limitations of RNAseq data where comparison across experiments is confounded by batch effects.

[0353] As a continuous score, the gene signature score is significantly associated with progression-free interval after adjusting for age and overall TNM (as shown in Table 10), which are the clinical variables that are currently used to predict risk of papillary thyroid cancer at the time of diagnosis in a multivariable cox-proportional hazards model.

Table 10

*aHR: adjusted hazard ratio.

[0354] A machine learning approach based on recursive partitioning and decision-tree techniques was used to identify score cut-off thresholds for separating patients into low vs. high-risk groups. This in turn generates a dichotomized gene signature score where a high score represents low-risk disease while low score represents high-risk disease. [0355] As a dichotomized score, after adjusting for age and overall TNM stage, the gene signature score was significantly associated with progression-free interval (as shown in Table

11). Patients with low gene signature score (high risk) had an approximately 2.5 times higher risk of developing recurrence, progression, or death due to papillary thyroid carcinoma, compared to those with a high gene signature score. Analyses were performed using multivariable cox-proportional hazards models.

Table 11

|

*aHR: hazard ratio adjusted for age and overall TNM stage

C. Example 3 - Gene Signature Score in a Validation Cohort

[0356] Baseline characteristics of patients in the independent validation cohort are shown in Table 12 below.

Table 12

[0357] As shown in FIG. 2, a Kaplan-Meier analysis was performed to show that in the validation cohort, the dichotomized gene signature score generated in FIG. 1 (based on the genes in Table 1) can separate patients who have high risk of recurrence, progression, or death due to papillary thyroid cancer from those that have a low risk for recurrence, progression, or death due to papillary thyroid cancer. Using the same cut off generated in the discovery cohort using machine learning, an independent cohort of patients with PTC was evaluated. The gene signature score was again able to separate patients into high risk and low risk groups. In this cohort of 391 patients, 43 were categorized as high risk (low score, the bottom curve in FIG. 2) with significantly decreased progression-free interval values in comparison to the low risk group (high score, the top curve in FIG. 2).

[0358] As a continuous score, the gene signature score is significantly associated with progression-free interval after adjusting for age and overall TNM in the independent validation cohort in multivariable cox proportional hazards model analysis (as shown in Table 13).

Table 13

*aHR: adjusted hazard ratio

[0359] We also showed that the gene signature score used as a dichomotized score, was significantly associated with the risk of recurrence, progression, or death due to papillary thyroid cancer after adjusting for age and overall TNM stage (as shown in Table 14). Compared to patients with a high gene signature score, patients with a low gene signature score had an approximately 2.9x higher risk of developing recurrence, progression, or death due to papillary thyroid cancer in the independent validation cohort.

Table 14

*aHR: hazard ratio adjusted for age and overall TNM stage

D. Example 4 - Gene Signature Score Is Associated with Different Gene Expression Profiles

[0360] Using a dichotomized gene signature score, a high score represents preserved differentiation in the tumor and is associated with a low risk of recurrence, progression, or death due to papillary thyroid cancer. A low score on the other hand, represents a loss of differentiation, and is associated with a higher risk of recurrence, progression, or death due to papillary thyroid cancer.

[0361] In FIG. 3A, using differential gene expression analyses, we showed that in the discovery cohort, patients categorized as having a low-risk (TFC_H) of recurrence, progression, or death due to papillary thyroid cancer demonstrate a different gene expression profile compared to patients categorized as high-risk (TFC_L). In general, high-risk patients have loss of expression in key genes compared to patients who are low-risk. In FIG. 3A, the gene names listed in the column to the right are, from top to bottom: NR4A1, IER2, CYR61, MAFB, MT2A, CHCHD10, GADD45B, ZBTB16, NDUFB1, SNRPN, SORD, ID4, METTL7A, CLIC3, GBP1, FHL1, OTOS, RGS16, GCSH, CALR, CITED2, EGR1, KLF6, MTRNR2L8, ID1, PEBP1, ATP1A1, TFF3, MT1G, CRABP1, NDUFB2, SORBS2, IYD, TNFRSF11B, AAK1.

[0362] In FIG. 3B, Using principal components analyses, we also found that low-risk patients (TFC_H) have a different gene expression profile compared to patients who are high- risk (TFC_L). In FIG. 3B, the majority of the dots on the left of the 0.0 axis belong to TFC_H, and the majority of the dots on the right of the 0.0 axis belong to TFC_L.

E. Example 5 - Prognostic RNA Expression Cell-specific Integrated SignaturE (PRECISE) Study

[0363] In Example 5, subsets of genes are used for various clinical scenarios. Subset 1 includes 41 genes (shown in Table 4 above). Subset 2 includes 40 genes (shown in Table 5 above). Using genes from subset 2, a score was developed and named as Prognostic RNA Expression Cell-specific Integrated SignaturE (PRECISE). PRECISE score converts gene expression values into a positive score where higher score is associated with higher risk disease (e.g., higher risk of having a poor outcome such as recurrence, progression, or death due to disease; and higher risk of not responding to treatment). Subset 3 includes 23 genes (shown in Table 6 above). Subset 4 includes 17 genes (shown in Table 7 above), respectively. The Parent Set used for Example 5 includes 71 genes (shown in Table 8 above).

[0364] In FIG. 4, the variation is shown across cohorts including 1) TCGA PTC cohort, 2) MDACC PTC cohort, and 3) GATCI cohort of patients with anaplastic thyroid cancer. In the TCGA cohort, it shows the distribution of PRECISE across adjacent normal thyroid tissue and PTC tumors. In the MDACC PTC cohort, patients are further stratified into low, intermediate, and high risk based on their survival outcomes. It is shown that 1) the highest risk tumors, which are the anaplastic thyroid cancers in the GATCI cohort, have the highest PRECISE score; and 2) adjacent normal tissue in the TCGA PTC cohort have the lowest score. PRECISE score also increases as risk increases in the MDACC PTC cohort.

[0365] FIG. 5 shows that PRECISE score predicts response to radioactive iodine (RAI). In FIG. 5, PRECISE score is significantly associated with response to radioactive iodine (RAI) in the MDACC PTC cohort. Y axis shows the PRECISE score. X axis shows response to RAI. On average, patients that are refractory to RAI have higher PRECISE scores compared to those that are sensitive to RAI.

[0366] FIG. 6 shows that PRECISE score is associated with survival outcomes in PTC. In FIG. 6, Kaplan-Meier curve shows probability of recurrence/progression or death (PFS) due to PTC on the Y axis and time in years on the X axis. When separated into three categories, low, intermediate, or high, PRECISE score is associated with recurrence/progression or death due to PTC where high PRECISE score is associated with the worst prognosis. In the upper panel of FIG. 6, the top curve reflects the low PRECISE score category, the medium curve reflects the intermediate PRECISE score category, and the bottom curve reflects the high PRECISE score category. In the lower panel of FIG. 6, the top row reflects the low PRECISE score category, the medium row reflects the intermediate PRECISE score category, and the bottom row reflects the high PRECISE score category.

[0367] Table 15 below compares the association between gene signature scores and survival outcomes. All gene set scores were generated by converting gene expression values into a positive score where higher score is associated with higher risk disease. C-index shows the ability of each gene set score to predict survival outcome. Compared to the full gene set, C-index and effective size is preserved within the smaller gene subsets. For Table 15, progression-free survival defined as recurrence, progression, and/or death due to PTC. Diseasespecific survival defined as death due to PTC.

Table 15. Univariable analyses of the association between risk scores and survival

*HR: hazard ratio.

[0368] Table 16 below shows the results of multivariable survival analyses adjusting for known predictors of survival outcome in PTC including age, ATA risk category (currently used clinically to predict risk of recurrence), and TERT promoter mutation status (known to be associated with higher risk of recurrence but not currently used clinically to determine risk of recurrence). Model 1 adjusts for age, ATA risk category. Model 2 adjusts for age, ATA risk category, and TERT promoter mutation status. PRECISE score remains independently associated with progression-free survival after adjusting for currently known predictors of PTC recurrence.

Table 16. Multivariable analyses of the association between risk scores and progression- free survival

*aHR: adjusted hazard ratio.

[0369] Table 17 below shows the results of multivariable survival analyses adjusting for known predictors of disease- specific survival in PTC including age, TNM stage (currently used clinically to predict risk death due to PTC), and TERT promoter mutation status (known to be associated with higher risk of death but not currently used clinically to determine risk of recurrence). Model 1 adjusts for age, overall TNM stage. Model 2 adjusts for age, overall TNM stage, and TERT promoter mutation status. All gene set scores remain significantly associated with disease- specific survival after adjusting for known prognostic variables.

Table 17. Multivariable analyses of the association between risk scores and disease specific survival

*aHR: adjusted hazard ratio.

[0370] Table 18 below shows the predictive performance of multivariable cox -regression models for progression-free and disease- specific survival. For each survival outcome, evaluation was done for the predictive performance of a base model that contains currently known prognostic information available at the time of diagnosis. The C-index shows that for progression-free survival, the model containing currently known prognostic information has approximately 70% predictive accuracy. For disease-specific survival, the base model containing currently known prognostic information has approximately 89% predictive accuracy. In comparison, with the addition of the PRECISE score (shown in Tables 16 and 17 above), the C-index increases to 0.803 (reflecting about 80% predictive accuracy) and 0.930 (reflecting about 93% predictive accuracy), for progression-free survival and disease-specific survival, respectively.

Table 18. Survival outcome prediction using currently available known clinicogenomic information

[0371] In sum, the results in Example 5 show that the addition of PRECISE score improves the ability to predict survival outcomes in PTC beyond currently known/used information.

F. Example 6 - Exemplary Clinical Implementation

[0372] Any of the biomarkers or gene signatures encompassed herein, such as expressionbased biomarkers or gene signatures, may be used for risk stratification at the time of diagnosis and/or after surgery. At the time of diagnosis, one or more of the expression-based biomarkers or gene signatures encompassed herein may have valuable utility as it can be used to help determine initial treatment (e.g., surgery extent, such as partial thyroidectomy or total thyroidectomy, and need for additional adjuvant treatment, such as radioactive iodine). There are no currently available biomarkers to help guide this clinical decision making process for initial treatment, and available clinical information, such as staging, may not be sufficient. One or more of the expression-based biomarkers or gene signatures encompassed herein may also be used after surgery (e.g., test performed on surgically resected tissues) to help guide adjuvant treatment (e.g., predicting response to radioactive iodine) and to help plan surveillance frequency, timing, and/or duration of follow-up.

* * *

[0373] All of the methods encompassed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the systems, compositions, and methods of this disclosure have been described in terms of exemplary embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating thyroid cancer in an individual comprising administering one or more treatments to the individual after measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

2. The method of claim 1, wherein the method further comprises: generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; and determining risk level of the individual based on the comparison.

3. The method of claim 1, wherein the expression levels of the one or more genes are measured by a RNA-based assay.

4. The method of claim 3, wherein the RNA-based assay comprises RNA sequencing (RNA- Seq), single cell RNA-Seq, and/or microarray.

5. The method of any one of claims 1 to 4, wherein the sample from the individual comprises a thyroid biopsy tissue and/or cells.

6. The method of any one of claims 1 to 5, wherein the one or more genes comprise ten or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DI01, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

7. The method of any one of claims 1 to 5, wherein the one or more genes comprise twenty or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SEC26A7, MTRNR2E12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SEC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PECG2, SEC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2E8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2E1, GEUE, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

8. The method of any one of claims 1 to 5, wherein the one or more genes comprise thirty or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2E8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2E1, GEUE, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPE17, CEIC3, NDUFB1, C16orf89, IPCEF1, SEEENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

9. The method of any one of claims 1 to 5, wherein the one or more genes comprise forty or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SEC26A7, MTRNR2E12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SEC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PECG2, SEC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2E8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2E1, GEUE, CITED2, FHE1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SEC25A29, AC007952.4, KRT7, PEA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPE17, CEIC3, NDUFB1, C16orf89, IPCEF1, SEEENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

10. The method of any one of claims 1 to 5, wherein the one or more genes comprise fifty or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

11. The method of any one of claims 1 to 5, wherein the one or more genes comprise sixty or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

12. The method of any one of claims 1 to 5, wherein the one or more genes comprise seventy or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

13. The method of any one of claims 1 to 5, wherein the one or more genes comprise eighty or more genes selected from the group consisting of TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, and ZBTB16.

14. The method of any one of claims 1 to 5, wherein the one or more genes comprise TPO, DIO2, DIO1, IYD, FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof.

15. The method of any one of claims 2 to 14, wherein risk level of the individual is determined further based on one or more clinical variables of the individual.

16. The method of claim 15, wherein the one or more clinical variables of the individual comprise histopathology, radioactive iodine uptake, age, stage, and/or clinical behavior.

17. The method of any one of claims 1 to 16, wherein the one or more treatments comprise one or more of active surveillance and/or one or more cancer treatments.

18. The method of claim 17, wherein the one or more cancer treatments comprise a surgery, cervical lymph node dissection, observation, determination of extent of cervical lymph node dissection, or a combination thereof.

19. The method of claim 18, wherein the surgery comprises partial thyroidectomy or total thyroidectomy.

20. The method of claim 17, wherein the one or more cancer treatments comprise one or more of radioactive iodine, adjuvant therapy, radiation therapy, proton therapy, hormone therapy, chemotherapy, immunotherapy, bisphosphate therapy, cryotherapy, ultrasound, and/or palliative care.

21. The method of claim 20, wherein the adjuvant therapy comprises radioactive iodine.

22. The method of claim 17, wherein the active surveillance comprises a determination of frequency, timing, and/or duration of follow-up visits of the individual.

23. The method of claim 14, wherein the one or more treatments comprise radioactive iodine.

24. The method of any one of claims 1 to 23, wherein the thyroid cancer comprises thyroid cancers derived from thyroid follicular cells.

25. The method of claim 24, wherein the thyroid cancers derived from thyroid follicular cells comprise follicular thyroid cancer, papillary thyroid cancer, follicular thyroid cancer, poorly differentiated thyroid cancers, oncocytic thyroid cancer, medullary thyroid cancer, and/or anaplastic thyroid cancers.

26. The method of any one of claims 1 to 23, wherein the thyroid cancer is papillary thyroid cancer.

27. A method of administering a thyroid cancer therapy to an individual having modulation of expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

28. A method of treating an individual for thyroid cancer comprising: measuring expression levels of one or more genes from a sample from the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DI02, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof; and administering one or more treatments to the individual based on the levels of expression of the one or more genes.

29. The method of claim 28, wherein the method further comprises: generating a gene signature score based on the expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; and determining risk level of the individual based on the comparison.

30. The method of claim 28, wherein the method further comprises generation of a threshold to identify a prognostically efficacious threshold that can be applied across cohorts.

31. The method of claim 30, wherein the generation comprises machine learning.

32. The method of claim 28, wherein the method further comprises summarizing gene expression across sets of genes that can be applied across patient cohorts.

33. A method of determining prognosis of thyroid cancer comprising measuring expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SEC26A7, MTRNR2E12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SEC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PECG2, SEC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2E8, SOD3, OTOS, RGS16, AEDH1A1, PCP4, GBP1, TFCP2E1, GEUE, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPE17, CEIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

34. The method of claim 33, wherein the method further comprises: generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; and determining risk level of the individual based on the comparison.

35. The method of claim 33, wherein the method further comprises determining prognosis of thyroid cancer by analyzing a sample from an individual.

36. A method of determining recurrence and/or progression of thyroid cancer comprising measuring expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KEF6, TNFRSF11B, TSHR, CPQ, METTE7A, CAER, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

37. The method of claim 36, wherein the method further comprises: generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; and determining risk level of the individual based on the comparison.

38. The method of claim 36, wherein the method further comprises determining recurrence and/or progression of thyroid cancer by analyzing a sample from an individual.

39. A method of determining grade, clinical behavior, and/or aggressiveness of thyroid cancer comprising measuring expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

40. The method of claim 39, wherein the method further comprises: generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; and determining risk level of the individual based on the comparison.

41. The method of claim 39, wherein the method further comprises determining grade, clinical behavior, and/or aggressiveness of thyroid cancer by analyzing a sample from an individual.

42. A method of determining stage and/or spread of thyroid cancer comprising measuring expression levels of one or more genes, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

43. The method of claim 42, wherein the method further comprises: generating a gene signature score based on the measured expression levels of the one or more genes; comparing the gene signature score to a preset threshold value; and determining risk level of the individual based on the comparison.

44. The method of claim 42, wherein the method further comprises determining stage and/or spread of thyroid cancer by analyzing a sample from an individual.

45. A method of treating an individual in need thereof comprising measuring expression levels of one or more genes and treating the individual in need thereof with at least one thyroid cancer therapy, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

46. A method of identifying an individual for treatment of thyroid cancer comprising: measuring expression levels of one or more genes from a sample of the individual; predicting thyroid cancer status of the individual; and determining treatment for the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

47. A method of monitoring efficacy of one or more thyroid cancer therapies in an individual comprising measuring expression levels of one or more genes before and after the one or more thyroid cancer therapies are administered one or more times to the individual, wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

48. The method of claim 47, wherein the measuring occurs after the one or more thyroid cancer therapies are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.

49. A method of predicting or measuring thyroid cancer treatment response of an individual to one or more treatments comprising: measuring expression levels of one or more genes from a sample of the individual; and predicting or measuring whether or not the individual is responsive to the one or more treatments; wherein the one or more genes comprise TFF3, MT1G, TPO, MT1F, MT1X, TG, SLC26A7, MTRNR2L12, MT1H, GPX3, IYD, ID3, NUPR1, MT1E, SLC26A4-AS1, CRYAB, DIO2, CD24, CHCHD10, PLCG2, SLC26A4, ID4, SYNE1, SORBS2, ID1, FCGBP, CRABP1, MTRNR2L8, SOD3, OTOS, RGS16, ALDH1A1, PCP4, GBP1, TFCP2L1, GLUL, CITED2, FHL1, PAX8, NR4A1, PEBP1, CYR61, SORD, MAFB, EGR1, CKB, SLC25A29, AC007952.4, KRT7, PLA2R1, ATP1A1, GCSH, KRT8, DIO1, IER2, FOSB, ID2, RPL17, CLIC3, NDUFB1, C16orf89, IPCEF1, SELENBP1, MT2A, NDUFB2, EGR2, PRDX1, GADD45B, ADIRF, KLF6, TNFRSF11B, TSHR, CPQ, METTL7A, CALR, AAK1, SNRPN, RAP1GAP, ZFP36, ZBTB16, and/or a combination thereof.

50. The method of claim 49, wherein the one or more genes comprise TPO, DIO2, DIO1, IYD,

FHL1, IPCEF1, RAP1GAP, NDUFB2, GBP1, PRDX1, NR4A1, MT1G, MT2A, FOSB, ZFP36, SNRPN, SYNE1, SORD, GCSH, CYR61, SLC26A7, PLA2R1, SORBS2, TFF3, CITED2, TNFRSF11B, ALDH1A1, CLIC3, MT1E, KRT8, ID4, NUPR1, PCP4, NDUFB1, MT1X, PLCG2, MT1F, MAFB, CHCHD10, RPL17, and/or a combination thereof.

51. The method of claim 49 or 50, wherein the one or more treatments comprise one or more of active surveillance and/or one or more cancer treatments.

52. The method of claim 51, wherein the one or more cancer treatments comprise a surgery, cervical lymph node dissection, observation, determination of extent of cervical lymph node dissection, or a combination thereof.

53. The method of claim 52, wherein the surgery comprises partial thyroidectomy or total thyroidectomy.

54. The method of claim 51, wherein the one or more cancer treatments comprise one or more of radioactive iodine, adjuvant therapy, radiation therapy, proton therapy, hormone therapy, chemotherapy, immunotherapy, bisphosphate therapy, cryotherapy, ultrasound, and/or palliative care.

55. The method of claim 54, wherein the adjuvant therapy comprises radioactive iodine.

56. The method of claim 51, wherein the active surveillance comprises a determination of frequency, timing, and/or duration of follow-up visits of the individual.

57. The method of claim 49 or 50, wherein the one or more treatments comprise radioactive iodine.