US20230073731A1

US20230073731A1 - Gene expression analysis techniques using gene ranking and statistical models for identifying biological sample characteristics

Info

Publication number: US20230073731A1
Application number: US17/948,763
Authority: US
Inventors: Zoia Antysheva; Viktor Svekolkin; Nikita Kotlov; Anton Karelin; Ekaterina Postovalova
Original assignee: BostonGene Corp
Current assignee: BostonGene Corp
Priority date: 2019-12-05
Filing date: 2022-09-20
Publication date: 2023-03-09
Also published as: JP7684303B2; US11482301B2; IL293374A; CA3163904A1; US20210174899A1; EP4070317A1; WO2021113784A1; JP2023504555A; AU2020398660A1

Abstract

Techniques for determining one or more characteristics of a biological sample using rankings of gene expression levels in expression data obtained using one or more sequencing platforms is described. The techniques may include obtaining expression data for a biological sample of a subject. The techniques further include ranking genes in a set of genes based on their expression levels in the expression data to obtain a gene ranking and determining using the gene ranking and a statistical model, one or more characteristics of the biological sample.

Description

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 120 and is a continuation of U.S. patent application Ser. No. 17/113,008, filed Dec. 5, 2020, titled “GENE EXPRESSION ANALYSIS TECHNIQUES USING GENE RANKINGS AND STATISTICAL MODELS FOR IDENTIFYING BIOLOGICAL SAMPLE CHARACTERISTICS,” which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 62/943,976, filed Dec. 5, 2019, titled “MACHINE LEARNING TECHNIQUES FOR GENE EXPRESSION ANALYSIS” and U.S. Provisional Patent Application Ser. No. 63/060,512, filed Aug. 3, 2020, titled “MACHINE LEARNING TECHNIQUES FOR DETERMINING PERIPHERAL T-CELL LYMPHOMA (PTCL) SUBTYPE USING GENE EXPRESSION DATA”, the entire contents of each of which are incorporated by reference herein.

FIELD

Aspects of the technology described herein relate to determining characteristics of a biological sample obtained from a subject known to have, suspected of having, or at risk of having cancer by sequencing the biological sample using one or multiple sequencing platforms and analyzing the resulting gene expression data using machine learning techniques. In particular, the technology described herein involves using gene expression data from one or multiple sequencing platforms to determine characteristics of the biological sample, such as tissue of origin and cancer grade.

BACKGROUND

Characteristics of a biological cell may relate to the expression levels of certain genes. For example, a cancerous cell may have some genes upregulated and other genes downregulated relative to a normal, healthy cell. This relationship between cell characteristics and gene expression levels may be utilized in analyzing gene expression data for biological cells, such as data obtained using a gene expression microarray or by performing next generation sequencing, to determine characteristics of the biological cells.

SUMMARY

Some embodiments are directed to a computer-implemented method, comprising using at least one computer hardware processor to perform: obtaining expression data obtained at least in part by sequencing a biological sample of a subject having, suspected of having or at risk of having cancer, the expression data comprising expression levels for a plurality of genes, the plurality of genes comprising a set of genes; ranking at least some genes in the set of genes, based on their expression levels in the expression data to obtain a gene ranking; and determining, using the gene ranking and a statistical model trained using training data indicating a plurality of gene rankings of at least some of the genes in the set of genes obtained, at least one characteristic of the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the set of genes.
The at least one characteristic may be selected from cancer grade for cells in the biological sample (e.g., breast cancer grade, kidney clear cell cancer grade, lung adenocarcinoma grade), tissue of origin for cells in the biological sample (e.g., lung, pancreas, stomach, colon, liver, bladder, kidney, thyroid, lymph nodes, adrenal gland, skin, breast, ovary, prostrate, or cell of origin in a tissue such as e.g. germinal center B-cell (GCB) or activated B-cell (ABC)), histological information (tissue type, such as e.g. adenocarcinoma, squamous cell carcinoma, carcinoma, cystadenocarcinoma, sarcoma, and glioma) for cells in the biological sample, and cancer subtype (e.g. PTCL subtype such as, anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL)), viral status (e.g., HPV status, such as HPV-positive or HPV-negative for head and neck squamous cell carcinoma) for cells in the biological sample.
In some embodiments, the at least one characteristic of the biological sample is a physiological characteristic of cells in the biological sample or a tissue from which the cells originate. In some embodiments, the at least one characteristic is selected from cancer grade for cells in the biological sample, tissue of origin for cells in the biological sample, tissue type for cells in the biological sample, and cancer subtype for cells in the biological sample.
In some embodiments, the method further comprises performing sequencing of the biological sample using a gene expression microarray prior to obtaining the expression data. In some embodiments, the method further comprises performing next generation sequencing of the biological sample prior to obtaining the expression data.
In some embodiments, the at least one characteristic includes cancer grade for cells in the biological sample. In some embodiments, the at least one characteristic includes tissue of origin for cells in the biological sample.
In some embodiments, the subject has, is suspected of having, or is at risk of having breast cancer. In some embodiments, the set of genes is selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 3 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 5 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 20 genes selected from the group of genes listed in Table 1.
In some embodiments, the subject has, is suspected of having, or is at risk of having kidney cancer. In some embodiments, the subject has, is suspected of having, or is at risk of having clear cell kidney cancer. In some embodiments, the set of genes is selected from the group of genes listed in Table 2. In some embodiments, the set of genes comprises at least 3 genes selected from the group of genes listed in Table 2. In some embodiments, the set of genes comprises at least 5 genes selected from the group of genes listed in Table 2. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 2. In some embodiments, the set of genes comprises at least 20 genes selected from the group of genes listed in Table 2.
In some embodiments, the subject has, is suspected of having, or is at risk of having lymphoma. In some embodiments, the set of genes is selected from the group of genes listed in Table 3. In some embodiments, the set of genes comprises at least 3 genes selected from the group of genes listed in Table 3. In some embodiments, the set of genes comprises at least 5 genes selected from the group of genes listed in Table 3. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 3. In some embodiments, the set of genes comprises at least 20 genes selected from the group of genes listed in Table 3.
In some embodiments, the subject has, is suspected of having, or is at risk of having head and neck squamous cell carcinoma. In some embodiments, the set of genes is selected from the group of genes listed in Table 6. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 6.
In some embodiments, the at least one characteristic includes human papillomavirus status for cells in a biological sample. In some embodiments, the set of genes is selected from the group of genes listed in Table 8. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 8.
In some embodiments, the method further comprises ranking at least some genes in a second set of genes based on their expression levels in the expression data to obtain a second gene ranking; and determining, using the second gene ranking and a second statistical model trained using second training data indicating a plurality of rankings for the at least some of the genes in the second set of genes, at least one second characteristic of the biological sample.
In some embodiments, the at least one second characteristic includes cancer grade for cells in the biological sample. In some embodiments, the at least one second characteristic includes tissue of origin for cells in the biological sample.
In some embodiments, determining the gene ranking comprises determining a relative rank for each gene in the set of genes based on the expression levels. In some embodiments, determining the at least one characteristic further comprises providing the gene ranking as input to the statistical model and obtaining an output indicating the at least one characteristic. In some embodiments, the statistical model comprises a gradient boosted decision tree classifier. In some embodiments, the statistical model comprises a classifier selected from the group consisting of: a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
In some embodiments, the set of genes includes at least 5 genes. In some embodiments, the set of genes consists of 5-50 genes. In some embodiments, the set of genes consists of 5-300 genes.
In some embodiments, the method further comprises presenting, to a user, an indication of the at least one characteristic. In some embodiments, presenting the indication of the at least one characteristic further comprises displaying the at least one characteristic to the user in a graphical user interface (GUI).
In some embodiments, the at least one characteristic includes cancer grade for cells in the biological sample, and the cancer grade is selected from the group consisting of Grade 1, Grade 2, Grade 3, Grade 4, and Grade 5. In some embodiments, the at least one characteristic includes tissue of origin for cells in the biological sample, and the tissue of origin is selected from the group consisting of lung tissue, pancreas tissue, stomach tissue, colon tissue, liver tissue, bladder tissue, kidney tissue, thyroid tissue, lymph node tissue, adrenal gland tissue, skin tissue, breast tissue, ovary tissue, prostate tissue, urothelial tissue, cervical tissue, esophagus tissue, brain tissue, soft tissue, connective tissue, head tissue, and neck tissue. In some embodiments, the at least one characteristic includes tissue type for cells in the biological sample, and the tissue type is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, carcinoma, cystadenocarcinoma, sarcoma, and glioma.
In some embodiments, the at least one characteristic includes human papillomavirus (HPV) status for cells in the biological sample, and wherein the set of genes includes at least 5 genes selected from the group of genes listed in Table 8. In some embodiments, the at least one characteristic includes a subtype of peripheral T-cell lymphoma (PTCL) for cells in the biological sample, and wherein the set of genes includes at least 5 genes selected from the group of genes listed in Table 10. In some embodiments, the subtype of PTCL is selected from the group consisting of: anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL).
In some embodiments, the subject has, is suspected of having, or is at risk of having breast cancer, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 1. In some embodiments, the subject has, is suspected of having, or is at risk of having kidney cancer, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 2. In some embodiments, the subject has, is suspected of having, or is at risk of having lymphoma, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 3. In some embodiments, the subject has, is suspected of having, or is at risk of having Diffuse Large B-Cell Lymphoma (DLBCL), the set of genes comprises at least 10 genes selected from the group of genes listed in Table 3, and the at least one characteristic is a cell of origin selected from the group consisting of germinal center B-cell (GCB) and activated B-cell (ABC). In some embodiments, the subject has, is suspected of having, or is at risk of having lung adenocarcinoma, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 6.
In some embodiments, the at least one characteristic is selected from the group consisting of cancer grade for cells in the biological sample, tissue of origin for cells in the biological sample, tissue type for cells in the biological sample, and cancer subtype for cells in the biological sample.
In some embodiments, determining the at least one characteristic further comprises providing the gene ranking as an input to the statistical model and obtaining an output indicating the at least one characteristic. In some embodiments, the at least one characteristic is selected from the group consisting of cancer grade for cells in the biological sample, tissue of origin for cells in the biological sample, tissue type for cells in the biological sample, and cancer subtype for cells in the biological sample.
In some embodiments, the subject has, is suspected of having, or is at risk of having head and neck squamous cell carcinoma, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 8. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 8.
In some embodiments, the at least one characteristic includes human papillomavirus (HPV) status for cells in a biological sample. In some embodiments, the at least one characteristic includes a subtype of peripheral T-cell lymphoma (PTCL) for cells in the biological sample, and wherein the set of genes includes at least 5 genes selected from the group of genes listed in Table 10. In some embodiments, the set of genes comprises at least 10 genes selected from the groups of genes listed in Table 10. In some embodiments, the subtype of PTCL is selected from the group consisting of: anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL).
Some embodiments are directed to a system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform a method. The method comprises obtaining expression data obtained at least in part by sequencing a biological sample of a subject having, suspected of having or at risk of having cancer, the expression data comprising expression levels for a plurality of genes, the plurality of genes comprising a set of genes; ranking at least some genes in the set of genes, based on their expression levels in the expression data to obtain a gene ranking; and determining, using the gene ranking and a statistical model trained using training data indicating a plurality of gene rankings of at least some of the genes in the set of genes obtained, at least one characteristic of the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the set of genes.
Some embodiments are directed to at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform: obtaining expression data obtained at least in part by sequencing a biological sample of a subject having, suspected of having or at risk of having cancer, the expression data comprising expression levels for a plurality of genes, the plurality of genes comprising a set of genes; ranking at least some genes in the set of genes, based on their expression levels in the expression data to obtain a gene ranking; and determining, using the gene ranking and a statistical model trained using training data indicating a plurality of gene rankings of at least some of the genes in the set of genes obtained, at least one characteristic of the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the set of genes.
Some embodiments are directed to a method, comprising using at least one computer hardware processor to perform: obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in at least one set of genes based on their expression levels in the expression data to obtain at least one gene ranking; and determining, using the at least one gene ranking and at least one statistical model trained using training data indicating a plurality of rankings for at least some genes in the at least one set of genes, tissue of origin for at least some of the cells in the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the at least one set of genes.
In some embodiments, the expression data was obtained using a gene expression microarray. In some embodiments, the expression data was obtained by performing next generation sequencing. In some embodiments, the tissue of origin is selected from the group consisting of lung tissue, pancreas tissue, stomach tissue, colon tissue, liver tissue, bladder tissue, kidney tissue, thyroid tissue, lymph node tissue, adrenal gland tissue, skin tissue, breast tissue, ovary tissue, prostate tissue, urothelial tissue, cervical tissue, esophagus tissue, brain tissue, soft tissue, connective tissue, head tissue, and neck tissue.
In some embodiments, determining, using the at least one gene ranking and the at least one statistical model, tissue type for at least some of the cells in the biological sample. In some embodiments, the tissue type is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, carcinoma, cystadenocarcinoma, sarcoma, and glioma. In some embodiments, a combination of the tissue of origin and the tissue type is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast carcinoma, colorectal adenocarcinoma, ovarian serous cystadenocarcinoma, phenochromocytoma, bladder urothelial carcinoma, cervical squamous cell carcinoma, glioblastoma multiforme, head squamous cell carcinoma, neck squamous cell carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, paraganglioma, prostate adenocarcinoma, sarcoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrial carcinoma.
In some embodiments, the subject has, is suspected of having, or is at risk of having lymphoma. In some embodiments, the subject has, is suspected of having, or is at risk of having Diffuse Large B-Cell Lymphoma (DLBCL). In some embodiments, the tissue of origin is a cell of origin selected from the group consisting of germinal center B-cell (GCB) and activated B-cell (ABC). In some embodiments, a set of genes of the at least one set of genes is selected from the group of genes listed in Table 3. In some embodiments, a set of genes of the at least one set of genes comprises at least 3 genes selected from the group of genes listed in Table 3. In some embodiments, a set of genes of the at least one set of genes comprises at least 5 genes selected from the group of genes listed in Table 3. In some embodiments, a set of genes of the at least one set of genes comprises at least 10 genes selected from the group of genes listed in Table 3.
In some embodiments, a set of genes of the at least one set of genes includes at least 5 genes. In some embodiments, a set of genes of the at least one set of genes consists of 5-100 genes. In some embodiments, a set of genes of the at least one set of genes consists of 10-200 genes. In some embodiments, a set of genes of the at least one set of genes consists of 20-100 genes. In some embodiments, a set of genes of the at least one set of genes consists of 50-100 genes.
In some embodiments, the expression data includes values, each representing an expression level for a gene in the at least one set of genes, and determining a gene ranking of the at least one gene ranking comprises determining a relative rank for each gene in one of the at least one set of genes based on the values. In some embodiments, determining the at least one characteristic further comprises using the at least one gene ranking as an input to the at least one statistical model and obtaining an output indicating the tissue of origin.
In some embodiments, the at least one statistical model comprises a gradient boosted decision tree classifier. In some embodiments, the at least one statistical model comprises at least one classifier selected from the group consisting of: a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
In some embodiments, the at least one set of genes comprises a first set of genes associated with predicting a first type of tissue and a second set of genes associated with predicting a second type of tissue.
Some embodiments are directed to a system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform a method. The method comprises obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in at least one set of genes based on their expression levels in the expression data to obtain at least one gene ranking; and determining, using the at least one gene ranking and at least one statistical model trained using training data indicating a plurality of rankings for at least some genes in the at least one set of genes, tissue of origin for at least some of the cells in the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the at least one set of genes.
Some embodiments are directed to at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform: obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in at least one set of genes based on their expression levels in the expression data to obtain at least one gene ranking; and determining, using the at least one gene ranking and at least one statistical model trained using training data indicating a plurality of rankings for at least some genes in the at least one set of genes, tissue of origin for at least some of the cells in the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the at least one set of genes.
Some embodiments are directed to a method, comprising using at least one computer hardware processor to perform: obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in a set of genes based on their expression levels in the expression data to obtain a gene ranking; and determining, using the gene ranking and a statistical model trained using training data indicating a plurality of rankings for at least some genes in the set of genes, cancer grade for at least some of the cells in the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the set of genes.
In some embodiments, the expression data was obtained using a gene expression microarray. In some embodiments, the expression data was obtained by performing next generation sequencing. In some embodiments, the cancer grade is selected from the group consisting of at least Grade 1, Grade 2, and Grade 3. In some embodiments, the cancer grade is selected from the group consisting of at least Grade 1, Grade 2, Grade 3, and Grade 4. In some embodiments, the cancer grade is selected from the group consisting of Grade 1, Grade 2, Grade 3, Grade 4, and Grade 5.
In some embodiments, the subject has, is suspected of having, or is at risk of having breast cancer. In some embodiments, the set of genes is selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 3 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 5 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 1.
In some embodiments, the subject has, is suspected of having, or is at risk of having kidney cancer. In some embodiments, the subject has, is suspected of having, or is at risk of having clear cell kidney cancer. In some embodiments, the set of genes is selected from the group of genes listed in Table 2. In some embodiments, the set of genes comprises at least 3 genes selected from the group of genes listed in Table 2. In some embodiments, the set of genes comprises at least 5 genes selected from the group of genes listed in Table 2. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 2.
In some embodiments, the subject has, is suspected of having, or is at risk of having lung adenocarcinoma. In some embodiments, the set of genes is selected from the group of genes listed in Table 6. In some embodiments, the set of genes comprises at least 10 genes selected from the group of genes listed in Table 6. In some embodiments, the set of genes includes at least 50 genes. In some embodiments, the set of genes consists of 10-100 genes. In some embodiments, the set of genes consists of 10-30 genes.
In some embodiments, the expression data includes values, each representing an expression level for a gene in the set of genes, and determining the gene ranking comprises determining a relative rank for each gene in the set of genes based on the values. In some embodiments, determining that at least one characteristic further comprises using the gene ranking as an input to the statistical model and obtaining an output indicating the cancer grade.
In some embodiments, the statistical model comprises a gradient boosted decision tree classifier. In some embodiments, the statistical model comprises a classifier selected from the group consisting of: a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
Some embodiments are directed to a system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform a method. The method comprises obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in a set of genes based on their expression levels in the expression data to obtain a gene ranking; and determining, using the gene ranking and a statistical model trained using training data indicating a plurality of rankings for at least some genes in the set of genes, cancer grade for at least some of the cells in the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the set of genes.
Some embodiments are directed to at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform: obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in a set of genes based on their expression levels in the expression data to obtain a gene ranking; and determining, using the gene ranking and a statistical model trained using training data indicating a plurality of rankings for at least some genes in the set of genes, cancer grade for at least some of the cells in the biological sample, wherein each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the set of genes.
Some embodiments are directed to a method, comprising using at least one computer hardware processor to perform using at least one computer hardware processor to perform: obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in at least one set of genes based on their expression levels in the expression data to obtain at least one gene ranking; and determining, using the at least one gene ranking and at least one statistical model, a subtype of peripheral T-cell lymphoma (PTCL) for at least some of the cells in the biological sample.
In some embodiments, the at least one statistical model was trained using training data indicating a plurality of rankings of expression levels for at least some genes in the at least one set of genes. In some embodiments, each of the plurality of gene rankings is obtained based on respective expression levels for the at least some genes in the at least one set of genes.
In some embodiments, the expression data was obtained using a gene expression microarray. In some embodiments, the expression data was obtained by performing next generation sequencing. In some embodiments, the expression data was obtained using a hybridization-based expression assay.
In some embodiments, the subtype of PTCL is selected from the group consisting of: anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL). In some embodiments, the subtype of PTCL is selected from the group consisting of: Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS), anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), Natural killer/T-cell lymphoma (NKTCL), Sezary syndrome, adult T-cell leukemia/lymphoma (ATLL), enteropathy-type T-cell lymphoma, nasal NK/T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell lymphomas of Follicular T-cell (TFH) origin, T-cell lymphomas of the gastrointestinal tract, and cutaneous T-cell lymphomas.
In some embodiments, a set of genes of the at least one set of genes is selected from the group of genes listed in Table 10. In some embodiments, a set of genes of the at least one set of genes comprises at least 3 genes selected from the group of genes listed in Table 10. In some embodiments, a set of genes of the at least one set of genes comprises at least 5 genes selected from the group of genes listed in Table 10. In some embodiments, a set of genes of the at least one set of genes comprises at least 10 genes selected from the group of genes listed in Table 10. In some embodiments, a set of genes of the at least one set of genes comprises at least 50 genes selected from the group of genes listed in Table 10.
In some embodiments, a set of genes of the at least one set of genes includes at least one up-regulated in AITL gene. In some embodiments, a set of genes of the at least one set of genes includes at least one down-regulated in AITL gene. In some embodiments, a set of genes of the at least one set of genes includes at least one MF profile gene.
In some embodiments, the subject has, is suspected of having, or is at risk of having lymphoma. In some embodiments, the subject has, is suspected of having, or is at risk of having peripheral T-cell lymphoma (PTCL).
In some embodiments, a set of genes of the at least one set of genes includes at least 5 genes. In some embodiments, a set of genes of the at least one set of genes consists of 5-100 genes. In some embodiments, a set of genes of the at least one set of genes consists of 10-200 genes. In some embodiments, a set of genes of the at least one set of genes consists of 20-100 genes. In some embodiments, a set of genes of the at least one set of genes consists of 50-100 genes.
In some embodiments, the expression data includes values, each representing an expression level for a gene in the at least one set of genes, and determining a gene ranking of the at least one gene ranking comprises determining a relative rank for each gene in one of the at least one set of genes based on the values.
In some embodiments, determining the subtype of PTCL further comprises using the at least one gene ranking as an input to the at least one statistical model and obtaining an output indicating the subtype of PTCL.
In some embodiments, the at least one statistical model comprises a gradient boosted decision tree classifier. In some embodiments, the at least one statistical model comprises at least one classifier selected from the group consisting of: a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
In some embodiments, the at least one statistical model includes a multi-class classifier. In some embodiments, the multi-class classifier has at least four outputs each corresponding to a different subtype of PTCL. In some embodiments, the at least four outputs include a first output corresponding to anaplastic large cell lymphoma (ALCL), a second output corresponding to angioimmunoblastic T-cell lymphoma (AITL), a third output corresponding to natural killer/T-cell lymphoma (NKTCL), and a fourth output corresponding to adult T-cell leukemia/lymphoma (ATLL).
In some embodiments, the at least one statistical model comprises a plurality of classifiers corresponding to different subtypes of PTCL. In some embodiments, the plurality of classifiers includes a first classifier, a second classifier, a third classifier, and a fourth classifier, wherein the first classifier corresponds anaplastic large cell lymphoma (ALCL), a second classifier corresponds to angioimmunoblastic T-cell lymphoma (AITL), a third classifier corresponds to natural killer/T-cell lymphoma (NKTCL), and a fourth classifier corresponds to adult T-cell leukemia/lymphoma (ATLL). In some embodiments, the at least one set of genes includes a first set of genes associated with a first classifier of the plurality of classifiers and a second set of genes associated with a second classifier of the plurality of classifiers.
In some embodiments, the subject has, is suspected of having, or is at risk of having lymphoma. In some embodiments, the subject has, is suspected of having, or is at risk of having PTCL.
In some embodiments, the method further comprises presenting, to a user, an indication of the subtype of PTCL. In some embodiments, presenting the indication of the subtype of PTCL further comprises displaying the subtype of PTCL to the user in a graphical user interface (GUI).
Some embodiments are directed to a system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform a method. The method comprises obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in at least one set of genes based on their expression levels in the expression data to obtain at least one gene ranking; and determining, using the at least one gene ranking and at least one statistical model, a subtype of peripheral T-cell lymphoma (PTCL) for at least some of the cells in the biological sample.
Some embodiments are directed to at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform: obtaining expression data for cells in a biological sample of a subject having, suspected of having, or at risk of having cancer; ranking at least some genes in at least one set of genes based on their expression levels in the expression data to obtain at least one gene ranking; and determining, using the at least one gene ranking and at least one statistical model, a subtype of peripheral T-cell lymphoma (PTCL) for at least some of the cells in the biological sample.

BRIEF DESCRIPTION OF DRAWINGS

Various aspects and embodiments will be described with reference to the following figures. The figures are not necessarily drawn to scale.

FIG. 1 is a diagram of an illustrative process for determining one or more characteristics of a biological sample based on one or more respective gene rankings for the biological sample using the machine learning techniques described herein.

FIG. 2 is a diagram of an illustrative process for determining a characteristic of a biological sample based on using multiple statistical models to obtain multiple characteristic predictions and aggregating the characteristic predictions using the machine learning techniques described herein.

FIG. 3 is a flow chart of an illustrative process for determining a characteristic of a biological sample using a gene ranking and a statistical model, using the machine learning techniques described herein.

FIG. 4 is a flow chart of an illustrative process for determining tissue of origin for cells in a biological sample using the machine learning techniques described herein.

FIG. 5 is a flow chart of an illustrative process for determining cancer grade for cells in a biological sample using the machine learning techniques described herein.

FIG. 6A shows example different data sets, associated clinical cancer grade for samples of the data sets, and predicted cancer grade obtained using the machine learning techniques described herein, for determining breast cancer grade.

FIG. 6B shows example the enrichment signatures for different pathways, illustrating gene expression profiles associated with breast cancer Grade 1 and Grade 3.

FIG. 6C shows example different data sets, associated clinical cancer grade for samples of the data sets, and predicted cancer grade, using the machine learning techniques described herein, for determining breast cancer grade.

FIG. 6D shows example the enrichment signatures for different pathways, illustrating gene expression profiles associated with breast cancer Grade 1 and Grade 3.

FIG. 7 is an illustrative plot of true positive rate versus false positive rate for predicting breast cancer grade of different biological samples using the machine learning techniques described herein.

FIG. 8A is a flowchart of an illustrative process for selecting a gene set, using the machine learning techniques described herein.

FIG. 8B is a flowchart of an illustrative process for selecting a gene set, using the machine learning techniques described herein.

FIG. 9A is an exemplary plot of quality score versus number of genes used for determining tissue of origin, using the machine learning techniques described herein.

FIG. 9B is an exemplary plot of F1 score versus number of genes used for determining tissue of origin for Diffuse Large B-Cell Lymphoma (DLBCL), such as germinal center B-cell (GCB) and activated B-cell (ABC), using the machine learning techniques described herein.

FIG. 10 is a block diagram of an illustrative computer system that may be used in implementing the machine learning techniques described herein.

FIG. 11 is a block diagram of an illustrative environment 1100 in which the machine learning techniques described herein may be implemented.

FIG. 12 is an exemplary distribution of molecular cancer grade among PAM50 subtypes.

FIG. 13 are illustrative data sets and enrichment signatures showing how progeny process scores correspond to given and predicted cancer grades in TCGA BRCA.

FIG. 14 are exemplary plots comparing different protein expression levels for different predicted cancer grades.

FIG. 15 is an exemplary plot of cytolitic score for different predicted cancer grades.

FIG. 16 are illustrative plots showing the difference in mutations between different cancer grades, according to WES data.

FIG. 17 shows example segments that are differentially amplified or deleted between predicted cancer grades, according to WES data.

FIG. 18 are illustrative data sets and enrichment signatures showing how progeny process scores correspond to given and predicted cancer grades in TCGA KIRC.

FIG. 19 is a plot illustrating chromosomal instability for different cancer grades.

FIG. 20 are plots comparing different protein expression for different predicted cancer grades.

FIG. 21 illustrates genes, according to WES data, that are differentially amplified or deleted between predicted cancer grades.

FIG. 22 illustrates genes, according to WES data, that are differentially amplified or deleted between predicted cancer grades.

FIG. 23A shows example validation data sets, associated cancer grade reported for samples of the data sets, predicted cancer grade obtained using the machine learning techniques described herein, for determining lung adenocarcinoma cancer grade, and the enrichment signatures for different pathways, illustrating gene expression profiles associated with grade 1 and grade 3.

FIG. 23B shows example results of applying validation data sets to a lung adenocarcinoma cancer grade classifier, using the machine learning techniques described herein.

FIG. 23C is an example plot of true positive rate versus false positive rate for predicting cancer grade of different biological samples using the machine learning techniques described herein.

FIG. 24A shows example validation data sets, associated cell of origin reported for samples of the data sets, predicted cell of origin obtained using the machine learning techniques described herein, for determining DLBCL subtype, and the enrichment signatures for ABC and GCB subtypes.

FIG. 24B shows example validation data sets, associated cell of origin reported for samples of the data sets, predicted cell of origin obtained using the machine learning techniques described herein, for determining DLBCL subtype, and the enrichment signatures for ABC and GCB subtypes.

FIGS. 24C and 24D are example plots of survival rates for different groups (ABC, GCB).

FIG. 24E is an example plot of true positive rate versus false positive rate for predicting DLBCL subtype of different biological samples using the machine learning techniques described herein.

FIG. 25A shows example validation data sets, associated HPV status reported for samples of the data sets, predicted HPV status obtained using the machine learning techniques described herein, for determining HPV status, and the enrichment signatures for different pathways, illustrating gene expression profiles associated with HPV status.

FIGS. 25B and 25C are example plots of survival rates for different groups of HPV status (positive HPV and negative HPV).

FIG. 25D is an example plot of true positive rate versus false positive rate for predicting HPV status of different biological samples using the machine learning techniques described herein.

FIG. 25E is an example plot of true positive rate versus false positive rate for predicting HPV status of different biological samples using the machine learning techniques described herein.

FIG. 25F is an example plot illustrating the performance of a classifier for different HPV strains, using the machine learning techniques described herein.

FIG. 26 is a diagram of an illustrative process for determining peripheral T-cell lymphoma (PTCL) subtype of a biological sample using the machine learning techniques described herein.

FIG. 27 is a diagram of an illustrative process for determining peripheral T-cell lymphoma (PTCL) subtype of a biological sample using the machine learning techniques described herein.

FIG. 28 is a diagram of an illustrative process for determining a characteristic of a biological sample based on using multiple statistical models to determine peripheral T-cell lymphoma (PTCL) subtype of the biological sample using the machine learning techniques described herein.

FIG. 29 is a flow chart of an illustrative process for determining a subtype of peripheral T-cell lymphoma (PTCL) for a biological sample using a gene ranking and a statistical model using the machine learning techniques described herein.

FIG. 30 is an example plot of survival rates for the different peripheral T-cell lymphoma (PTCL) subtypes.

DETAILED DESCRIPTION

Characteristics of a biological cell may relate to the expression levels of certain genes. For example, a cancerous cell may have some genes upregulated and other genes downregulated relative to a normal, healthy cell. This relationship between cell characteristics and gene expression levels may be utilized in analyzing gene expression data for biological cells. In particular, such a relationship may provide certain benefits in analyzing characteristics of biological cells that are considered histological characteristics, including tissue of origin and cancer grade, which generally relate to features of biological cells that are observed visually by a person (e.g., pathologist). In some instances, the gene expression data may provide a more consistent assessment of a certain cell characteristic than by using histological techniques, which may be subject to variation between differences in assessment among pathologists.
Large amounts of gene expression data can be obtained through different platforms, including by using a gene expression microarray and by performing next generation sequencing, and is now available or can be generated to characterize biological cells. However, the inventors have recognized that information that is derivable from these data is compromised by differences among different gene sequencing platforms which may lead to variation in gene expression data produced by the sequencing platforms, even if they are used to sequence the same biological sample. For example, microarrays and next generation sequencing (NGS) techniques, may produce gene expression data where the particular values representing gene expression levels may vary among the platforms, even if obtained from the same biological sample. This variation in the expression values across different sequencing platforms may occur because of how the expression data is obtained. The processes and devices used to obtain gene expression data using a particular type of sequencing platform (e.g., next generation sequencing, microarray) may impact the specific values for the expression levels obtained. In turn, the values for the expression levels depend on which sequencing platform was used to obtain the gene expression data. This variation may occur not only across different types of sequencing platforms, but may also occur where the different sequencing platforms are of the same type (e.g., next generation sequencing) and involve different systems (e.g., optical systems, detectors) and processes (e.g., biological sample preparation), or even the same devices in different locations (e.g., due to differences in calibration, use, environment, etc.).
The inventors have recognized that such variation in expression level values presents significant challenges in analyzing gene expression data to characterize cells, especially when using gene expression data obtained using different sequencing platforms. For some expression data, it may be a challenge to normalize the expression level values in such a way so that expression data obtained using different sequencing platforms may be analyzed using the same or similar techniques.
Conventional techniques for analyzing expression data are generally applicable only to analyzing expression data that was obtained using a single sequencing platform and to the specific conditions used in preparing and sequencing the sample. Such conventional techniques are not applicable to analyzing expression data obtained from multiple sequencing platforms, even when the sequencing platforms are of the same type (e.g., next generation sequencing, microarray). For example, conventional techniques for analyzing gene expression data may involve different data analysis pipelines for expression data obtained using different next generation sequencing devices. In addition, some conventional techniques involve implementing different data analysis pipelines depending on how the expression data was obtained even if the same sequencing device was used. For example, conventional techniques for analyzing gene expression data may differ for different sequencing conditions or different sample processing methods. As a result, conventional techniques for analyzing expression data cannot be implemented across different sequencing platforms, sample preparation techniques, and sequencing conditions. This significantly impacts the usability of gene expression data to determine characteristics of cells.
One important group of techniques for analyzing expression data include statistical models (e.g., machine learning models) that are configured to receive expression level values (or a derivative thereof) as input to produce an output of interest such as a prediction or classification. Examples of such statistical models, developed by the inventors, are provided herein. Prior to being used such statistical models are trained on training data comprising pairs of inputs/outputs. If the training data inputs include expression level values (or a derivative thereof) that comes from one type of sequencing platform, then a statistical model trained with such data will exhibit poor performance (on the task for which it is trained) when being provided with expression level values that come from another type of sequencing platform. Indeed, variation across expression level values from different sequencing platforms makes it difficult or impossible to design a single statistical model trained to perform a task using data from any one of multiple types of sequencing platforms. Instead, a separate statistical model would have to be trained for each particular sequencing platform using training data obtained for that particular sequencing platform, which is difficult because it requires training multiple models for each platform and this requires not only additional computational resources, but may simply not be possible as there may not be sufficient training data available for each type of platform.
The inventors have recognized the need for common techniques that can be used for analyzing expression data obtained across different sequencing platforms, despite differences in the type of expression level data generated by the platforms. Such techniques would ease analysis of gene expression data across different subjects, which conventional gene expression level analysis techniques would not allow. For example, techniques described herein for analyzing gene expression data may involve using the same or similar data analysis pipeline (which pipeline may include one or more statistical models, examples of which are provided herein) for expression data obtained using the same type of sequencing platform (e.g., next generation sequencing, microarray) for multiple subjects. Such a data analysis pipeline may allow for expression data to be analyzed in the same or similar manner regardless of sample processing (e.g., DNA extraction, amplification), sequencing conditions (e.g., temperature, pH), data processing (e.g., data processing for next generation sequencing, microarrays) used in obtaining the expression data.
To address some of the difficulties that arise with conventional techniques for analyzing expression data, the inventors have developed improved techniques in analyzing expression data that are independent of the sequencing platform and data processing used to obtain the expression data. In particular, the inventors have recognized that variation of the expression levels among sequencing platforms may be accounted for by using the ranking of a set of genes, rather than the specific values of the expression levels in the data, in subsequent data analysis. For example, the inventors have developed various statistical models for determining various characteristics of a biological sample (e.g., tissue of origin, cancer grade, cancer type for a tissue sample). Each such statistical model is trained to determine a respective characteristic of the biological sample using a ranking of a respective set of genes, rather than using expression levels themselves, which allows the statistical model to operate on expression data obtained from different types of sequencing platforms.
Accordingly, in some embodiments, a statistical model may be used to predict the characteristic(s) of a biological sample based on an input ranking of genes, ranked based on their respective expression levels, for a sequencing platform. Using the input ranking(s), instead of the specific values for the expression levels, allows for the same or similar data processing pipeline to be used across different expression data regardless of the specific manner in which the expression levels were obtained (e.g., regardless of which sequencing platform, sequencing conditions, sample preparation, data processing to obtain expression levels, etc.). As described herein, the statistical model may be specific to the particular characteristic being determined. A statistical model according to the techniques described here may be used to predict one or more characteristics, including cancer grade for cells in the biological sample (e.g., breast cancer grade, kidney clear cell cancer grade, lung adenocarcinoma grade), tissue of origin for cells in the biological sample (e.g., lung, pancreas, stomach, colon, liver, bladder, kidney, thyroid, lymph nodes, adrenal gland, skin, breast, ovary, prostrate, or cell of origin in a tissue such as e.g. germinal center B-cell (GCB) or activated B-cell (ABC)), histological information (tissue type, such as e.g. adenocarcinoma, squamous cell carcinoma, carcinoma, cystadenocarcinoma, sarcoma, and glioma) for cells in the biological sample, and cancer subtype (e.g. PTCL subtype such as, anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL)), viral status (e.g., HPV status, such as HPV-positive or HPV-negative for head and neck squamous cell carcinoma) for cells in the biological sample.
For example, in some embodiments, rankings of genes based on the gene expression levels (in a biological sample) as determined by a sequencing platform may be provided as input to a statistical model trained to predict tissue of origin for the biological sample. As another example, in some embodiments, rankings of genes based on the gene expression levels (in a biological sample) as determined by a sequencing platform may be provided as input to a statistical model trained to predict cancer grade for the biological sample. In some embodiments, the set of genes being ranked depends on the particular biological characteristic of interest. For example, one set of genes may be used for determining the tissue of origin and another set of genes may be used for determining the cancer grade.
The machine learning techniques that involve using rankings of genes as described herein are an improvement of conventional machine learning technology because they improve over conventional machine learning techniques that use gene expression values directly to analyze gene expression data. For instance, training data obtained using different sequencing platforms may be used in training the statistical models described herein because of the benefits provided by using gene rankings in allowing a common statistical model to be implemented regardless of how the expression data was generated. In contrast, conventional machine learning techniques that involve using gene expression values require individual separate statistical models depending on how the expression data was generated, such as when using different sequencing platforms, sample preparation techniques, etc. Accordingly, the machine learning techniques described herein reduce the need for collecting training data across different sequencing platforms in order to train multiple statistical models required to analyze expression data generated in different ways. In addition, the statistical models described herein may have better performance in contrast to conventional techniques. For instance, a statistical model according to the techniques described herein can be trained using training data obtained from different sources, and thus more training data in general, which improves overall performance of the statistical model being used. In contrast, sources of training data for conventional machine learning models may be limited to a particular sequencing platform, sample preparation technique, etc. and performance may depend on the amount of training data available using a particular way of generating the expression data.
In addition, having a statistical model that is independent of the sequencing platform, sample preparation, and sequencing conditions used may make deployment and use of such a statistical model more practical. In clinical practice, data from different patients is likely to originate from multiple sources, such as expression data generated using different sample preparation techniques and sequencing platforms. As discussed above, the techniques described herein allow for the ability to handle patient data originating from these different sources in a uniform manner by using a common statistical model. The ability to analyze patient data in this way provides improvements to bioinformatics technology that depends on the number of patients represented by the patient data because a larger pool of patients can be analyzed using a common statistical model. These benefits extend to applications where bioinformatics analysis may be used, including predicting characteristic(s) of cells in a biological sample, where being able to use a larger sample size, across many patients, is beneficial.
Moreover, the machine learning techniques described herein may streamline handling of different formats for storing expression data. Different types of sequencing platforms output expression data using different data formats. As discussed herein, a ranking process is used to generate gene rankings, which are then input to a common statistical model. The ranking process may allow for expression data originating from sources that use different data formats to have a similar type of input to the statistical model. This may improve handling of expression data obtained from different sequencing platforms in comparison to conventional analysis techniques where different data processing pipelines are required for different input data formats.
Some embodiments described herein address all of the above-described issues that the inventors have recognized with determining characteristics of a biological sample using gene expression data. However, not every embodiment described herein addresses every one of these issues, and some embodiments may not address any of them. As such, it should be appreciated that embodiments of the technology described herein are not limited to addressing all or any of the above-discussed issues with determining characteristics of a biological sample using gene expression data.
Some embodiments involve obtaining gene expression data for a biological sample of a subject, ranking genes in set(s) of genes based on their expression levels in the expression data to obtain one or more gene rankings. The one or more gene rankings may be used, along with a statistical model, to determine one or more characteristics of the biological sample, including tissue of origin and cancer grade. The statistical model may be trained using rankings of expression levels for some or all genes in the set(s) of genes.
The gene ranking(s) may be obtained by ranking genes in one or more sets of genes based on their expression levels in the expression data. In some embodiments, the expression data includes values, each representing an expression level for a gene in the set(s) of genes. Determining the gene ranking(s) may involve determining a relative rank for each gene in the set(s) of genes based on the values. For example, a first gene ranking may be obtained by ranking genes in a first set of genes based on their expression levels and a second gene ranking may be obtained by ranking genes in a second set of genes based on their expression levels. In some embodiments, the first set of genes and the second set of genes may share some or all genes. Determining the one or more characteristics may involve using the first gene ranking, the second gene ranking, and the statistical model, where the statistical model is trained using training data indicating gene rankings of expression levels for some or all genes in the first set of genes and the second set of genes. Different gene sets may correspond to predicting particular characteristics of the biological sample, and a gene ranking for a specific gene set may be used to determine the characteristic associated with the gene set. For example, a gene ranking where expression levels for a gene set associated with predicting cancer grade may be used to predict cancer grade for cells in the biological sample from which the expression data is obtained.
In some embodiments, the expression data may be obtained for cells in the biological sample, where the subject has or is suspected of having cancer. In the context where tissue of origin is a characteristic being determined, the tissue of origin is for the cells in the biological sample. The tissue of origin may refer to a particular tissue type from which the cells originate, such as lung, pancreas, stomach, colon, liver, bladder, kidney, thyroid, lymph nodes, adrenal gland, skin, breast, ovary, and prostrate.
For example, some embodiments involve using a gene set for predicting tissue of origin, which may include cell of origin, for Diffuse Large B-Cell Lymphoma (DLBCL), such as germinal center B-cell (GCB) and activated B-cell (ABC). Genes in the gene set may be selected from the group consisting of: ITPKB, MYBL1, LMO2, BATF, IRF4, LRMP, CCND2, SLA, SP140, PIM1, CSTB, BCL2, TCF4, P2RX5, SPINK2, VCL, PTPN1, REL, FUT8, RPL21, PRKCB1, CSNK1E, GPR18, IGHM, ACP1, SPIB, HLA-DQA1, KRT8, FAM3C, and HLA-DMB.
In the context where cancer grade is a characteristic being determined, the cancer grade is for the cells in the biological sample. The cancer grade may refer to proliferation and differentiation characteristics of the cells in the biological sample and refer to a numerical grade that is generally determined by visual observation of cells using microscopy, such as Grade 1, Grade 2, Grade 3, and Grade 4. For example, a pathologist may examine a biopsied tissue under a microscope and determine a cancer grade for the tissue. Cancer grades generally depend on the amount of abnormality of the cells in tissue and may depend on the type cancer. For Grade 1, tumor cells and the organization of the tumor tissue appears close to normal, healthy tissue. Grade 1 tumors tend to grow and spread slowly. In contrast, cells and tissue of Grade 3 and Grade 4 tumors do not look like normal cells and tissue. Grade 3 and Grade 4 tumors tend to grow rapidly and spread faster than tumors with a lower grade. An example grading system for cancer tissue is described in American Joint Committee on Cancer AJCC Cancer Staging Manual. 7th ed. New York, N.Y.: Springer; 2010, which is incorporated by reference in its entirety. This grading system applies the following definitions: Grade X (GX) is an undetermined grade and applies when the grade of a tissue cannot be assessed; Grade 1 (G1) is a low grade and applies when the cells are well differentiated; Grade 2 (G2) is an intermediate grade and applies when the cells are moderately differentiated; Grade 3 (G3) is a high grade and applies when the cells are poorly differentiated; and Grade 4 (G4) is a high grade and applies when the cells are undifferentiated.
For example, some embodiments involve using a gene set for predicting breast cancer grade. Genes in the gene set may be selected from the group consisting of: UBE2C, MYBL2, PRAME, LMNB1, CXCL9, KPNA2, TPX2, PLCH1, CCL18, CDK1, MELK, CCNB2, RRM2, CCNB1, NUSAP1, SLC7A5, TYMS, GZMK, SQLE, C1orf106, CDC25B, ATAD2, QPRT, CCNA2, NEK2, IDO1, NDC80, ZWINT, ABCA12, TOP2A, TDO2, S100A8, LAMP3, MMP1, GZMB, BIRC5, TRIP13, RACGAP1, ASPM, ESRP1, MAD2L1, CENPF, CDC20, MCM4, MK167, PBK, CKS2, KIF2C, MRPL13, TTK, BUB1, TK1, FOXM1, CEP55, EZH2, ECT2, PRC1, CENPU, CCNE2, AURKA, HMGB3, APOBEC3B, LAGE3, CDKN3, DTL, ATP6V1C1, KIAA0101, CD2, KIF11, KIF20A, CDCA8, NCAPG, CENPN, MTFR1, MCM2, DSCC1, WDR19, SEMA3G, KCND3, SETBP1, KIF13B, NR4A2, NAV3, PDZRN3, MAGI2, CACNA1D, STC2, CHAD, PDGFD, ARMCX2, FRY, AGTR1, MARCH8, ANG, ABAT, THBD, RAI2, HSPA2, ERBB4, ECHDC2, FST, EPHX2, FOSB, STARD13, ID4, FAM129A, FCGBP, LAMA2, FGFR2, PTGER3, NME5, LRRC17, OSBPL1A, ADRA2A, LRP2, C1orf115, COL4A5, DIXDC1, KIAA1324, HPN, KLF4, SCUBE2, FMO5, SORBS2, CARD10, CITED2, MUC1, BCL2, RGS5, CYBRD1, OMD, IGFBP4, LAMB2, DUSP4, PDLIM5, IRS2, and CX3CR1.
As another example, some embodiments involve using a gene set for predicting kidney clear cell cancer grade. Genes in the gene set may be selected from the group consisting of: PLTP, CIS, LY96, TSKU, TPST2, SERPINF1, SRPX2, SAA1, CTHRC1, GFPT2, CKAP4, SERPINA3, CFH, PLAU, BASP1, PTTG1, MOCOS, LEF1, SLPI, PRAME, STEAP3, LGALS2, CD44, FLNC, UBE2C, CTSK, SULF2, TMEM45A, FCGR1A, PLOD2, C19orf80, PDGFRL, IGF2BP3, SLC7A5, PRRX1, RARRES1, LHFPL2, KDELR3, TRIB3, IL20RB, FBLN1, KMO, C1R, CYP1B1, KIF2A, PLAUR, CKS2, CDCP1, SFRP4, HAMP, MMP9, SLC3A1, NAT8, FRMD3, NPR3, NAT8B, BBOX1, SLC5A1, GBA3, EMCN, SLC47A1, AQP1, PCK1, UGT2A3, BHMT, FMO1, ACAA2, SLC5A8, SLC16A9, TSPAN18, SLC17A3, STK32B, MAP7, MYLIP, SLC22A12, LRP2, CD34, PODXL, ZBTB42, TEK, FBP1, and BCL2.
As another example, some embodiments involve using a gene set for predicting cancer grade for lung adenocarcinoma. Genes in the gene set may be selected from the group consisting of: AADAC, ALDOB, ANXA10, ASPM, BTNL8, CEACAM8, CENPA, CHGB, CHRNA9, COL11A1, CRABP1, F11, GGTLC1, HJURP, IGF2BP3, IHH, KCNE2, KIF14, LRRC31, MYBL2, MYOZ1, PCSK2, PI15, SCTR, SHH, SLC22A3, SLC7A5, SPOCK1, TM4SF4, TRPM8, YBX2.
Some embodiments involve using the machine learning techniques described herein to predict cell of origin for diffuse large B-cell lymphoma (DLBCL) for a biological sample. Such embodiments may involve using a gene set for predicting cell of origin, such as germinal center B-cell (GCB) and activated B-cell (ABC). Genes in the gene set may be selected from the group consisting of: ITPKB, MYBL1, LMO2, BATF, IRF4, LRMP, CCND2, SLA, SP140, PIM1, CSTB, BCL2, TCF4, P2RX5, SPINK2, VCL, PTPN1, REL, FUT8, RPL21, PRKCB1, CSNKIE, GPR18, IGHM, ACP1, SPIB, HLA-DQA1, KRT8, FAM3C, and HLA-DMB.
Some embodiments involve using the machine learning techniques described herein to predict a subtype of peripheral T-cell lymphoma (PTCL) for a biological sample. Such embodiments may involve using a gene set for predicting PTCL subtype, such as, anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL). Genes in the gene set may be selected from the group consisting of: EFNB2, ROBO1, SlPR3, ANK2, LPAR1, SNAP91, SOX8, RAMP3, TUBB2B, ARHGEF10, NOTCH1, ZBTB17, CCNE1, FGF18, MYCN, PTHLH, SMARCA2, WNK1, NKX2-1, CYP26A1, HPSE, CTLA4, PELI1, PRKCB, SPAST, ALS2, KIF3B, ZFYVE27, GF18, FNTB, REL, DMRT1, SLC19A2, STK3, PERP, TNFRSF8, TMOD1, BATF3, CDC14B, WDFEY3, AGT, ALK, ANXA3, BTBD11, CCNA1, DNER, GAS1, HS6ST2, IL1RAP, PCOLCE2, PDE4DIP, SLC16A3, TIAM2, TUBB6, WNT7B, SMOX, TMEM158, NLRP7, ADRB2, GALNT2, HRASLS, CD244, FASLG, KIR2DL4, LOC100287534, KLRD1, SH2D1B, KLRC2, NCAMI, CXCR5, IL6, ICOS, CD40LG, CD84, IL21, BCL6, MAF, SH2D1A, IL4, PTPN1, PIM1, ENTPD1, IRF4, CCND2, IL16, ETV6, BLNK, SH3BP5, FUT8, CCR4, GATA3, IL5, IL10, IL13, MMEITPKB, MYBL1, LRMP, KIAA0870, LMO2, CR1, LTBR, PDPN, TNFRSF1A, FCER2, ICAM1, FCGR2B, IKZF2, CCR8, TNFRSF18, IKZF4, FOXP3, IL2, TBX21, IFNG, GZMH, GNLY, EOMES, NCRI, GZMB, NKG7, FGFBP2, KLRF1, CD160, KLRK1, CD226, NCR3, TNFRSF8, BATF3, TMOD1, TMEM158, MSC, POPDC3.
Some embodiments involve using the machine learning techniques described herein to predict a viral status for a biological sample. In some embodiments the viral status is human papillomavirus (HPV) status (e.g., HPV-positive status, HPV-negative status) for a biological sample. In some embodiments, the HPV status may be determined for a subject having, suspected of having, or at risk of having head and neck squamous cell carcinoma. Genes in the gene set may be selected from the group consisting of: APOBEC3B, ATAD2, BIRC5, CCL20, CCND1, CDC45, CDC7, CDK1, CDKN2A, CDKN2C, CDKN3, CENPF, CENPN, CXCL14, DCN, DHFR, DKK3, DLGAP5, EPCAM, FANCI, FEN1, GMNN, GPX3, ID4, IGLC1, IL18, IL1R2, KIF18B, KIF20A, KIF4A, KLK13, KLK7, KLK8, KNTC1, KRT19, LAMP3, LMNB1, MCM2, MCM4, MCM5, ME1, MELK, MKI67, MLF1, MMP12, MTHFD2, NDN, NEFH, NEK2, NUP155, NUP210, NUSAP1, PDGFD, PLAGL1, PLOD2, PPPIR3C, PRIM1, PRKDC, PSIP1, RAD51AP1, RASIPI, RFC5, RNASEH2A, RPA2, RPL39L, RSRC1, RYR1, SLC35G2, SMC2, SPARCL1, STMN1, SYCP2, SYNGR3, TIMELESS, TMPO, TPX2, TRIP13, TYMS, UCP2, UPF3B, USP1, ZSCAN18.
It should be appreciated that the various aspects and embodiments described herein be used individually, all together, or in any combination of two or more, as the technology described herein is not limited in this respect.
FIG. 1 is a diagram of an illustrative processing pipeline 100 for determining one or more characteristics (e.g. tissue of origin, cancer grade, PTCL subtype) of a biological sample based on one or more respective gene rankings for the biological sample, which may include ranking genes based on their gene expression levels and using the ranking(s) and one or more statistical models to determine the one or more characteristics, in accordance with some embodiments of the technology described herein. Processing pipeline 100 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, processing pipeline 100 may be performed by a desktop computer, a laptop computer, a mobile computing device. In some embodiments, processing pipeline 100 may be performed within one or more computing devices that are part of a cloud computing environment.
As shown in FIG. 1 , gene expression data 102 may be obtained for a biological sample of a subject. The subject may have, be suspected of having, or be at risk of having cancer (e.g., breast cancer, kidney cancer, clear cell kidney cancer, lymphoma). A subject having, suspected of having, or at risk of having cancer may be a subject exhibiting one or more signs or symptoms of cancer, subject that is diagnosed as having cancer, a subject that has a family history and/or a genetic predisposition to having cancer, and/or a subject that has one or more other risk factors for cancer (e.g., age, exposure to carcinogens, environmental exposure, exposure to a virus associated with a higher likelihood of developing cancer, etc.). Expression data 102 may be obtained using any suitable sequencing platform (e.g., gene expression microarray, next generation sequencing, hybridization-based expression assay), resulting in expression data (e.g., microarray data, RNAseq data, hybridization-based expression assay data) for the biological sample. Some embodiments involve performing a sequencing process of the biological sample (e.g., a gene expression microarray, next generation sequencing) prior to obtaining expression data 102. In some embodiments, obtaining gene expression data 102 may involve obtaining gene expression data 102 in silico, such as by accessing, using a computing device, expression data (e.g., expression data that has been previously obtained from a biological sample) in one or more data stores, receiving the expression data from one or more other device, or any other way. In some embodiments, obtaining gene expression data 102 may involve analyzing a biological sample (in vitro) and accessing (e.g., by a computing device, by a processor) the expression data. Further aspects relating to obtaining expression data are provided in the section titled “Obtaining Expression Data”.
As shown in FIG. 1 , expression data 102 includes expression level values for N different genes, “gene1”, “gene2”, “gene3”, . . . “geneN” of “sample 1.” Different sequencing platforms may be used to obtain expression data 102. In some embodiments, expression data 102 may be obtained using a gene expression microarray (e.g., by determining an amount of RNA that binds to different probes on a microarray). A gene expression microarray may detect expression of thousands of genes at a time. Expression data 102 associated with using a gene expression microarray may be associated with 1,000, at least 10,000, or at least 100,000 gene detection events. In some embodiments, expression data 102 may be obtained by performing next generation sequencing. Such expression data may be associated with obtaining sequence reads using next generation sequencing, aligning the sequencing reads to a reference (e.g., by using one or more sequence alignment algorithms), determine expression level values for certain genes based on the alignment, etc. Expression data 102 associated with performing next generation sequencing may be associated with at least 10,000, at least 100,000, at least 1,000,000, or at least 10,000,000 sequence reads. In some embodiments, expression data 102 may be obtained by using a hybridization-based expression assay (e.g., labeled probe to target a region of interest in a biological sequence). Expression data 102 associated with using a hybridization-based expression may be associated with 1,000, at least 10,000, or at least 100,000 gene detection events.
In some embodiments, expression data 102 includes RNA Seq data. In such embodiments, expression data 102 may involve obtaining RNA expression levels obtained by performing RNA sequencing. In some embodiments, expression data 102 is obtained by performing whole genome sequencing (WGS). In some embodiments, expression data 102 is obtained by performing whole exome sequencing (WES). In some embodiments, expression data 102 includes a combination of RNA Seq data and WGS data. In some embodiments, expression data 102 includes a combination of RNA Seq data and WES data.
In some embodiments, expression data 102 includes values for the N different genes, where a value represents an expression level for a particular gene. For example, first expression data 102 includes a value of 10.455 representing the expression level for gene2 and a value of 0.0001 representing the expression level for geneN, which indicates that gene2 has a higher expression level in sample 1 than geneN. As discussed above, the sequencing platform used to obtain expression data 102 may impact the specific values of the expression data and the relative values among the genes.
According to some embodiments, ranking process 108 may involve ranking genes based on their expression levels in expression data 102 to obtain gene ranking(s) 110. Ranking process 108 may involve ranking genes in a set of genes based on numerical values of their expression levels. In some embodiments, ranking process 108 may involve ranking some or all of the genes in expression data 102 to obtain gene ranking(s) 110. Different gene rankings may be obtained by ranking expression levels for different gene sets. Determining a gene ranking may involve determining a relative rank for each gene in the set of genes. As shown in FIG. 1 , genes in expression data 102 may be ranked based on their expression levels using ranking process 108 for gene set 1 106 a to obtain first gene ranking 110 a. Similarly, genes in expression data 102 may be ranked based on their expression levels using ranking process 108 for gene set 2 106 b to obtain second gene ranking 110 b. Gene ranking 110 a and gene ranking 110 b have relative ranks for the different genes. As shown in FIG. 1 , gene ranking 110 a has the relative ranks of 30, N−1, 2, and 1, for gene1, gene2, gene3, and geneN, respectively, and gene ranking 110 b has the relative ranks of 15, 21, 2, and 1, for gene1, gene2, gene3, and geneN, respectively. A gene ranking may include values identifying the relative ranks for genes in the gene ranking. In some embodiments, the values identifying the relative ranks may include ordinal numbers. In some embodiments, the values identifying the relative ranks may include whole numbers, such as shown in FIG. 1 . In some embodiments, the values identifying the relative ranks may be used as an input (e.g., a vector of the relative ranks) to a statistical model for predicting a characteristic using the techniques described herein. In some embodiments, a gene ranking may include a sorted list of genes according to the relative ranks of the genes. In such embodiments, the sorted list of genes may be used as an input (e.g., a vector with the sorted list of genes) to a statistical model for predicting a characteristic using the techniques described herein. For example, a gene set may include gene list A=[x1, x2, x3, . . . xN−1, xN] and ranking process 108 may output a sorted list of genes [x2, x15, xN−1 . . . x1, xN] with their corresponding relative ranks as [1, 2, 3, . . . N−1, N]. The sorted list of genes [x2, x15, xN−1 . . . x1, xN] and their relative ranks [1, 2, 3, . . . N−1, N] may be used as input to a statistical model.
In some embodiments, ranking process 108 may involve ordering genes in the gene set from the lowest to highest expression level and labeling the list of genes with the rank for individual genes. For example, lowest expression level values are ordered first on the list of genes and their corresponding labels are lowest (e.g., 1, 2, 3, etc.) while the highest expression level values have corresponding higher labels. In some embodiments, ranking process 108 may involve ordering genes in descending order so that genes in the gene set are ranked from highest to lowest expression level values. In some embodiments, ranking process 108 may involve one or more pre-processing steps prior to ranking genes, including binning gene expression values, rounding gene expression values. For example, in some embodiments, gene expression values may be sorted into bins and then ranked. As another example, in some embodiments, gene expression values may be truncated and then ranked. Other pre-processing steps may be applied to the expression levels and the ranking may be performed on the pre-processed values, as aspects of the technology described herein are not limited to ranking only by sorting on the exact gene expression levels that were obtained.
In instances where a group of genes have equal or substantially similar expression level values, the genes in the group may have a common rank and a label indicating the common rank. In some embodiments, the common rank may be determined as being the average of the ranks for the genes in the group. For example, one gene in the gene set may have an expression level value of 30 and is ranked as 4 and the next genes in the ordered list have expression level values of 35, 35, and 35, which are ranked as 5, 6, and 7, respectively, then these genes are all ranked as 6 (which is the average of 5, 6, and 7). In some embodiments, a gene ranking may include two or more genes having a common rank. In some embodiments, a gene ranking where a group of genes have a common rank may include consecutive ranking labels (e.g., 1, 2, 2, 2, 3, 4, 5, etc.). In some embodiments, a gene ranking where a group of genes have a common rank may include ranking labels that skip one or more values (e.g., 1, 2, 2, 2, 5, 6, 6, 8, etc.). In some embodiments, a group of genes having equal or substantially similar expression level values may be ranked according to the minimum rank or maximum rank in the group of genes.
To determine a particular characteristic of a biological sample (e.g., tissue of origin, cancer grade, tissue type, tissue subtype, such as e.g., PTCL subtype, viral status, such as e.g., HPV status), a selected set of genes may be used in ranking process 108 to obtain gene ranking(s) 110. As shown in FIG. 1 , gene set 1 106 a is used to obtain gene ranking 110 a, which is then used to determine characteristic 1 114 a. Similarly, gene set 2 106 b is used to obtain gene ranking 110 b, which is then used to determine characteristic 2 114 b. For example, one set of genes may be used for determining tissue of origin for the biological sample and another set of genes may be used for determining cancer grade.
The number of genes in a set of genes may be in the range of 3 to 1,000 genes, 5 to 500 genes, 5 to 200 genes, 5 to 100 genes, 3 to 50 genes, 20 to 100 genes, 50 to 100 genes, 50 to 200 genes, 50 to 300 genes, 100 to 300 genes, and 50 to 500 genes. The set of genes may include at least 3 genes, at least 5 genes, at least 10 genes, or at least 20 genes. The set of genes may consist of 5-50 genes, 5-100 genes, 20-100 genes, 50-100 genes, 5-200 genes, 5-300 genes, 10-200 genes, 50-300 genes, 5-500 genes, or 50-500 genes.
A gene ranking and a statistical model may be used to determine a particular characteristic of the biological sample. In particular, a gene ranking may be used as an input to the statistical model and an output indicating the characteristic may be obtained. To obtain different characteristics, different gene sets and different statistical models are used where determining a particular characteristic involves using a specific gene set and a statistical model trained using training data indicating rankings of expression levels for some or all genes in the set of genes. For example, statistical model 112 a is specific for determining characteristic 1 114 a and was trained using training data indicating rankings of expression levels for some or all of the genes in gene set 1 106 a. Similarly, statistical model 112 b is specific for determining characteristic 2 114 b and was trained using training data indicating rankings of expression levels for some or all of the genes in gene set 2 106 b. For example, statistical model 112 a and gene set 1 106 a may be used for determining cancer grade for cells in the biological sample and statistical model 112 b and gene set 2 106 b may be used for determining tissue of origin for cells in the biological sample.
The training data may include rankings of expression levels associated with multiple samples, including samples associated with the characteristic being determined using the statistical model. For example, in embodiments where the statistical model is used to predict cancer grade, the training data may include rankings of expression levels associated with samples of multiple cancer grades (e.g., Grade 1, Grade 2, Grade 3). As another example, in embodiments where the statistical model is used to predict tissue of origin, the training data may include rankings of expression levels associated with samples from multiple tissue of origins (e.g., thyroid tissue, lymph node tissue, adrenal gland tissue, skin tissue, breast tissue, ovary tissue, prostate tissue, urothelial tissue, cervical tissue, esophagus tissue, brain tissue, soft tissue, connective tissue, head tissue, and neck tissue). As another example, in embodiments where the statistical model is used to predict HPV status, the training data may include rankings of expression levels associated with samples from both HPV-positive status and HPV-negative status. As another example, in embodiments where the statistical model is used to predict PTCL subtype, the training data may include rankings of expression levels associated with samples from different PTCL subtypes (e.g., adult T-cell leukemia/lymphoma (ATLL), angioimmunoblastic T-cell lymphoma (AITL), NK/T-cell lymphoma (NKTCL), anaplastic large cell lymphoma (ALCL), and cases belong to the Not Otherwise Specified (PTCL-NOS)).
It should be appreciated that a statistical model, such as statistical model 112 a and statistical model 112 b, may be used determining one or more characteristics for different biological samples obtained from different subjects. In some instances, the number of subjects that may use the same statistical model may be at least 50, 100, 200, 300, 500, 1,000, 2,000, 5,000, 10,000, or more. Using the statistical model for different subjects may ease analysis of expression data across the different subjects because the same data processing pipeline may be implemented for the individual subjects.
In some embodiments, ranking process 108 may only rank genes included in a set of genes such that not all of the genes in the expression data may obtain a rank or be included in a gene ranking. In such embodiments, the ranking is specific to the set of genes and may be used as an input to statistical model 112.
In some embodiments, ranking process 108 may involve ranking all the genes in expression data 102, such that each gene has a respective rank. In such embodiments, the ranking includes genes outside the set of genes. In some embodiments, an input to a statistical model may include the ranks, determined by ranking process 108, for the set of genes. In some embodiments, an input to a statistical model may include the ranking obtained by ranking process 108 and a statistical model may selectively use the ranks for the set of genes in the ranking as part of determining the one or more characteristics.
A statistical model may involve using one or more suitable machine learning algorithms, including one or more classifiers. Examples of classifiers that a statistical model may include are a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier. In some embodiments, a statistical model may involve using a gradient boosted decision tree classifier. In some embodiments, a statistical model may involve using a decision tree classifier. In some embodiments, a statistical model may involve using a gradient boosted classifier. In some embodiments, a statistical model may involve using a random forest classifier. In some embodiments, a statistical model may involve using a clustering-based classifier. In some embodiments, a statistical model may involve using a Bayesian classifier. In some embodiments, a statistical model may involve using a Bayesian network classifier. In some embodiments, a statistical model may involve using a neural network classifier. In some embodiments, a statistical model may involve using a kernel-based classifier. In some embodiments, a statistical model may involve using a support vector machine classifier.
In some embodiments, a statistical model may perform binary classification of one or more features as an output of the statistical model. For example, such a statistical model may perform classification of one or more cancer grades (e.g., Grade 1, Grade 2, Grade 3) and an output of the statistical model may include a prediction for each of the one or more cancer grades indicating whether a biological sample is categorized as being a particular cancer grade.
In some embodiments, a statistical model may involve using a machine learning algorithm that implements of a gradient boosting framework, such as a gradient boosting decision tree (GBDT) and a gradient boosted regression tree (GBRT). An example of a machine learning algorithm that implements a gradient boosting decision tree is the LightGBM package, which is further described in Guolin Ke, Qi Meng, Thomas Finley, Taifeng Wang, Wei Chen, Weidong Ma, Qiwei Ye and Tie-Yan Liu, LightGBM: A highly efficient gradient boosting decision tree, Advances in Neural Information Processing Systems, pp. 3149-3157, 2017, which is incorporated by reference herein in its entirety. An example of a machine learning algorithm that implements a gradient boosting framework is the XGBoost package, which is further described in Tiangi Chen and Carlos Guestrin. XGBoost: A scalable tree boosting system, In Proceedings of the 22Nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 785-794, ACM, 2016, which is incorporated by reference herein in its entirety. An example of a machine learning algorithm that implements a gradient boosted regression tree is the pGBRT package, which is further described in Stephen Tyree, Kilian Q Weinberger, Kunal Agrawal, and Jennifer Paykin, Parallel boosted regression trees for web search ranking, In Proceedings of the 20th international conference on World wide web, pp. 387-396, ACM, 2011, which is incorporated by reference herein in its entirety.
A statistical model may be trained using multiple rankings of expression levels for some or all of the genes in the set of genes. Training data may include available expression data obtained through research organizations, including the National Cancer Institute (NCI) (e.g., Gene Expression Omnibus (GEO)), National Center for Biotechnology Information (NCBI) (e.g., Sequence Reach archive (SRA)), The Cancer Genome Atlas Program (TCGA), ArrayExpress Archive of Functional Genomics Data (by the European Molecular Biology Laboratory), and International Cancer Genome Consortium.
For example, a statistical model used for determining cancer grade for breast cancer may be trained using data from Series GSE96058 available through the NCI. As another example, a statistical model used for determining cancer grade for kidney clear cell cancer may be trained using data from The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) data collection. As yet another example, a statistical model used for determining tissue of origin for DLBCL (e.g., ABC, GCB) may be trained using data from one or more of Series GSE117556, Leipzig Lymphoma data set (10.1186/s13073-019-0637-7), Series GSE31312, Series GSE10846, Series GSE87371, Series GSE11318, Series GSE32918, Series GSE23501, Lymphoma/Leukemia Molecular Profiling Project (LLMPP), and Series GSE93984. As another example, a statistical model used for determining tissue of origin and histological information (e.g., tissue type) for cancer may be trained using data from The Cancer Genome Atlas Program (TCGAP).
One characteristic that may be determined using the techniques described herein is cancer grade for cells in the biological sample. Cancer grade may include Grade 1, Grade 2, Grade 3, Grade 4, and Grade 5. It should be appreciated that some cancer grading systems may include any suitable number of grades, or other scores, and that the techniques described herein may be used for determining any number of cancer grades regardless of the cancer grading system being implemented. For example, some cancer grading systems may have a number of cancer grades in the range of 1 to 10. Another characteristic is tissue of origin for cells in the biological sample. Tissue of origin may include lung tissue, pancreas tissue, stomach tissue, colon tissue, liver tissue, bladder tissue, kidney tissue, thyroid tissue, lymph node tissue, adrenal gland tissue, skin tissue, breast tissue, ovary tissue, prostate tissue, urothelial tissue, cervical tissue, esophagus tissue, brain tissue, soft tissue, connective tissue, head tissue, and neck tissue. In some instances, tissue of origin may refer to cell of origin. For example, where the subject has, is suspected of having, or is at risk of having Diffuse Large B-Cell Lymphoma (DLBCL), the tissue of origin is a cell of origin may include germinal center B-cell (GCB) and activated B-cell (ABC).
Another characteristic is histological information for cells in the biological sample. The histological information may correspond to a determination made by a physician (e.g., pathologist) using microscopy to visually inspect the biological sample. Histological information may include a tissue type. Examples of tissue types include adenocarcinoma, squamous cell carcinoma, carcinoma, cystadenocarcinoma, sarcoma, and glioma. In some embodiments, a statistical model may output a combination of tissue of origin and histological information. The combination of tissue of origin and histological information may include lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast carcinoma, colorectal adenocarcinoma, ovarian serous cystadenocarcinoma, phenochromocytoma, bladder urothelial carcinoma, cervical squamous cell carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, paraganglioma, prostate adenocarcinoma, sarcoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrial carcinoma.
A characteristic (e.g., cancer grade, tissue of origin, PTCL subtype) may be output to a user, such as a physician or clinician, by displaying the characteristic to the user in a graphical user interface (GUI), including the characteristic in a report, sending an email to the user, and/or in any other suitable way. The subject's characteristic may be used for various clinical purposes, including assessing the efficacy of a treatment for cancer, identifying a treatment for the subject, administering a treatment for the subject, determining a prognosis for the subject, and/or evaluating suitability of the subject for participating in a clinical trial. In some embodiments, the subject's characteristic may be used in identifying a treatment for the subject. For example, in embodiments where a tissue of origin is determined for cells in the biological sample, the determined tissue of origin may be used to identify a treatment for the subject associated with treating cancers of the determined tissue of origin. As yet another example, in embodiments where a cancer grade is determined for cells in the biological sample, the determined cancer grade may be used to identify a treatment for the subject associated with treating cancers having the determined cancer grade. As yet another example, in embodiments where a PTCL subtype is determined for cells in the biological sample, the determined PTCL subtype may be used to identify a treatment for the subject suitable for treating lymphomas of the determined PTCL subtype. In turn, the identified treatment may be administered.
In some embodiments, the subject's characteristic may be used for administering a treatment for the subject. For example, in embodiments where a tissue of origin is determined for cells in the biological sample, a physician may administer a treatment for the subject associated with treating cancers of the determined tissue of origin. As yet another example, in embodiments where a cancer grade is determined for cells in the biological sample, a physician may administer a treatment for the subject associated with treating cancers having the determined cancer grade. As yet another example, in embodiments where a PTCL subtype is determined for cells in the biological sample, a physician may administer a treatment for the subject suitable for treating lymphomas of the determined PTCL subtype. Further examples where characteristics of a biological sample determined using the techniques described herein are used for administering a treatment are provided in the section titled “Methods of Treatment”.
In some embodiments, the subject's characteristic may be used in determining a prognosis for the subject. In embodiments where the subject has, is suspected of having, or is at risk of having cancer (e.g., kidney cancer, clear cell kidney cancer, lymphoma, head and neck squamous cell carcinoma, lung adenocarcinoma), the determined subject's characteristic may be used to determine a prognosis for the subject. For example, in embodiments, where the subject's characteristic is cancer grade, the determined cancer grade (e.g., Grade 1, Grade2, Grade3) may be used to determine a prognosis for the subject. Further aspects relating to other applications where characteristics of a biological sample determined using the techniques described herein are provided in the section titled “Applications”.
In some embodiments, the determined characteristic of the biological sample may include cancer grade for cells in the biological sample. In such embodiments, the set of genes used to obtain a gene ranking may include genes associated with biological features, expression pathways, or otherwise associated with determining cancer grade. Some embodiments involve using a gene set for determining cancer grade for breast cancer. Examples of genes that may be included in such a gene set are listed in Table 1, below.

TABLE 1

Grade Classifier for Breast Cancer

	NCBI
Gene	Gene ID	NCBI Accession Number(s)

UBE2C	11065	NM_001281741; NM_001281742; NM_007019; NM_181799;
		NM_181800; NM_181801
MYBL2	4605	NM_001278610; NM_002466
PRAME	23532	NM_001291715; NM_001291716; NM_006115; NM_206953;
		NM_206954; NM_206955; NM_206956; NM_001291717;
		NM_001291719; NM_001318126; NM_001318127
LMNB1	4001	NM_005573
CXCL9	4283	NM_002416
KPNA2	3838	NM_001320611; NM_002266
TPX2	22974	NM_012112; XM_011528697; XM_011528699
PLCH1	23007	NM_001130960; NM_001130961; NM_014996; XM_011512561;
		XM_017005923; XM_017005926; NM_001349251; XM_011512560;
		XM_011512562; XM_011512565; XM_017005925; NM_001349250;
		NM_001349252; XM_005247238; XM_005247239; XM_011512567;
		XM_017005927; XM_011512566
CCL18	6362	NM_002988
CDK1	983	NM_001320918; NM_001786; NM_033379; XM_005270303
MELK	9833	NM_001256685; NM_001256687; NM_001256688; NM_001256689;
		NM_001256690; NM_001256692; NM_001256693; NM_014791;
		XM_011518076; XM_011518077; XM_011518078; XM_011518079;
		XM_011518081; XM_011518082; XM_011518083; XM_011518084
CCNB2	9133	NM_004701
RRM2	6241	NM_001034; NM_001165931
CCNB1	891	NM_031966
NUSAP1	51203	NM_001243142; NM_001243143; NM_001243144; NM_001301136;
		NM_016359; NM_018454; XM_005254430
SLC7A5	8140	NM_003486
TYMS	7298	NM_001071
GZMK	3003	NM_002104
SQLE	6713	NM_003129
C1orf106	55765	NM_001142569; NM_018265; XM_011509754; XM_011509755
CDC25B	994	NM_001287519; NM_001287520; NM_004358; NM_021872;
		NM_021873; NM_001287516; NM_001287522; NM_001287518;
		NM_001287524
ATAD2	29028	NM_014109
QPRT	23475	NM_001318249; NM_001318250; NM_014298
CCNA2	890	NM_001237
NEK2	4751	NM_001204182; NM_001204183; NM_002497; XM_005273147
IDO1	3620	NM_002164
NDC80	10403	NM_006101
ZWINT	11130	NM_001005413; NM_007057; NM_032997
ABCA12	26154	NM_015657; NM_173076
TOP2A	7153	NM_001067
TDO2	6999	NM_005651
S100A8	6279	NM_001319197; NM_001319198; NM_001319201; NM_002964
LAMP3	27074	NM_014398
MMP1	4312	NM_002421
GZMB	3002	NM_001346011; NM_004131
BIRC5	332	NM_001168; NM_001012271; NM_001012270
TRIP13	9319	NM_004237; XM_011514163
RACGAP1	29127	NM_001126103; NM_001126104; NM_001319999; NM_001320000;
		NM_001320001; NM_001320002; NM_001320003; NM_001320004;
		NM_013277; XM_006719359; XM_011538238; XM_017019220;
		NM_001320005; NM_001320006; NM_001320007
ASPM	259266	NM_001206846; NM_018136
ESRP1	54845	NM_001122827; NM_001034915; NM_001122825; NM_001122826;
		NM_017697; XM_005250991
MAD2L1	4085	NM_002358
CENPF	1063	NM_016343; XM_017000086
CDC20	991	NM_001255
MCM4	4173	NM_005914; NM_182746
MKI67	4288	NM_001145966; NM_002417
PBK	55872	NM_001278945; NM_018492; NM_001363040
CKS2	1164	NM_001827
KIF2C	11004	NM_001297655; NM_001297656; NM_001297657; NM_006845
MRPL13	28998	NM_014078
TTK	7272	NM_001166691; NM_003318; XM_011536099; XM_011536100
BUB1	699	NM_001278617; NM_004336
TK1	7083	NM_001346663; NM_003258
FOXM1	2305	NM_001243088; NM_202003; NM_202002; NM_001243089;
		NM_021953
CEP55	55165	NM_001127182; NM_018131
EZH2	2146	NM_004456; XM_011515884; NM_001203247; NM_001203248;
		NM_001203249; NM_152998
ECT2	1894	NM_001258315; NM_001258316; NM_018098; XM_011512514
PRC1	9055	NM_003981; NM_001267580
CENPU	79682	NM_024629
CCNE2	9134	NM_057749; XM_011517366; NM_004702; XM_017013959;
		XM_017013958; NM_057735
AURKA	6790	NM_001323303; NM_001323304; NM_001323305; NM_003600;
		NM_198433; NM_198434; NM_198435; NM_198436; NM_198437;
		XM_017028035
HMGB3	3149	NM_001301228; NM_001301229; NM_001301231; NM_005342
APOBEC3B	9582	NM_001270411; NM_004900
LAGE3	8270	NM_006014
CDKN3	1033	NM_001258
DTL	51514	NM_001286229; NM_016448
ATP6V1C1	528	NM_001695
KIAA0101	9768	NM_001029989; NM_014736
CD2	914	NM_001328609; NM_001767
KIF11	3832	NM_004523
KIF20A	10112	NM_005733
CDCA8	55143	NM_001256875; NM_018101
NCAPG	64151	NM_022346
CENPN	55839	NM_001100624; NM_001100625; NM_001270473; NM_001270474;
		NM_018455; XM_006721236; XM_017023456
MTFR1	9650	NM_014637; NM_001145838
MCM2	4171	NM_004526
DSCC1	79075	NM_024094
WDR19	57728	NM_001317924; NM_025132
SEMA3G	56920	NM_020163
KCND3	3752	NM_172198; XM_011541425; XM_006710632; XM_011541428;
		NM_001378969; NM_004980; NM_001378970; XM_006710629;
		XM_006710631; XM_017001245; XM_011541426; XM_011541427;
		XM_017001244
SETBP1	26040	NM_001130110; NM_015559
KIF13B	23303	NM_015254
NR4A2	4929	NM_006186; XM_017004219; XM_017004220; XR_001738751;
		XR_001738752; NM_173173; NM_173171; XM_011511246;
		NM_173172; XR_427087; XM_005246621; XM_006712553
NAV3	89795	XM_017020172; NM_001024383; NM_014903; XM_011538944
PDZRN3	23024	NM_001303140; NM_001303142; NM_001303139; NM_001303141;
		NM_015009
MAGI2	9863	NM_001301128; NM_012301
CACNA1D	776	XM_005265448; NM_000720; NM_001128839; NM_001128840
STC2	8614	NM_003714
CHAD	1101	NM_001267
PDGFD	80310	NM_025208; NM_033135
ARMCX2	9823	NM_001282231; NM_014782; NM_177949; XM_005278109;
		XM_005278110; XM_005278111; XM_005278113; XM_005278114;
		XM_005278115; XM_005278116; XM_005278117; XM_011531071;
		XM_011531072; XM_017029987; XM_017029988; XM_017029989;
		XM_017029990; XM_017029991; XM_017029992; XM_017029993;
		XM_017029994; XM_017029995; XM_017029996; XM_017029997
FRY	10129	NM_023037
AGTR1	185	NM_032049; NM_004835; NM_031850; NM_000685; NM_009585
MARCH8	220972	NM_001002266; NM_145021; XM_011539495; XM_006717704;
		NM_001002265; XR_246519; NM_001282866; XM_005271804;
		XM_011539492
ANG	283	NM_001097577; NM_001145
ABAT	18	NM_000663; NM_001127448; NM_020686; NM_001386602;
		NM_001386609; NM_001386611; NM_001386612; NM_001386613;
		NM_001386605; NM_001386610; NM_001386616; NM_001386601;
		NM_001386603; NM_001386604; NM_001386606; NM_001386615;
		NM_001386600; NM_001386607; NM_001386614; NM_001386608
THBD	7056	NM_000361
RAI2	10742	NM_001172739; NM_001172743; NM_021785; NM_001172732;
		XM_006724459; XM_006724460; XM_011545439; XM_011545440;
		XM_011545441
HSPA2	3306	NM_021979
ERBB4	2066	NM_001042599; NM_005235; XM_005246376; XM_005246377
ECHDC2	55268	NM_001198962; NM_001198961; NM_018281; XM_011541722;
		XM_011541726; XM_017001638; XM_024448158; XM_011541719;
		XM_011541723; XM_024448164; XR_002957011; NM_001319958;
		XR_002957012; XM_011541709; XM_024448153; XM_024448157;
		XR_002957014; XM_011541713; XM_024448163; XM_011541715;
		XM_017001640; XM_024448160; XM_011541720; XM_011541727;
		XM_024448159; XM_024448161; XR_002957013; XM_017001639;
		XM_024448152
FST	10468	NM_013409; NM_006350
EPHX2	2053	NM_001256482; NM_001256483; NM_001256484; NM_001979
FOSB	2354	NM_006732; XM_005258691; NM_001114171
STARD13	90627	NM_178006; NM_052851; NM_001243466; NM_001243474;
		NM_001243476; NM_178007
ID4	3400	NM_001546
FAM129A	116496	NM_052966
FCGBP	8857	NM_003890
LAMA2	3908	NM_000426; NM_001079823
FGFR2	2263	NM_001144919; NM_023029; NM_000141; NM_001144913;
		NM_001144914; NM_001144915; NM_001144916; NM_001144917;
		NM_001144918; NM_001320654; NM_001320658; NM_022970
PTGER3	5733	NM_198718; NM_001126044; NM_198714; NM_198715; NM_198716;
		NM_198717; NM_198719
NME5	8382	NM_003551
LRRC17	10234	NM_001031692; NM_005824; XM_005250108
OSBPL1A	114876	NM_080597; NM_018030; XM_017025533; XR_002958162;
		XM_006722380; XM_017025530; NM_133268; XM_017025532;
		XR_001753139; NM_001242508; XM_006722382; XM_017025531
ADRA2A	150	NM_000681
LRP2	4036	NM_004525
C1orf115	79762	NM_024709
COL4A5	1287	NM_000495; NM_033380
DIXDC1	85458	NM_001037954; NM_001278542; NM_033425
KIAA1324	57535	NM_001267048; NM_020775; XM_011541825
HPN	3249	NM_002151; NM_182983; XM_017026732; NM_001384133;
		XM_017026731; NM_001375441
KLF4	9314	NM_001314052; NM_004235
SCUBE2	57758	NM_001170690; NM_001330199; NM_020974
FMO5	2330	NM_001461; XM_005272946; XM_005272947; XM_005272948;
		XM_011509350; NM_001144829; NM_001144830; XM_006711245
SORBS2	8470	NM_001145670; NM_001145671; NM_001145672; NM_001145673;
		NM_001145674; NM_001270771; NM_003603; NM_021069;
		XM_005263312; XM_006714390; XM_017008771
CARD10	29775	NM_014550
CITED2	10370	NM_001168389; NM_001168388; NM_006079
MUC1	4582	NM_001204292; NM_001204286; NM_001204291; NM_001204285;
		NM_001204287; NM_001204288; NM_001204289; NM_001204290;
		NM_001204295; NM_001204297; NM_001204296; NM_001018016;
		NM_001018017; NM_001044390; NM_001044391; NM_001044392;
		NM_001044393; NM_001204293; NM_001204294; NM_002456
BCL2	596	NM_000633; NM_000657
RGS5	8490	NM_003617; NM_001195303; NM_001254748; NM_001254749
CYBRD1	79901	NM_001127383; NM_001256909; NM_024843
OMD	4958	NM_005014
IGFBP4	3487	NM_001552
LAMB2	3913	NM_002292; XM_005265127
DUSP4	1846	NM_001394; NM_057158; XM_011544428
PDLIM5	10611	NM_006457; NM_001011515; NM_001011516; NM_001256429
IRS2	8660	NM_003749
CX3CR1	1524	NM_001171171; NM_001171172; NM_001171174; NM_001337

Some embodiments involve using a gene set for determining cancer grade for kidney clear cell cancer. Examples of genes that may be included in such a gene set are listed in Table 2, below.

TABLE 2

Grade Classifier for Kidney Clear Cell

	NCBI
Gene	Gene ID	NCBI Accession Number(s)

PLTP	5360	NM_001242920; NM_001242921; NM_006227; NM_182676
C1S	716	NM_001346850; NM_001734; NM_201442; XM_005253760
LY96	23643	NM_001195797; NM_015364
TSKU	25987	NM_001258210; NM_001318477; NM_001318478; NM_001318479;
		NM_015516
TPST2	8459	NM_001008566; NM_003595
SERPINF1	5176	NM_001329903; NM_002615
SRPX2	27286	NM_014467
SAA1	6288	NM_000331; NM_001178006; NM_199161
CTHRC1	115908	NM_001256099; NM_138455
GFPT2	9945	NM_005110
CKAP4	10970	NM_006825
SERPINA3	12	NM_001085
CFH	3075	NM_001014975; NM_000186
PLAU	5328	NM_002658; NM_001145031; NM_001319191
BASP1	10409	NM_001271606; NM_006317
PTTG1	9232, 10744	NM_001282382; NM_001282383; NM_004219
MOCOS	55034	NM_017947
LEF1	51176	NM_001130713; NM_001130714; NM_001166119; NM_016269
SLPI	6590	NM_003064
PRAME	23532	NM_001291715; NM_001291716; NM_006115; NM_206953; NM_206954;
		NM_206955; NM_206956; NM_001291717; NM_001291719; NM_001318126;
		NM_001318127
STEAP3	55240	NM_001008410; NM_018234; XM_006712614; XM_006712615;
		XM_011511403; NM_138637; NM_182915
LGALS2	3957	NM_006498
CD44	960	NM_000610; NM_001001389; NM_001001390; NM_001001391;
		NM_001001392; NM_001202555; NM_001202556; NM_001202557;
		XM_011520488
FLNC	2318	NM_001127487; NM_001458
UBE2C	11065	NM_001281741; NM_001281742; NM_007019; NM_181799; NM_181800;
		NM_181801
CTSK	1513	NM_000396
SULF2	55959	NM_001161841; NM_018837; NM_198596; NM_001387053; XM_006723830;
		NM_001387051; NM_001387055; NM_001387048; NM_001387049;
		NM_001387054; XM_011528914; NM_001387050; NM_001387052
TMEM45A	55076	XM_005247569; NM_018004; XM_024453614; XM_024453615;
		NM_001363876
FCGR1A	2209, 100132417	NM_000566
PLOD2	5352	NM_000935; NM_182943
C19orf80	55908	NM_018687
PDGFRL	5157	NM_006207
IGF2BP3	10643	NM_006547
SLC7A5	8140	NM_003486
PRRX1	5396	NM_006902; NM_022716
RARRES1	5918	NM_002888; NM_206963
LHFPL2	10184	NM_005779; XM_006714515
KDELR3	11015	NM_006855; NM_016657
TRIB3	57761	NM_001301201; XM_017027989; NM_001301188; NM_001301190;
		NM_001301193; NM_001301196; NM_021158
IL20RB	53833	NM_144717
FBLN1	2192	NM_001996; NM_006485; NM_006486; NM_006487
KMO	8564	NM_003679
C1R	715	NM_001733
CYP1B1	1545	NM_000104
KIF2A	3796	NM_001098511; NM_001243952; NM_004520
PLAUR	5329	NM_001301037; NM_001005376; NM_001005377; NM_002659
CKS2	1164	NM_001827
CDCP1	64866	NM_022842; NM_178181
SFRP4	6424	NM_003014
HAMP	57817	NM_021175
MMP9	4318	NM_004994
SLC3A1	6519	NM_000341; XM_011533047
NAT8	9027, 51471	NM_003960
FRMD3	257019	NM_001244959; NM_001244960; NM_001244961; NM_001244962;
		NM_174938
NPR3	4883	NM_000908; NM_001204375; NM_001204376
NAT8B	9027, 51471	NM_016347
BBOX1	8424	NM_003986; NM_001376258; NM_001376259; NM_001376260;
		NM_001376261; XM_011520402
SLC5A1	6523	NM_000343; NM_001256314
GBA3	57733	NM_020973; NM_001128432; NM_001277225
EMCN	51705	NM_016242; XM_011532024; NM_001159694
SLC47A1	55244	NM_018242
AQP1	358	NM_198098
PCK1	5105	NM_002591
UGT2A3	79799	NM_024743
BHMT	635	NM_001713
FMO1	2326	NM_001282693; NM_002021; NM_001282692; NM_001282694
ACAA2	10449, 648603	NM_006111
SLC5A8	160728	NM_145913
SLC16A9	220963	NM_001323981; NM_194298; XM_017015884
TSPAN18	90139	NM_130783; XM_006718373; XM_011520459
SLC17A3	10786	NM_006632; NM_001098486
STK32B	55351	NM_001306082; NM_018401
MAP7	9053	NM_001198609; NM_001198608; NM_001388350; NM_001198614;
		NM_001198617; NM_001388331; NM_001388333; NM_001388336;
		NM_001388340; NM_001388344; NM_001388345; NM_001388347;
		NM_001388348; NM_001388349; XM_011536246; NM_001198616;
		NM_001388330; NM_001388335; NM_003980; XM_011536243;
		NM_001388341; NM_001388342; NM_001388343; NM_001198611;
		NM_001388338; NM_001388346; NM_001198615; NM_001198618;
		NM_001198619; NM_001388329; NM_001388334; NM_001388339;
		NM_001388328; NM_001388332; NM_001388337; NM_001388351;
		NM_001388352; NM_001388353
MYLIP	29116	XM_005249033; NM_013262
SLC22A12	116085	NM_001276326; NM_001276327; NM_144585; NM_153378
LRP2	4036	NM_004525
CD34	947	NM_001025109; NM_001773
PODXL	5420	NM_001018111; NM_005397
ZBTB42	100128927	NM_001137601; NM_001370342
TEK	7010	NM_000459; NM_001290077; NM_001290078
FBP1	2203	NM_000507; NM_001127628
BCL2	596	NM_000633; NM_000657

In some embodiments, the determined characteristic of the biological sample may include tissue of origin for cells in the biological sample. In such embodiments, the set of genes used to obtain a gene ranking may include genes associated with biological features, expression pathways, or otherwise associated with determining tissue of origin. Some embodiments involve using a gene set for predicting tissue of origin for Diffuse Large B-Cell Lymphoma (DLBCL), such as germinal center B-cell (GCB) and activated B-cell (ABC). Examples of genes that may be included in such a gene set are listed in Table 3, below.

TABLE 3

Tissue of origin classifier for DLBCL

	NCBI
Gene	Gene ID	NCBI Accession Number(s)

ITPKB	3707	NM_002221; NM_001388404; XM_017001211
MYBL1	4603	NM_001080416; NM_001144755; NM_001294282
LMO2	4005	NM_001142315; NM_001142316; NM_005574; XM_005252921;
		XM_017017727; XM_017017728; XM_017017729;
		XM_017017730; XM_017017731; XM_017017732; XM_017017733
BATF	10538	NM_006399
IRF4	3662	NM_001195286; NM_002460
LRMP	4033	NM_001204126; NM_001204127; NM_006152; NM_001366543;
		NM_001366544; NM_001366546; NM_001366549;
		NM_001366545; NR_159367; NR_159368; NM_001366541;
		NR_159366; NM_001366540; NM_001366542; NM_001366547;
		NR_159369; NM_001366548
CCND2	894	NM_001759
SLA	6503	NM_001045556; NM_001045557; NM_001282964;
		NM_001282965; NM_006748; XM_017013739
SP140	11262	NM_001278452; NM_001005176; NM_001278451;
		NM_001278453; NM_007237
PIM1	5292	NM_002648; NM_001243186
CSTB	1476	NM_000100
BCL2	596	NM_000633; NM_000657
TCF4	6925	NM_003199; XM_017025956; NM_001348220; NM_001369581;
		NM_001369582; NM_001369585; XM_005266749;
		XM_005266761; XM_017025950; NM_001083962;
		NM_001243235; NM_001348211; NM_001348214;
		NM_001369583; XM_017025951; XM_024451241;
		NM_001369578; NM_001243227; NM_001243231;
		NM_001348218; NM_001369571; NM_001369575;
		NM_001369586; XM_005266755; XM_006722538;
		XM_017025954; NM_001243230; NM_001243233;
		NM_001306207; NM_001306208; NM_001330604;
		NM_001348212; XM_005266752; NM_001243236;
		NM_001330605; NM_001348213; NM_001348215;
		NM_001348217; NM_001348219; NM_001369577;
		NM_001369580; XM_017025952; XM_017025953;
		NM_001243226; NM_001348216; NM_001369567;
		NM_001369569; NM_001369573; NM_001369574;
		NM_001369579; NM_001243228; NM_001243232;
		NM_001243234; NM_001369568; NM_001369570;
		NM_001369572; NM_001369576; NM_001369584
P2RX5	5026	NM_001204519; NM_001204520; NM_002561; NM_175080
SPINK2	6691	NM_021114; NM_001271722; NM_001271720; NM_001271718;
		NM_001271721
VCL	7414	NM_003373; NM_014000
PTPN1	5770	NM_002827; NM_001278618
REL	5966	NM_001291746; NM_002908
FUT8	2530	NM_004480; NM_178155; NM_178156
RPL21	6144	NM_000982
PRKCB1	5579	NM_002738; NM_212535
CSNK1E	1454	NM_001289912; NM_001894; NM_152221
GPR18	2841	NM_001098200; NM_005292; XM_006719946
IGHM	3500, 3492, 28396,	NG_001019.6
	3502, 3507, 28450,
	28452
ACP1	52	NM_004300; NM_007099
SPIB	6689	NM_003121; NM_001243998; NM_001243999; NM_001244000
HLA-DQA1	3117, 731682,	NM_002122
	100133678
KRT8	390601, 149501, 3856	NM_001256293; NM_002273
FAM3C	10447	NM_001040020; NM_014888; XM_011515736; XM_011515737
HLA-DMB	3109	NM_002118
GOT2	2806	NM_001286220; NM_002080
PIM2	11040	NM_006875
PLEK	5341	NM_002664

Some embodiments may involve determining a characteristic of a biological sample by using different gene sets and statistical models corresponding to the different gene sets to obtain characteristic predictions, which are used to determine the characteristic. FIG. 2 is a diagram of an illustrative processing pipeline 200 for determining a characteristic of a biological sample, which may include ranking genes based on their gene expression levels and using the rankings and statistical models to determine the characteristic, in accordance with some embodiments of the technology described herein. Processing pipeline 200 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, processing pipeline 200 may be performed by a desktop computer, a laptop computer, a mobile computing device. In some embodiments, processing pipeline 200 may be performed within one or more computing devices that are part of a cloud computing environment.
In some embodiments, gene expression data 102 is used to rank genes in different sets of genes based on their expression levels in gene expression data 102 to obtain multiple gene rankings. For example, a gene ranking may be obtained for each gene set and the gene ranking may be input to a statistical model trained using training data indicating rankings of expression levels for some or all genes in the gene set. As shown in FIG. 2 , ranking process 108 may involve using expression data 102 to rank genes in different gene sets, including Gene Set 1 106 a, Gene Set 2 106 b, Gene Set 3 106 c, and Gene Set 4 106 d, to obtain Gene Ranking 1 110 a, Gene Ranking 2 110 b, Gene Ranking 3 110 c, and Gene Ranking 4 110 d, respectively. Ranking process 108 may involve ranking genes in a set of genes based on numerical values of their expression levels. Different gene rankings may be obtained by ranking expression levels for different gene sets, and each gene ranking may be input to its respective statistical model to obtain a characteristic prediction. As shown in FIG. 2 , Gene Ranking 1 110 a, Gene Ranking 2 110 b, Gene Ranking 3 110 c, and Gene Ranking 4 110 d is provided as input to Statistical Model 1 112 a, Statistical Model 2 112 b, Statistical Model 3 112 c, and Statistical Model 4 112 d, respectively.
In some embodiments, the different statistical models and their respective gene sets may correspond to a particular characteristic of the biological sample. In such embodiments, each of the statistical models may output a prediction of the biological sample having a particular characteristic. In some instances, the prediction output by a statistical model may include a probability of the biological sample having the characteristic.
As shown in FIG. 2 , Statistical Model 1 112 a outputs Characteristic Prediction 1 116 a, Statistical Model 2 112 b outputs Characteristic Prediction 2 116 b, Statistical Model 3 112 c outputs Characteristic Prediction 3 116 c, and Characteristic Prediction 4 116 d outputs Characteristic Prediction 4 116 d. The predictions output by the different statistical models may be analyzed using prediction analysis process 118 to determine characteristic 114 for the biological sample. Prediction analysis process 118 may involve aggregating the different predictions and selecting a particular characteristic for the biological sample from among the different characteristic predictions. In some embodiments, a characteristic prediction may include a probability that the biological sample has the particular characteristic. In such embodiments, prediction analysis process 118 may involve aggregating the probabilities for the different characteristic predictions and selecting a characteristic based on the probabilities. In some embodiments, selecting the characteristic may involve selecting the characteristic having the highest probability as being characteristic 114.
Although four gene sets and four statistical models are shown in FIG. 2 , it should be appreciated that any suitable number of gene sets and corresponding statistical models may be implemented using the techniques described above in determining characteristic predictions and aggregating the characteristic predictions to obtain a characteristic of a biological sample. In some embodiments, the number of gene sets and corresponding statistical models may be in the range of 3 to 100, 3 to 70, 3 to 50, 3 to 40, 3 to 30, 5 to 50, 10 to 60, or 10 to 70.
In some embodiments, the number of gene sets and corresponding statistical models is equal to or less than the number of classes for the characteristic being predicted using processing pipeline 200. For instance, in embodiments where the characteristic being predicted is tissue of origin, the number of classes may correspond to the different types of tissue that can be determined using processing pipeline 200. Such embodiments may involve a different gene set and corresponding statistical model for each type of tissue. For example, Gene Set 1 106 a and Statistical Model 1 112 a may be used for generating a prediction of the biological sample being lung tissue (as Characteristic Prediction 1 116 a), Gene Set 2 106 b and Statistical Model 2 112 b may be used for generating a prediction of the biological sample being stomach tissue (as Characteristic Prediction 2 116 b), Gene Set 3 106 c and Statistical Model 3 112 c may be used for generating a prediction of the biological sample being liver tissue (as Characteristic Prediction 3 116 c), and Gene Set 4 106 d and Statistical Model 4 112 d may be used for generating a prediction of the biological sample being bladder tissue (as Characteristic Prediction 4). It should be appreciated that additional gene sets and their corresponding statistical models may be implemented for different tissue types. In some embodiments, there may be 21 gene sets and corresponding statistical models, allowing processing pipeline 200 to predict 21 types of tissue.
FIG. 3 is a flow chart of an illustrative process 300 for determining one or more characteristics of a biological sample using a gene ranking and a statistical model, in accordance with some embodiments of the technology described herein. Process 300 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, ranking process 108 and statistical model 112 may perform some or all of process 300 to determine one or more characteristics, such as characteristic(s) 114.
Process 300 begins at act 310, where expression data for a biological sample of a subject is obtained. In some embodiments, the expression data may be obtained using a gene expression microarray. In some embodiments, the expression data may be obtained by performing next generation sequencing. Some embodiments involve performing a sequencing process of the biological sample (e.g., a gene expression microarray, next generation sequencing) prior to obtaining expression data 102. In some embodiments, obtaining gene expression data 102 may involve obtaining gene expression data 102 in silico, such as by accessing, using a computing device, expression data (e.g., expression data that has been previously obtained from a biological sample) in one or more data stores, receiving the expression data from one or more other device, or any other way. In some embodiments, obtaining gene expression data 102 may involve analyzing a biological sample (in vitro) and accessing (e.g., by a computing device, a processor) the expression data. Further aspects relating to obtaining expression data are provided in the section titled “Obtaining Expression Data”.
Next, process 300 proceeds to act 320, where genes in a set of genes are ranked based on their expression levels in the expression data to obtain a gene ranking, such as by using ranking process 108. The expression data may include values, each representing an expression level for a gene in the set of genes, and determining the gene ranking may involve determining a relative rank for each gene in the set of genes based on the values.
In some embodiments, the subject has, is suspected of having, or is at risk of having breast cancer. The set of genes may be selected from the group of genes listed in Table 1. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes may include all the genes listed in Table 1. In some embodiments, the set of genes may include 3-100 genes, 5-100 genes, 20-100 genes, 50-100 genes, 80-100 genes listed in Table 1. In some embodiments, the set of genes may include 100 or fewer genes, 80 or fewer genes, 50 or fewer genes, 20 or fewer genes listed in Table 1.
In some embodiments, the subject has, is suspected of having, or is at risk of having clear cell kidney cancer. The set of genes may be selected from the group of genes listed in Table 2. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 2. In some embodiments, the set of genes may include all the genes listed in Table 2. In some embodiments, the set of genes may include 3-80 genes, 5-80 genes, 20-80 genes, 50-80 genes, 70-80 genes listed in Table 2. In some embodiments, the set of genes may include 80 or fewer genes, 50 or fewer genes, 20 or fewer genes listed in Table 2.
In some embodiments, the subject has, is suspected of having, or is at risk of having lymphoma. The set of genes may be selected from the group of genes listed in Table 3. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 3. In some embodiments, the set of genes may include all the genes listed in Table 3. In some embodiments, the set of genes may include 3-25 genes, 5-25 genes, 10-25 genes, 20-25 genes listed in Table 3. In some embodiments, the set of genes may include 25 or fewer genes, 20 or fewer genes, 15 or fewer genes, 10 or fewer genes listed in Table 3.
Next process 300 proceeds to act 320, where one or more characteristics of the biological sample is determined using the gene ranking and a statistical model, such as statistical model 112. In some embodiments, a characteristic determined by process 300 may include cancer grade for cells in the biological sample. In some embodiments, a characteristic determined by process 300 may include tissue of origin for cells in the biological sample. The statistical model may be trained using rankings of expression levels for one or more genes in the set of genes. In some embodiments, the gene ranking may be used as an input to the statistical model to obtain an output indicating the one or more characteristics. In some embodiments, the statistical model comprises a classifier selected from the group consisting of: a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
In some embodiments, process 300 may include ranking genes in a second set of genes based on their expression levels in the expression data to obtain a second gene ranking. The second gene ranking and a second statistical model may be used to determine one or more second characteristics of the biological sample. The second statistical model may be trained using second training data indicating rankings of expression levels for some or all of the genes in the second set of genes. The one or more second characteristics of the biological sample may be different than a characteristic determined by act 330. For example, in some embodiments, a characteristic determined by act 330 may include cancer grade for cells in the biological sample and the second characteristic may include tissue of origin for cells in the biological sample.
In some embodiments, process 300 may include outputting the one or more characteristics to a user (e.g., physician), such as by displaying the one or more characteristics to the user on a graphical user interface (GUI), including the one or more characteristics in a report, sending an email to the user, and in any other suitable way.
In some embodiments, process 300 may include administering a treatment to the subject based on the determined one or more characteristics of the biological sample. For example, in embodiments where tissue of origin is determined for cells in the biological sample, a physician may administer a treatment for the subject associated with treating cancers of the determined tissue of origin. As yet another example, in embodiments where cancer grade is determined for cells in the biological sample, a physician may administer a treatment for the subject associated with treating cancers having the determined cancer grade. Further examples where characteristics of a biological sample determined using the techniques described herein are used for administering a treatment are provided in the section titled “Methods of Treatment”.
In some embodiments, process 300 may include identifying a treatment for the subject based on the determined characteristic(s) of the biological sample. For example, in embodiments where tissue of origin is determined for cells in the biological sample, the determined tissue of origin may be used to identify a treatment for the subject associated with treating cancers of the determined tissue of origin. As yet another example, in embodiments where cancer grade is determined for cells in the biological sample, the determined cancer grade may be used to identify a treatment for the subject associated with treating cancers having the determined cancer grade.
In some embodiments, process 300 may include determining a prognosis for the subject based on the determined one or more characteristics of the biological sample. For example, in embodiments where tissue of origin is determined for cells in the biological sample, the determined tissue of origin may be used to determine a prognosis for the subject associated with treating cancers of the determined tissue of origin. As yet another example, in embodiments where cancer grade is determined for cells in the biological sample, the determined cancer grade may be used to determine a prognosis for the subject associated with treating cancers having the determined cancer grade. Further aspects relating to other applications where characteristics of a biological sample determined using the techniques described herein are provided in the section titled “Applications”.
FIG. 4 is a flow chart of an illustrative process 400 for determining tissue of origin for cells in a biological sample, in accordance with some embodiments of the technology described herein. Process 400 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, ranking process 108 and statistical model 112 may perform some or all of process 400 to determine a tissue of origin.
Process 400 begins at act 410, where expression data for cells in a biological sample of a subject having, suspected of having, or is at risk of having cancer is obtained. In some embodiments, the expression data was obtained using a gene expression microarray. In some embodiments, the expression data was obtained by performing next generation sequencing. Some embodiments involve performing a sequencing process of the biological sample (e.g., a gene expression microarray, next generation sequencing) prior to obtaining expression data 102. In some embodiments, obtaining gene expression data 102 may involve obtaining gene expression data 102 in silico, such as by accessing, using a computing device, expression data (e.g., expression data that has been previously obtained from a biological sample) in one or more data stores, receiving the expression data from one or more other device, or any other way. In some embodiments, obtaining gene expression data 102 may involve analyzing a biological sample (in vitro) and accessing (e.g., by a computing device, processor) the expression data. Further aspects relating to obtaining expression data are provided in the section titled “Obtaining Expression Data”.
Next, process 400 proceeds to act 420, where genes in one or more sets of genes are ranked based on their expression levels in the expression data to obtain one or more gene rankings, such as by using ranking process 108. The expression data may include values, each representing an expression level for a gene in the one or more sets of genes, and determining a gene ranking may involve determining a relative rank for each gene in a set of genes based on the values.
In some embodiments, the subject has, is suspected of having, or is at risk of having breast cancer. The set of genes may be selected from the group of genes listed in Table 1. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 1. The set of genes may consist of 5-100 genes, 10-200 genes, 20-100 genes, or 50-100 genes. In some embodiments, the set of genes may include all the genes listed in Table 1. In some embodiments, the set of genes may include 3-100 genes, 5-100 genes, 20-100 genes, 50-100 genes, 80-100 genes listed in Table 1. In some embodiments, the set of genes may include 100 or fewer genes, 80 or fewer genes, 50 or fewer genes, 20 or fewer genes listed in Table 1.
Next, process 400 proceeds to act 430, where tissue of origin for some or all of the cells in the biological sample is determined using the one or more gene rankings and one or more statistical models, such as statistical model 112. A statistical model may be trained using rankings of expression levels for some or all genes in a set of genes. Each of the gene rankings may be obtained based on respective expression levels for the one or more genes in the set of genes. In some embodiments, one or more gene rankings may be used as an input to the one or more statistical models to obtain an output indicating the tissue of origin. The tissue of origin may include lung tissue, pancreas tissue, stomach tissue, colon tissue, liver tissue, bladder tissue, kidney tissue, thyroid tissue, lymph node tissue, adrenal gland tissue, skin tissue, breast tissue, ovary tissue, prostate tissue, urothelial tissue, cervical tissue, esophagus tissue, brain tissue, soft tissue, connective tissue, head tissue, and neck tissue.
In some embodiments, the one or more statistical models comprises one or more classifiers selected from the group consisting of: a statistical model may include are a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
In some embodiments, process 400 may further include determining, using the gene ranking and the one or more statistical models, histological information (e.g., tissue type) for at least some of the cells in the biological sample. The histological information may include adenocarcinoma, squamous cell carcinoma, carcinoma, cystadenocarcinoma, sarcoma, and glioma. A combination of the tissue of origin and the histological information may be selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast carcinoma, colorectal adenocarcinoma, ovarian serous cystadenocarcinoma, phenochromocytoma, bladder urothelial carcinoma, cervical squamous cell carcinoma, glioblastoma multiforme, head squamous cell carcinoma, neck squamous cell carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, paraganglioma, prostate adenocarcinoma, sarcoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrial carcinoma.
FIG. 5 is a flow chart of an illustrative process 500 for determining a cancer grade for cells in a biological sample, in accordance with some embodiments of the technology described herein. Process 500 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, ranking process 108 and statistical model 112 may perform some or all of process 500 to determine a cancer grade.
Process 500 begins at act 510, where expression data for cells in a biological sample of a subject having, suspected of having, or is at risk of having cancer is obtained. In some embodiments, the expression data was obtained using a gene expression microarray. In some embodiments, the expression data was obtained by performing next generation sequencing. Some embodiments involve performing a sequencing process of the biological sample (e.g., a gene expression microarray, next generation sequencing) prior to obtaining expression data 102. In some embodiments, obtaining gene expression data 102 may involve obtaining gene expression data 102 in silico, such as by accessing, using a computing device, expression data (e.g., expression data that has been previously obtained from a biological sample) in one or more data stores, receiving the expression data from one or more other device, or any other way. In some embodiments, obtaining gene expression data 102 may involve analyzing a biological sample (in vitro) and accessing (e.g., by a computing device, processor) the expression data. Further aspects relating to obtaining expression data are provided in the section titled “Obtaining Expression Data”.
Next, process 500 proceeds to act 520, where genes in a set of genes are ranked based on their expression levels in the expression data to obtain a gene ranking, such as by using ranking process 108. The expression data may include values, each representing an expression level for a gene in the set of genes, and determining the gene ranking may involve determining a relative rank for each gene in the set of genes based on the values. The set of genes may consist of 5-500 genes, 5-200 genes, 50-500 genes, or 50-300 genes.
In some embodiments, the subject has, is suspected of having, or is at risk of having breast cancer. The set of genes may be selected from the group of genes listed in Table 1. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 1. In some embodiments, the set of genes may include all the genes listed in Table 1. In some embodiments, the set of genes may include 3-100 genes, 5-100 genes, 20-100 genes, 50-100 genes, 80-100 genes listed in Table 1. In some embodiments, the set of genes may include 100 or fewer genes, 80 or fewer genes, 50 or fewer genes, 20 or fewer genes listed in Table 1.
In some embodiments, the subject has, is suspected of having, or is at risk of having clear cell kidney cancer. The set of genes may be selected from the group of genes listed in Table 2. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 2. In some embodiments, the set of genes may include 3-80 genes, 5-80 genes, 20-80 genes, 50-80 genes, 70-80 genes listed in Table 2. In some embodiments, the set of genes may include 80 or fewer genes, 50 or fewer genes, 20 or fewer genes listed in Table 2.
Next, process 500 proceeds to act 530, where cancer grade for the cells in the biological sample is determined using the gene ranking and a statistical model, such as statistical model 112. The statistical model may be trained using gene rankings of expression levels for one or more genes in the set of genes. Each of the gene rankings may be obtained based on respective expression levels for the one or more genes in the set of genes. In some embodiments, the gene ranking may be used as an input to the statistical model to obtain an output indicating the cancer grade. The cancer grade may include Grade 1, Grade 2, Grade 3, Grade 4, and Grade 5. In some embodiments, the statistical model comprises a classifier selected from the group consisting of: a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
An example of how the techniques described herein may be implemented in predicting breast cancer grade are discussed in connection with FIGS. 6A, 6B, 6C, 6D, and 7 . FIG. 6A shows different data sets (data sets that vary in sample preparation, sequencing platform, data processing used to obtain expression data), associated clinical cancer grade for samples of the data sets, and predicted cancer grade obtained using the machine learning techniques described herein, for determining breast cancer grade. In particular, FIG. 6A shows different data sets (top panel) where each vertical line corresponds to a different sample, where the shading of the line corresponds to different data sets. FIG. 6A also shows the clinical grade associated with samples of the data sets, where the lighter shade indicates Grade 1 (“G1”) and the darker shade indicates Grade 3 (“G3”). The clinical grade may be a determination by a physician (e.g., pathologist) using microscopy to visually inspect the samples. FIG. 6B shows the enrichment signatures for different pathways, illustrating gene expression profiles associated with breast cancer Grade 1 and Grade 3. Genes in one or more of these pathways may be used for determining breast cancer grade according to the techniques described herein. As an example, the HALLMARK_G2M_CHECKPOINT signature is shown in the top panel and has a majority of upregulated genes for the right portion of samples and a majority of downregulated genes for the left portion of samples. Other examples of pathways associated with cancer grade classification for breast cancer are in Table 4, below. In particular the different pathways that are enriched in a set of genes that are upregulated for Grade 3 (“G3”) and pathways that are enriched in a set of genes that are upregulated for Grade 1 (“G1”) are listed in Table 4.

TABLE 4

Grade classifier for breast cancer gene set enrichment (according to MSigDB 6.1)
Pathway enrichment

			Genes in/all
Pathway	P-value	FDR	genes	Description

Molecular G3 upregulated

HALLMARK_G2M_CHECKPOINT	1.10E−60	1.40E−57	33/200	Genes defining early response
				to estrogen.
HALLMARK_E2F_TARGETS	4.40E−47	6.10E−44	27/200	Genes encoding cell cycle
				related targets of E2F
				transcription factors.
REACTOME_CELL_CYCLE_MITOTIC	3.80E−35	5.30E−32	24/325	Cell Cycle, Mitotic
REACTOME_CELL_CYCLE	4.80E−34	6.60E−31	25/421	Cell Cycle
HALLMARK_MITOTIC_SPINDLE	1.80E−28	2.40E−25	18/200	Genes important for mitotic
				spindle assembly.
PID_PLK1_PATHWAY	6.20E−24	8.60E−21	11/46	PLK1 signaling events
KEGG_CELL_CYCLE	4.30E−20	5.90E−17	12/128	Cell cycle
PID_AURORA_B_PATHWAY	5.10E−20	7.00E−17	9/39	Aurora B signaling
PID_FOXM1_PATHWAY	6.70E−20	9.30E−17	9/40	FOXM1 transcription factor
				network
REACTOME_DNA_REPLICATION	1.70E−19	2.30E−16	13/192	DNA Replication
REACTOME_MITOTIC_M_M_G1_PHASES	2.20E−18	3.10E−15	12/172	Genes involved in Mitotic M-
				M/G1 phases
REACTOME_MITOTIC_PROMETAPHASE	3.00E−16	4.10E−13	9/87	Mitotic Prometaphase
REACTOME_CELL_CYCLE_CHECKPOINTS	1.20E−14	1.60E−11	9/124	Cell Cycle Checkpoints
HALLMARK_SPERMATOGENESIS	2.80E−14	3.80E−11	9/135	Genes up-regulated during
				production of male gametes
				(sperm), as in spermatogenesis.
REACTOME_MITOTIC_G1_G1_S_PHASES	3.20E−14	4.40E−11	9/137	Mitotic G1-G1/S phases

Molecular G1 upregulated

NABA_MATRISOME	9.40E−09	1.30E−05	11/1028	Ensemble of genes encoding
				extracellular matrix and
				extracellular matrix-associated
				proteins
HALLMARK_ESTROGEN_RESPONSE_EARLY	9.50E−09	1.30E−05	6/200	Genes defining early response
				to estrogen.
HALLMARK_TNFA_SIGNALING_VIA_NFKB	9.50E−09	1.30E−05	6/200	Genes regulated by NF-kB in
				response to TNF (Gene ID:
				7124)
NABA_CORE_MATRISOME	8.40E−08	0.00012	6/275	Ensemble of genes encoding
				core extracellular matrix
				including ECM glycoproteins,
				collagens and proteoglycans
NABA_BASEMENT_MEMBRANES	2.50E−07	0.00034	3/40	Genes encoding structural
				components of basement
				membranes
HALLMARK_ESTROGEN_RESPONSE_LATE	2.90E−07	0.0004	5/200	Genes defining late response to
				estrogen.
KEGG_FOCAL_ADHESION	3.00E−07	0.00041	5/201	Focal adhesion
PID_INTEGRIN4_PATHWAY	3.60E−07	0.0005	2/11	Alpha6 beta4 integrin-ligand
				interactions
BIOCARTA_ACE2_PATHWAY	6.30E−07	0.00087	2/13	Angiotensin-converting enzyme
				2 regulates heart function
PID_INTEGRIN1_PATHWAY	1.90E−06	0.0026	3/66	Beta1 integrin cell surface
				interactions

FIGS. 12, 13, 14, 15, 16, and 17 illustrate relationships between biological features and different breast cancer grades. In particular, these figures describe the biology of molecular grades (Grade 1 and Grade 3) for breast cancer, where the data depicted is for TCGA BRCA, and the predicted breast cancer grades were obtained using the techniques described herein. FIG. 12 is a distribution of molecular cancer grade among PAM50 subtypes. FIG. 12 illustrates the majority of molecular Grade 1 samples belong to luminal subtypes. Further comparisons on breast cancer datasets for FIGS. 13-17 are for only luminal subtypes. FIG. 13 shows how progeny process scores correspond to given and predicted cancer grades in TCGA BRCA. The progeny process scores are calculated from expression data. FIG. 14 shows plots comparing different protein expression for different predicted cancer grades. The protein expression is according to RPPA data. FIG. 15 is a plot of cytolitic score (CYT) for different predicted cancer grades. FIG. 16 are plots showing the difference in mutations between different cancer grades. FIG. 16 illustrates genes, according to WES data, that are significantly differentially mutated between predicted cancer grades. FIG. 17 shows segments that are differentially amplified or deleted between predicted cancer grades. The segments shown in FIG. 17 are according to WES data.
To compare with the computational techniques described herein, FIG. 6A shows the predicted grade (lower panel) using the expression data and a statistical model according to the techniques described herein. The predicted grade shows how the different samples are predicted as being Grade 1 (“G1”) for the left portion of samples and as being Grade 3 (“G3”) for the right portion of samples. This is further shown in the plot of “G3 probability” over the different samples below the bottom panel of FIG. 6A, where the probability of the Grade 3 is higher for the right portion of samples than the left portion of samples. FIGS. 6C and 6D show similar data as that shown in FIGS. 6A and 6B, respectively, except that the samples and pathway signatures are associated with predicting breast cancer as being Grade 1 or Grade 3 for Grade 2 samples. Here, FIGS. 6C and 6D show how the biological features associated with Grade 2 is similar to the biological features associated with Grade 1 and Grade 3.
FIG. 7 is a plot of true positive rate versus false positive rate for a number of biological samples (shown in the solid line). The plot shows that the predicted cancer grade using the techniques described herein have a high true positive rate while maintaining a low false positive rate.
As another example, pathways associated with cancer grade classification for kidney clear cell are in Table 5, below. In particular the different pathways that are enriched in a set of genes that are upregulated for Grade 4 (“G4”) and for Grade 1 (“G1”) are listed in Table 5.

TABLE 5

Grade classifier for kidney clear cell cancer gene set enrichment (according to MsigDB 6-1)
Pathway enrichment

			Genes in/all
Pathway	P-value	FDR	genes	Description

Molecular G4 upregulated

HALLMARK_KRAS_SIGNALING_UP	1.70E−14	2.40E−11	9/200	Genes up-regulated by KRAS
				activation.
HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION	1.00E−12	1.40E−09	8/200	Genes defining epithelial-
				mesenchymal transition, as in
				wound healing, fibrosis and
				metastasis.
NABA_MATRISOME	3.20E−12	4.40E−09	13/1028	Ensemble of genes encoding
				extracellular matrix and
				extracellular matrix-associated
				proteins
NABA_ECM_REGULATORS	4.80E−12	6.60E−09	8/238	Genes encoding enzymes and
				their regulators involved in the
				remodeling of the extracellular
				matrix
KEGG_COMPLEMENT_AND_COAGULATION_CASCADES	1.40E−10	1.90E−07	5/69	Complement and coagulation
				cascades
HALLMARK_COAGULATION	1.70E−10	2.30E−07	6/138	Genes encoding components
				of blood coagulation system;
				also up-regulated in platelets.
REACTOME_IMMUNE_SYSTEM	4.10E−09	5.70E−06	10/933	Genes involved in Immune
				System
NABA_MATRISOME_ASSOCIATED	7.50E−09	1.00E−05	9/753	Ensemble of genes encoding
				ECM-associated proteins
				including ECM-affilaited
				proteins, ECM regulators and
				secreted factors
PID_AVB3_OPN_PATHWAY	3.80E−08	5.30E−05	3/31	Osteopontin-mediated events
PID_FRA_PATHWAY	8.00E−08	0.00011	3/37	Validated transcriptional
				targets of AP1 family
				members Fra1 and Fra2
HALLMARK_COMPLEMENT	8.50E−08	0.00012	5/200	Genes encoding components
				of the complement system,
				which is part of the innate
				immune system.
PID_AMB2_NEUTROPHILS_PATHWAY	1.20E−07	0.00017	3/41	amb2 Integrin signaling
PID_UPA_UPAR_PATHWAY	1.30E−07	0.00019	3/42	Urokinase-type plasminogen
				activator (uPA) and uPAR-
				mediated signaling
PID_FGF_PATHWAY	4.10E−07	0.00056	3/55	FGF signaling pathway
BIOCARTA_CLASSIC_PATHWAY	4.40E−07	0.0006	2/14	Classical Complement
				Pathway
REACTOME_INNATE_IMMUNE_SYSTEM	6.00E−07	0.00083	5/279	Innate Immune System
PID_INTEGRIN5_PATHWAY	8.20E−07	0.0011	2/17	Beta5 beta6 beta7 and beta8
				integrin cell surface
				interactions
BIOCARTA_COMP_PATHWAY	1.20E−06	0.0016	2/19	Complement Pathway
NABA_ECM_GLYCOPROTEINS	2.40E−06	0.0034	4/196	Genes encoding structural
				ECM glycoproteins
HALLMARK_INTERFERON_GAMMA_RESPONSE	2.70E−06	0.0037	4/200	Genes up-regulated in
				response to
				IFNG [GeneID = 3458].
HALLMARK_G2M_CHECKPOINT	2.70E−06	0.0037	4/200	Genes involved in the G2/M
				checkpoint, as in progression
				through the cell division cycle.
HALLMARK_GLYCOLYSIS	2.70E−06	0.0037	4/200	Genes encoding proteins
				involved in glycolysis and
				gluconeogenesis.
HALLMARK_HYPOXIA	2.70E−06	0.0037	4/200	Genes up-regulated in
				response to low oxygen levels
				(hypoxia).
HALLMARK_TNFA_SIGNALING_VIA_NFKB	2.70E−06	0.0037	4/200	Genes regulated by NF-kB in
				response to
				TNF [GeneID = 7124].
PID_TOLL_ENDOGENOUS_PATHWAY	2.70E−06	0.0038	2/25	Endogenous TLR signaling

Molecular G1 upregulated

REACTOME_TRANSMEMBRANE_TRANSPORT_OF_—	1.20E−08	1.70E−05	6/413
SMALL_MOLECULES
REACTOME_SLC_MEDIATED_TRANSMEMBRANE_—	1.60E−08	2.20E−05	5/241	SLC-mediated transmembrane
TRANSPORT				transport
REACTOME_TRANSPORT_OF_GLUCOSE_AND_OTHER_—	4.50E−07	0.00063	3/89
SUGARS_BILE_ SALTS_AND_ORGANIC_ACIDS_METAL_—
IONS_AND_AMINE_COMPOUNDS
KEGG_PROXIMAL_TUBULE_BICARBONATE_—	5.40E−07	0.00074	2/23	Proximal tubule bicarbonate
RECLAMATION				reclamation
REACTOME_GLUCONEOGENESIS	1.80E−06	0.0025	2/34	Gluconeogenesis

FIGS. 18, 19, 20, 21, and 22 illustrate relationships between biological features and different kidney clear cell grades. In particular, these figures describe the biology of molecular grades (Grade 1 and Grade 4) for kidney renal clear cell cancer, where the data depicted is for TCGA KIRC, and the predicted breast cancer grades were obtained using the techniques described herein. FIG. 18 shows how progeny process scores correspond to given and predicted cancer grades in TCGA KIRC. The progeny process scores are calculated from expression data. FIG. 19 is a plot illustrating chromosomal instability (CIN) for different cancer grades. FIG. 20 are plots comparing different protein expression, according to RPPA data, for different predicted cancer grades. FIGS. 21 and 22 illustrate genes, according to WES data, that are differentially amplified or deleted between predicted cancer grades.
Some embodiments involve using the techniques described herein for determining cancer grade for lung adenocarcinoma. Examples of genes that may be included in a gene set for determining cancer grade for lung adenocarcinoma are listed in Table 6, below. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 6. In some embodiments, the set of genes may include all the genes listed in Table 6. In some embodiments, the set of genes may include 3-25 genes, 5-25 genes, 10-25 genes, 20-25 genes listed in Table 6. In some embodiments, the set of genes may include 25 or fewer genes, 20 or fewer genes, 15 or fewer genes, 10 or fewer genes listed in Table 6.

TABLE 6

Cancer Grade Classifier for Lung Adenocarcinoma

	NCBI
Gene	Gene ID	NCBI Accession Number(s)

AADAC	13	NM_001086
ALDOB	229	NM_000035
ANXA10	11199	NM_007193
ASPM	259266	NM_001206846; NM_018136
BTNL8	79908	NM_001040462; NM_001159707; NM_001159708; NM_001159709;
		NM_001159710; NM_024850
CEACAM8	1088	NM_001816; XM_017026195; NM_001816
CENPA	1058	NM_001042426; NM_001809
CHGB
	1114	NM_001819
CHRNA9	55584	NM_017581
COL11A1	1301	NM_001190709; NM_001854; NM_080629; NM_080630
CRABP1	1381	NM_004378; NM_004378
F11	2160	NM_000128
GGTLC1	92086	NM_178311; NM_178312; XM_005260865; XM_017028126
HJURP	55355	NM_001282962; NM_001282963; NM_018410
IGF2BP3	10643	NM_006547
IHH	3549	NM_002181
KCNE2	9992	NM_172201
KIF14	9928	NM_001305792; NM_014875; XM_011510231; XM_011510232;
		XM_017003005
LRRC31	79782	NM_001277127; NM_001277128; NM_024727; XM_011513160
MYBL2	4605	NM_001278610; NM_002466
MYOZ1	58529	NM_021245
PCSK2	5126	NM_001201528; NM_001201529; NM_002594
PI15	51050	NM_001324403; NM_015886
SCTR	6344	NM_002980
SHH	6469	NM_000193
SLC22A3	6581	NM_021977
SLC7A5	8140	NM_003486
SPOCK1	6695	NM_004598
TM4SF4	7104	NM_004617
TRPM8	79054	NM_024080
YBX2	51087	NM_015982

The techniques described herein may be implemented in predicting cancer grade for lung adenocarcinoma and are discussed in connection with FIGS. 23A, 23B, and 23C. In particular, a cancer grade classifier for lung adenocarcinoma may distinguish between molecular grade 1 (mG1), a low grade, and molecular grade 3 (mG3), a high grade. Such a classifier may be developed by using samples from TCGA LUAD (from the National Cancer Institute) and CPTAC3 (from NCBI) lung adenocarcinoma expression data as training data. For the classifier discussed in connection with FIGS. 23A, 23B, and 23C 117 samples of TCGA LUAD were excluded from the training data set and included as validation data. An initial gene set was formed from differentially expressed genes between grade 1 and grade 3. A genomic grade index (DOI: 10.1093/jnci/djj052) based on the initial gene set was calculated and training data set samples were split into high and low cancer grade based on survival mode. Through selection of the gene set used for the classifier, the number of genes was reduced. For example, the classifier discussed in connection with FIGS. 23A, 23B, and 23C, the initial gene set included 321 genes and the gene set used in the classifier included 31 genes. Validation data sets included 117 samples from TCGA LUAD and Series GSE68465. After hyperparameter tuning, the classifier's performance on the validation data set reached a 0.89AUC score in distinguishing between grade 1 and grade 3. These results demonstrated the capability of lung molecular grades to be statistically significant in predicting survival.
FIG. 23A shows validation data sets, associated cancer grade reported for samples of the data sets, predicted cancer grade obtained using the machine learning techniques described herein, for determining lung adenocarcinoma cancer grade, and the enrichment signatures for different pathways, illustrating gene expression profiles associated with grade 1 and grade 3. The validation data sets shown in FIG. 23A vary in vary in sample preparation, sequencing platform, and data processing used to obtain expression data. FIG. 23A shows data sets (top panels) where each vertical line corresponds to a different sample, where the shading of the line corresponds to different data sets. The cancer grade associated with samples of the data sets is shown, where the lighter shade indicates grade 1 and the darker shade indicates grade 3. The cancer grade associated with the samples may be a determination by a physician (e.g., pathologist) using microscopy to visually inspect the samples. The probability of molecular grade 3 predicted using the cancer grade classifier is also shown. FIG. 23A also shows the enrichment signatures for different pathways, illustrating gene expression profiles associated with lung adenocarcinoma grade 1 and grade 3. Genes in one or more of these pathways may be used for determining lung adenocarcinoma grade according to the techniques described herein. As an example, the HALLMARK_G2M_CHECKPOINT signature is shown in the top panel and has a majority of upregulated genes for the right portion of samples and a majority of downregulated genes for the left portion of samples. FIG. 23B shows results of applying validation data sets to lung adenocarcinoma cancer grade classifier. FIG. 23C is a plot of true positive rate versus false positive rate for predicting cancer grade of different biological samples where the classifier had a 0.894 AUC score.
FIG. 8A is a flow chart of an illustrative process 800 for selecting a gene set, in accordance with some embodiments of the technology described herein. Process 800 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, ranking process 108 and statistical model 112 may perform some or all of process 800 to select a gene set, which may be implemented in determining one or more characteristics of a biological sample, such as a tissue of origin, a cancer grade, and a PTCL subtype.
Process 800 begins at act 810, where expression data is ranked to obtain a gene ranking for the genes represented by the expression levels in the expression data. Ranking process 108 may be used in ranking the expression data to obtain the gene ranking.
The expression data used in selecting a gene set may include available expression data obtained through research organizations, including the National Cancer Institute (NCI) (e.g., Gene Expression Omnibus (GEO)), National Center for Biotechnology Information (NCBI), and The Cancer Imaging Archive (TCIA). For example, a gene set used for predicting breast cancer grade may be obtained by using expression data from Series GSE2990 available through the NCI. As another example, a gene set used for determining cancer grade for kidney clear cell cancer may be obtained by using expression data from Series GSE40435. As another example, a gene set used for determining tissue of origin and histological information (e.g., tissue type) for cancer may be obtained by using expression data from The Cancer Genome Atlas Program (TCGAP). As another example, a gene set used for predicting PTCL subtype may be obtained using expression data listed in Table 9.
Next, process 800 proceeds to act 820, where the ranked expression data is input into a statistical model, such as statistical model 112. An output indicating one or more desired characteristics may be obtained as a result of inputting the ranked expression data into the statistical model. Process 800 may proceed to act 830, where a validation quality score is calculated based on the output obtained by inputting the ranked expression data into the statistical model of act 820. A validation quality score may be calculated using one or more suitable metrics, including negative log loss, AUC, F-score (micro, macro, weighted), accuracy, balanced accuracy, precision, and recall.
Next, process 800 proceeds to act 840, where importance value(s) for different genes included in the ranking are calculated. An example of an importance value is a Shapley Additive Explanations (SHAP) value, which is described in “A Unified Approach to Interpreting Model Predictions” by Scott M. Lundberg and Su-In Lee, which is incorporated by reference in its entirety. Example SHAP values are shown in Table 7 in connection with a cell of origin classifier for DLBCL.
Next, process 800 proceeds to act 850, where the N (e.g., 1, 2, 3, 4) least important genes are excluded based on the importance values. Next process 800 proceeds to act 860, where a gene set updated based on excluding the N least important genes. In some embodiments, at least the gene have the lowest importance value is removed from the gene set.
Process 800 may initialize with a larger number of genes (e.g., ˜3,000 genes) in the gene set and decrease the number of genes in the set through subsequent iterations. Process 800 may continue by repeating the acts with the gene set selected in act 860 of the prior iteration until a desired quality score is achieved (e.g., a quality score higher than a threshold value). In some instances, an initial gene set may be ranked at act 810 and narrowed by process 800 to achieve a limited gene set used for a classifier as described herein.
FIG. 8B is a flow chart of an illustrative process 800 for selecting a gene set, in accordance with some embodiments of the technology described herein. Process 900 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, ranking process 108 and statistical model 112 may perform some or all of process 900 to select a gene set, which may be implemented in determining one or more characteristics of a biological sample, such as a tissue of origin, a cancer grade, and a PTCL subtype.
Process 900 begins at act 910, where an initial gene set is selected. The initial gene set selected may include a set of genes selected from Table 1, Table 2, Table 3, Table 6, and Table 8. The number of initial genes may be at least 1,000 genes, at least 3,000 genes, or at least 5,000 genes.
Next, process 900 proceeds to act 810, as discussed above in connection with process 800. Next process 900 proceeds to act 920, where hyperparameters for the statistical model are selected and fit to the statistical model.
Next process proceeds to acts 840, 850, and 860 as discussed above in connection with process 800. As discussed in connection with process 800, the initial set of genes may decrease in number though subsequent iterations of these steps. As a result of these iterative steps, process 900 proceeds to act 925, where a minimum size of gene set is reached.
As part of these iterative steps, process 900 proceeds to act 930, where a cross validation score is calculated based on inputting the ranked expression data into the statistical model of act 820. A cross validation score may be calculated by performing k-fold cross validation.
Process 900 proceeds to act 940, where a gene set is selected based on the cross validation score calculated in act 930. In some embodiments, the gene set selected has the highest cross validation score from a group of gene sets.
Next process 900 proceeds to act 950, where expression data is ranked to obtain a gene ranking for the genes represented by the expression levels in the expression data. Ranking process 108 may be used in ranking the expression data to obtain the gene ranking.
Next process 900 proceeds to act 960, where hyperparameters for the statistical model are selected and fit to the statistical model for the gene set selected in act 940.
For example, FIG. 9A is a plot of quality score versus number of genes, which illustrates how decreasing the number of genes to 28 from 30 increases the quality score. FIG. 9B is an exemplary plot of F1 score versus number of genes used in ranking for ABC/GCB tissue of origin prediction, in accordance with some embodiments of the technology described herein.
Cell of Origin DLBCL Classifier
As discussed herein, some embodiments involve using the techniques described herein for determining cell of origin for DLBCL. In particular, a cell of origin DLBCL classifier may categorize samples as being either germinal center B-cell (GCB) and activated B-cell (ABC). Such a classifier may be developed by using samples from Series GSE117556, Leipzig Lymphoma data set (10.1186/s13073-019-0637-7), Series GSE31312, Series GSE10846, Series GSE87371, Series GSE11318, Series GSE32918, Series GSE23501, Lymphoma/Leukemia Molecular Profiling Project (LLMPP), and Series GSE93984 as training data. For each data set, samples were selected to have a balanced cell of origin ratio ABC:GCB ratio of 40:60 per data set. For example, this may involve selecting samples having cell of origin labeling followed by a round of random selection of samples to obtained a desired ABC:GCB ratio. An example cell of origin DLBCL classifier is discussed in connection with FIGS. 24A, 24B, 24C, 24D, and 24E. For this classifier, the training data set includes 1,968 samples.
Suitable data sets may be used to validate the trained cell of origin DLBCL classifier. Validation of a cell of origin DLBCL may involve using data from Series GSE34171 (GPL96+GPL97), Series GSE22898, Series GSE64555, Series GSE145043, Series GSE19246, and the National Cancer Institute Center for Cancer Research (NCICCR) “Genetics and Pathogenesis of Diffuse Large B Cell Lymphoma” data set. Validation of the classifier described in connection with FIGS. 24A, 24B, 24C, 24D, and 24E involved using a validation data set of 928 samples.
A classifier may be further validated using data sets of unknown and unclassified samples. A cell of origin DLBCL classifier may be validated using data from Series GSE69051, Series GSE69049, E-TABM-346, Series GSE68895, Series GSE38202, Series GSE2195, International Cancer Genome Consortium Malignant Lymphoma—DE (ICGC_MALY_DE) data set, and National Cancer Institute Cancer Genome Characterization Initiative (NCICGCI) Non-Hodgkin Lymphoma data set. For the cell of origin DLBCL classifier discussed in connection with FIGS. 24A, 24B, 24C, 24D, and 24E, 1,169 unknown and unclassified samples were used in validation of the classifier.
The cell of origin classifier discussed in connection with FIGS. 24A, 24B, 24C, 24D, and 24E may involve identifying a gene set, such as by process 800 shown in FIG. 8 . In particular, an initial gene set may be identified from genes discussed in Wright G, et al., A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma, PNAS, 2003; 100:9991-9996 (doi: 10.1073/pnas.1732008100), which is incorporated by reference herein in its entirety. The initial gene set was curated down to 30 genes to be used in the classifier. After hyperparameter tuning, the classifier's performance on a validation data set reached 0.93 f1-score and 0.978 AUC score.
In this example classifier, binary classification was performed using a gradient booster decision tree classifier in LightGBM. Feature selection was performed by estimating feature importance in the model using SHAP package. Example SHAP importance values calculated for possible genes to include in a cell of origin classifier for DLBCL are shown in Table 7, below.

TABLE 7

Genes for DLBCL cell of origin classifier
and SHAP importance values

	Cell of origin genes	SHAP importance

	ITPKB	1.198
	MYBL1	1.15
	LMO2	0.93
	IRF4	0.94
	LRMP	0.71
	CCND2	0.7
	BATF	0.66
	SP140	0.52
	SPINK2	0.54
	TCF4	0.41
	CSTB	0.41
	PIM1	0.32
	VCL	0.3
	GPR18	0.24
	FUT8	0.22
	BCL2	0.28
	SLA	0.24
	RPL21	0.2
	P2RX5	0.11
	REL	0.12
	HLA-DQA1	0.13
	CSNK1E	0.16
	PTPN1	0.05
	KRT8	0.15
	IGHM	0.13
	PRKCB1	0.11
	GOT2	0.11
	FAM3C	0.07
	SPIB	0.09
	ACP1	0.06
	PIM2	0.04
	PLEK	0.06

FIG. 24A shows validation data sets, associated cell of origin reported for samples of the data sets, predicted cell of origin obtained using the machine learning techniques described herein, for determining DLBCL subtype, and the enrichment signatures for ABC and GCB subtypes. FIG. 24B shows validation data sets, associated cell of origin reported for samples of the data sets, predicted cell of origin obtained using the machine learning techniques described herein, for determining DLBCL subtype, and the enrichment signatures for ABC and GCB subtypes. The validation data sets shown in FIGS. 24A and 24B vary in sample preparation, sequencing platform, and data processing used to obtain expression data. Both FIGS. 24A and 24B shows data sets (top panel) where each vertical line corresponds to a different sample, where the shading of the line corresponds to different data sets. The cell of origin associated with samples of the data sets is shown, where the lighter shade indicates GCB subtype and the darker shade indicates ABC subtype. The cell of origin associated with the samples may be a determination by a physician (e.g., pathologist) using microscopy to visually inspect the samples. The enrichment signatures for ABC signature and GCB signature are shown in FIGS. 24A and 24B. The ABC signature generally has a majority of upregulated genes on the right portion of samples and the GBC signature has a majority of upregulated genes for the left portion of samples. FIGS. 24C and 24D are plots of survival rates for different groups (ABC, GCB). FIG. 24E is a plot of true positive rate versus false positive rate for predicting DLBCL subtype of different biological samples where the classifier had a 0.978 AUC score.
Human Papillomavirus (HPV) Head and Neck Squamous Cell Carcinoma Classifier
Some embodiments involve using the techniques described herein for predicting HPV status (HPV-positive, HPV-negative). Such embodiments may involve determining a sample as having an HPV-positive status or HPV-negative status. In some embodiments, the HPV status may be determined for a subject having, suspected of having, or at risk of having head and neck squamous cell carcinoma. Examples of genes that may be included in a gene set for determining HPV status for head and neck squamous cell carcinoma are listed in Table 8, below. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 8. In some embodiments, the set of genes may include all the genes listed in Table 8. In some embodiments, the set of genes may include 3-130 genes, 5-130 genes, 20-130 genes, 50-130 genes, 80-130 genes listed in Table 8. In some embodiments, the set of genes may include 130 or fewer genes, 100 or fewer genes, 80 or fewer genes, 50 or fewer genes, 20 or fewer genes listed in Table 8.

TABLE 8

HPV Status Classifier for Head and Neck Squamous Cell Carcinoma

	NCBI
Gene	Gene ID	NCBI Accession Number(s)

APOBEC3B	9582	NM_001270411; NM_004900
ATAD2	29028	NM_014109; NM_014109
BIRC5	332	NM_001168; NM_001012271; NM_001012270; NM_001168
CCL20	6364	NM_001130046; NM_004591
CCND1	595	NM_053056; NM_053056
CDC45	8318	NM_001178010; NM_001178011; NM_003504; XM_011530417;
		XM_011530416; NR_161281; XM_017028966; ; XR_002958716;
		NM_001178010; XM_011530418; XM_017028967; XM_024452277;
		NM_001369291
CDC7	8317	NM_001134419; NM_001134420; NM_003503; XM_005271241
CDK1	983	NM_001320918; NM_001786; NM_033379; XM_005270303
CDKN2A	1029	NM_000077; NM_000077; NM_001195132; NM_058197;
		XM_005251343; XM_011517676; ; NM_058196; NM_058195;
		XM_011517675; NM_001363763; XR_929159
CDKN2C	1031	NM_001262; NM_078626; NM_001262
CDKN3	1018	NM_001258
CENPF	1063	NM_016343; XM_017000086
CENPN	55839	NM_001100624; NM_001100625; NM_001270473; NM_001270474;
		NM_018455; XM_006721236; XM_017023456
CXCL14	9547	NM_004887
DCN	1634	NM_133505; NM_001920; NM_133503; NM_133504; NM_133505;
		NM_133506; NM_133507;
DHFR	1719	NM_000791; NM_001290357; NM_000791; NM_001290354
DKK3	27122	NM_001330220; XM_017017554; XM_017017555; NM_001018057;
		NM_001330220; NM_013253; NM_015881; XM_006718178
DLGAP5	9787	NM_001146015; NM_014750; XM_017021840
EPCAM	4072	NM_002354
FANCI	55215	NM_001113378; NM_018193; XM_011521756; NM_001113378;
		NM_001376910; NM_001376911; ; XM_011521764
FEN1	2237	NM_004111; NM_004111
GMNN	51053	NM_001251989; NM_001251990; NM_001251991; NM_015895;
		XM_005249159; NM_001251989
GPX3	2878	NM_001329790; NM_002084
ID4	3400	NM_001546
IGLC1	3537	NG_000002.1
IL18	3606	NM_001243211; XM_011542805; NM_001243211; NM_001562; ;
		NM_001386420
IL1R2	7850	NM_001261419; NM_004633; XM_006712734; XM_011511804
KIF18B	146909	NM_001264573; NM_001265577; NM_001264573
KIF20A	10112	NM_005733
KIF4A	24137	NM_012310
KLK13	26085	NM_001348177; NM_001348178; NM_001348177; NM_015596
KLK7	5650	NM_005046; NM_139277; NM_001207053; NM_001243126;
		NM_005046
KLK8	11202	NM_001281431; NM_001281431; NM_007196; NM_144505;
		NM_144506; NM_144507
KNTC1	9735	NM_014708; XM_006719706
KRT19	3880	NM_002276
LAMP3	27074	NM_014398
LMNB1	4001	NM_005573
MCM2	4171	NM_004526
MCM4	4173	NM_005914; NM_182746
MCM5	4174	NM_006739; NM_006739
ME1	4199	NM_002395; XM_011535836
MELK	9833	NM_001256685; NM_001256687; NM_001256688; NM_001256689;
		NM_001256690; NM_001256692; NM_001256693; NM_014791;
		XM_011518076; XM_011518077; XM_011518078; XM_011518079;
		XM_011518081; XM_011518082; XM_011518083; XM_011518084
MKI67	4288	NM_001145966; NM_002417
MLF1	4291	NM_022443; NM_001130156; NM_001130157; NM_001195432;
		NM_001195433; NM_001195434; NM_022443; NM_001369782;
		NM_001378848; NM_001378853; ; NM_001369784;
		NM_001369785; NM_001369781; NM_001378846; NM_001378855;
		NM_001378845; NM_001378847; NM_001378850; NM_001378852;
		NM_001369783; NM_001378851
MMP12	4321	NM_002426
MTHFD2	10797	NM_006636; XM_006711924
NDN	4692	NM_002487; NM_002487
NEFH	4744	NM_021076
NEK2	4751	NM_001204182; NM_001204182; NM_001204183; NM_002497;
		XM_005273147
NUP155	9631	NM_153485; NM_001278312; NM_004298; NM_153485
NUP210	23225	NM_024923
NUSAP1	51203	NM_001243142; NM_001243143; NM_001243144; NM_001301136;
		NM_016359; NM_018454; XM_005254430
PDGFD	80310	NM_025208; NM_033135
PLAGL1	5325	NM_001080951; NM_001080952; NM_001080953; NM_001080954;
		NM_001289042; NM_001289043; NM_001289044; NM_001289047;
		NM_001289048; NM_001289049; NM_001317157; NM_001317159;
		NM_006718; NM_001080955; NM_001289037; NM_001289038;
		NM_001289039; NM_001289040; NM_001080951; NM_001080956;
		NM_001289041; NM_001289045; NM_001289046; NM_001317156;
		NM_001317158; NM_001317160; NM_001317161; NM_001317162
PLOD2	5352	NM_000935; NM_182943
PPP1R3C	5507	NM_005398
PRIM1	5557	NM_000946
PRKDC	5591	NM_001081640; NM_006904
PSIP1	11168	NM_001128217; NM_001317898; NM_001317900; NM_021144;
		NM_033222
RAD51AP1	10635	NM_001130862; NM_006479
RASIP1	54922	NM_017805; NM_017805
RFC5	5985	NM_001130112; NM_001130113; NM_007370; NM_181578;
		XM_011538643; XM_011538645
RNASEH2A	10535	NM_006397
RPA2	6118	NM_001286076; NM_001297558; NM_002946
RPL39L	116832	NM_052969
RSRC1	51319	NM_001271834; NM_001271838; NM_016625
RYR1	6261	NM_000540; NM_001042723
SLC35G2	80723	NM_001097599; NM_001097600; NM_025246; XM_006713773;
		XM_011513214; XM_017007289; XM_017007290; XM_017007291
SMC2	10592	NM_001265602; NM_001042550; NM_001042551; NM_001265602;
		NM_006444; XM_006716933; XM_011518148; XM_011518149;
		XM_011518153; XM_017014206; XM_017014207; XM_017014208
SPARCL1	8404	NM_001128310; NM_001291976; NM_001291977; NM_004684
STMN1	3925	NM_001145454; NM_005563; NM_203399; NM_203401
SYCP2	10388	NM_014258; XM_011528489
SYNGR3	9143	NM_004209
TIMELESS	8914	NM_001330295; NM_003920
TMPO	7112	NM_001032283; NM_001032284; NM_001307975; NM_003276;
		NM_001032283
TPX2	22974	NM_012112; XM_011528697; XM_011528699; NM_012112
TRIP13	9319	NM_004237; XM_011514163
TYMS	7298	NM_001071; NM_001071
UCP2	7351	NM_003355; NM_003355
UPF3B	65109	NM_023010; NM_080632
USP1	7398	NM_001017415; NM_001017416; NM_003368
ZSCAN18	65982	NM_001145542; NM_001145543; NM_001145544; NM_023926;
		XM_005259174; XM_006723335; XM_011527238; XM_011527239;
		XM_017027169; XM_017027170; XM_017027171
APOBEC3B	9582	NM_001270411; NM_004900
ATAD2	29028	NM_014109; NM_014109
BIRC5	332	NM_001168; NM_001012271; NM_001012270; NM_001168
CCL20	6364	NM_001130046; NM_004591
CCND1	595	NM_053056; NM_053056
CDC45	8318	NM_001178010; NM_001178011; NM_003504; XM_011530417;
		XM_011530416; NR_161281; XM_017028966; ; XR_002958716;
		NM_001178010; XM_011530418; XM_017028967; XM_024452277;
		NM_001369291
CDC7	8317	NM_001134419; NM_001134420; NM_003503; XM_005271241
CDK1	983	NM_001320918; NM_001786; NM_033379; XM_005270303
CDKN2A	1029	NM_000077; NM_000077; NM_ 001195132; NM_058197;
		XM_005251343; XM_011517676; ; NM_058196; NM_058195;
		XM_011517675; NM_001363763; XR_929159
CDKN2C	1031	NM_001262; NM_078626; NM_001262
CDKN3	1018	NM_001258
CENPF	1063	NM_016343; XM_017000086
CENPN	55839	NM_001100624; NM_001100625; NM_001270473; NM_001270474;
		NM_018455; XM_006721236; XM_017023456
CXCL14	9547	NM_004887
DCN	1634	NM_133505; NM_001920; NM_133503; NM_133504; NM_133505;
		NM_133506; NM_133507;
DHFR	1719	NM_000791; NM_001290357; NM_000791; NM_001290354
DKK3	27122	NM_001330220; XM_017017554; XM_017017555; NM_001018057;
		NM_001330220; NM_013253; NM_015881; XM_006718178
DLGAP5	9787	NM_001146015; NM_014750; XM_017021840
EPCAM	4072	NM_002354
FANCI	55215	NM_001113378; NM_018193; XM_011521756; NM_001113378;
		NM_001376910; NM_001376911; XM_011521764
FEN1	2237	NM_004111; NM_004111
GMNN	51053	NM_001251989; NM_001251990; NM_001251991; NM_015895;
		XM_005249159; NM_001251989
GPX3	2878	NM_001329790; NM_002084
ID4	3400	NM_001546
IGLC1	3537	NG_000002.1
IL18	3606	NM_001243211;XM_011542805; NM_001243211; NM_001562;
		NM_001386420
IL1R2	7850	NM_001261419; NM_004633; XM_006712734; XM_011511804
KIF18B	146909	NM_001264573; NM_001265577; NM_001264573
KIF20A	10112	NM_005733
KIF4A	24137	NM_012310
KLK13	26085	NM_001348177; NM_001348178; NM_001348177; NM_015596
KLK7	5650	NM_005046; NM_139277; NM_001207053; NM_001243126;
		NM_005046
KLK8	11202	NM_001281431; NM_001281431; NM_007196; NM_144505;
		NM_144506; NM_144507
KNTC1	9735	NM_014708; XM_006719706
KRT19	3880	NM_002276
LAMP3	27074	NM_014398
LMNB1	4001	NM_005573
MCM2	4171	NM_004526
MCM4	4173	NM_005914; NM_182746
MCM5	4174	NM_006739; NM_006739
ME1	4199	NM_002395; XM_011535836
MELK	9833	NM_001256685; NM_001256687; NM_001256688; NM_001256689;
		NM_001256690; NM_001256692; NM_001256693; NM_014791;
		XM_011518076; XM_011518077; XM_011518078; XM_011518079;
		XM_011518081; XM_011518082; XM_011518083; XM_011518084
MKI67	4288	NM_001145966; NM_002417
MLF1	4291	NM_022443; NM_001130156; NM_001130157; NM_001195432;
		NM_001195433; NM_001195434; NM_022443; NM_001369782;
		NM_001378848; NM_001378853; ; NM_001369784;
		NM_001369785; NM_001369781; NM_001378846; NM_001378855;
		NM_001378845; NM_001378847; NM_001378850; NM_001378852;
		NM_001369783; NM_001378851
MMP12	4321	NM_002426
MTHFD2	10797	NM_006636; XM_006711924
NDN	4692	NM_002487; NM_002487
NEFH	4744	NM_021076
NEK2	4751	NM_001204182; NM_001204182; NM_001204183; NM_002497;
		XM_005273147
NUP155	9631	NM_153485; NM_001278312; NM_004298; NM_153485
NUP210	23225	NM_024923
NUSAP1	51203	NM_001243142; NM_001243143; NM_001243144; NM_001301136;
		NM_016359; NM_018454; XM_005254430
PDGFD	80310	NM_025208; NM_033135
PLAGL1	5325	NM_001080951; NM_001080952; NM_001080953; NM_001080954;
		NM_001289042; NM_001289043; NM_001289044; NM_001289047;
		NM_001289048; NM_001289049; NM_001317157; NM_001317159;
		NM_006718; NM_001080955; NM_001289037; NM_001289038;
		NM_001289039; NM_001289040; NM_001080951; NM_001080956;
		NM_001289041; NM_001289045; NM_001289046; NM_001317156;
		NM_001317158; NM_001317160; NM_001317161; NM_001317162
PLOD2	5352	NM_000935; NM_182943

Such a classifier may be developed by using samples from Series GSE65858, Series GSE41613, E-TABM-302 (from EMBL-EBI), Series GSE25727, Series GSEE3292, Series GSE6791, Series GSE10300, TCGA HNSC (from The Cancer Imaging Archive (TCIA)) data sets as training data. For classifier discussed in connection with FIGS. 25A, 25B, 25C, 25D, 25E, and 25F, 60 samples of the TCGA HNSC data set were excluded from the training data and used in validation data sets. Validation data sets included the 60 samples from the TCGA HNSC data set and Series GSE40774. Series GSE74927 was used as an additional validation data set where different strains of HPV virus are represented, allowing for assessment of the classifier's performance across different HPV strains. A gene set for the classifier was identified from genes discussed in Chakravarthy et al., Human Papillomavirus Drives Tumor Development Throughout the Head and Neck: Improved Prognosis Is Associated With an Immune Response Largely Restricted to the Oropharynx, Journal of Clinical Oncology, 34, no. 34 (Dec. 1, 2016) 4132-4141 (DOI: 10.1200/JCO.2016.68.2955), which is incorporated by reference herein in its entirety. The initial gene set was curated down to 82 genes, such as by using process 800 shown in FIG. 8 . After hyperparameter tuning, the classifier's performance on the validation data set with the TCGA HNSC data set and Series GSE40774 reached a 0.975 AUC score and 0.9 f1 score. The classifier's performance on the validation data set with Series GSE74927 reached a 1.0 AUC score and 1.0 f1 score. It is noted that that classifier successfully recognized several HPV strains, including HPV16 strain, HPV18 strain, HPV33 strain, and HPV55.
FIG. 25A shows validation data sets, associated HPV status reported for samples of the data sets, predicted HPV status obtained using the machine learning techniques described herein, for determining HPV status, and the enrichment signatures for different pathways, illustrating gene expression profiles associated with HPV status. Both FIG. 25A shows data sets (top panel) where each vertical line corresponds to a different sample, where the shading of the line corresponds to different data sets. The HPV status associated with samples of the data sets is shown, where the lighter shade indicates negative HPV status and the darker shade indicates positive HPV status. The probability of the sample having a positive HPV status is shown in the middle panel of FIG. 25A. The enrichment signatures for different pathways, illustrating gene expression profiles associated with HPV status are shown in FIG. 25A (bottom panel). As an example, the HALLMARK_E2F_TARGETS signature is shown in FIG. 25A and has a majority of upregulated genes for the right portion of samples and a majority of downregulated genes for the left portion of samples. FIGS. 25B and 25C are plots of survival rates for different groups of HPV status (positive HPV and negative HPV). FIG. 25D is a plot of true positive rate versus false positive rate for predicting HPV status of different biological samples (from the TCGA HNSC data set and Series GSE40774 validation data) where the classifier had a 0.975 AUC score. FIG. 25E is a plot of true positive rate versus false positive rate for predicting HPV status of different biological samples (from the Series GSE74927 validation data) where the classifier had a 1.0 AUC score. FIG. 25F is a plot of illustrating the performance of the classifier for different HPV strains in the Series GSE74927 validation data.
Peripheral T-Cell Lymphoma (PTCL) Classifier
Aspects of the present application relate to techniques, developed by the inventors, for analyzing gene expression data to determine a subtype of peripheral T-cell lymphoma (PTCL) for a biological sample. These techniques involve ranking a set of genes based on gene expression levels and using the ranking and one or more statistical models to determine the PTCL subtype. The set of genes may be associated with biological features (e.g., cell morphology, cell migration, cell cycle), expression pathways, or otherwise associated with one or more subtypes of peripheral T-cell lymphoma (PTCL).
Peripheral T-cell lymphomas accounts for approximately 10% of all non-Hodgkin lymphomas. Peripheral T-cell lymphomas are a heterogeneous group of diseases, which includes more than 20 subtypes, the exact definition of which is limited to modern methods of laboratory diagnosis. Examples of PTCL subtypes include but are not limited to Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS), anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), Natural killer/T-cell lymphoma (NKTCL), Sezary syndrome, adult T-cell leukemia/lymphoma (ATLL), enteropathy-type T-cell lymphoma, nasal NK/T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell lymphomas of Follicular T-cell (TFH) origin, T-cell lymphomas of the gastrointestinal tract (e.g., EATL, MEITL), cutaneous T-cell lymphomas, etc.
The most frequent subgroups among PTCL are adult T-cell leukemia/lymphoma (ATLL), angioimmunoblastic T-cell lymphoma (AITL), NK/T-cell lymphoma (NKTCL), anaplastic large cell lymphoma (ALCL), and cases belong to the Not Otherwise Specified (PTCL-NOS), which correspond to approximately to 35% of the total PTCL patients. Other PTCL subtypes are rare and mostly represented by extranodal tumors. It is anticipated that more effective annotation of the PTCL will eventually lead to the design and implementation of personalized treatments. As discussed herein, the inventors have recognized certain benefits from using the ranking of a set of genes in contrast to particular values for gene expression levels. In some embodiments, the technology described herein involves determining a subtype of peripheral T-cell lymphoma (PTCL) for a biological sample.
For example, in some embodiments, rankings of genes based on the gene expression levels (in a biological sample) as determined by a sequencing platform may be provided as input to a statistical model trained to predict PTCL subtype for the biological sample. The statistical model may include a multi-class classifier and have multiple outputs corresponding to different PTCL subtypes. As another example, in some embodiments, rankings of genes based on the gene expression levels (in a biological sample) as determined by a sequencing platform may be provided as input to multiple statistical models trained to predict different PTCL subtypes. For example, one statistical model may be trained to predict anaplastic large cell lymphoma (ALCL) for the biological sample and another statistical model may be trained to predict angioimmunoblastic T-cell lymphoma (AITL) for the biological sample. In such embodiments, the statistical models may be binary classifiers, each being trained for a different PTCL subtype, or regression type classifiers estimating a likelihood of a particular PTCL subtype.
Different PTCL subtype(s) may have different molecular signatures. In some embodiments, the set of genes being ranked depends on the particular PTCL subtype(s) of interest. In some embodiments, one set of genes may be used for determining a group of PTCL subtype(s) and another set of genes may be used for determining a different group of PTCL subtype(s). For example, one set of genes may be used for determining a group of PTCL subtype(s) that include anaplastic large cell lymphoma (ALCL), and another set of genes may angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL). Another set of genes may be used for determining a group of PTCL subtype(s) that include enteropathy-type T-cell lymphoma, nasal NK/T-cell lymphoma, and hepatosplenic gamma-delta T-cell lymphoma. As another example, one set of genes may be used for determining anaplastic large cell lymphoma (ALCL), and another set of genes may be used for determining natural killer/T-cell lymphoma (NKTCL).
Some embodiments described herein address all of the above-described issues that the inventors have recognized with determining PTCL subtype of a biological sample using gene expression data. However, not every embodiment described herein addresses every one of these issues, and some embodiments may not address any of them. As such, it should be appreciated that embodiments of the technology described herein are not limited to addressing all or any of the above-discussed issues with determining PTCL subtype of a biological sample using gene expression data.
Some embodiments involve obtaining gene expression data for a biological sample of a subject, ranking genes in set(s) of genes based on their expression levels in the expression data to obtain one or more gene rankings. The one or more gene rankings may be used, along with one or more statistical models, to determine a subtype of PTCL for cells in the biological sample. The statistical model may be trained using rankings of expression levels for some or all genes in the set(s) of genes.
In some embodiments, the gene ranking(s) may be obtained by ranking genes in one or more sets of genes based on their expression levels in the expression data. In some embodiments, the expression data includes values, each representing an expression level for a gene in the set(s) of genes. Determining the gene ranking(s) may involve determining a relative rank for each gene in the set(s) of genes based on the values. For example, a first gene ranking may be obtained by ranking genes in a first set of genes based on their expression levels.
In some embodiments, the expression data may be obtained for cells in the biological sample, where the subject has or is suspected of having cancer. In some embodiments, the expression data may be obtained for cells in the biological sample, where the subject has or is suspected of having lymphoma. In some embodiments, the subject has or is suspected of having PTCL.
In some embodiments, processing pipeline 100 shown in FIG. 1 may be used for determining one or more PTCL subtypes. In such embodiments, a gene ranking and a statistical model may be used to determine one or more PTCL subtypes of a biological sample. In some embodiments, one set of genes may be used for determining PTCL subtype for the biological sample and another set of genes may be used for determining tissue of origin. For example, statistical model 112 a and gene set 1 106 a may be used for determining PTCL subtype for cells in the biological sample and statistical model 112 b and gene set 2 106 b may be used for determining tissue of origin for cells in the biological sample. In some embodiments, different gene sets may be used for determining different PTCL subtypes. For example, gene set 1 106 a may be used for determining whether the biological sample has the AITL subtype and gene set 2 106 b may be used for determining whether the biological sample has the ATLL subtype.
In some embodiments, different gene sets and different statistical models may be used for determining different PTCL subtypes. For example, statistical model 112 a and gene set 1 106 a may be used for determining one PTCL subtype (e.g., AITL) for cells in the biological sample and statistical model 112 b and gene set 2 106 b may be used for another PTCL subtype (e.g., ATLL) for cells in the biological sample.
A statistical model used for determining PTCL subtype may be trained using data from one or more of Series GSE58445, Series GSE45712, Series GSE1906, Series GSE90597, Series GSE6338, Series GSE36172, Series GSE65823, Series GSE118238, Series GSE78513, Series GSE51521, Series GSE14317, Series GSE80631, Series GSE19067, and Series GSE20874 available through the GEO database. As another example, a statistical model used for determining PTCL subtype may be trained using data from one or more of the cohorts listed in Table 9, below.

TABLE 9

Cohorts of patients with gene expression data for training
statistical model(s) for PTCL subtype classification.

Cohort	Database	Platform	Year

GSE58445	GEO	GPL570	2014
n = 191
GSE45712	GEO	GPL8432	2018
n = 101		GPL14591
GSE19069	GEO	GPL570	2015
n = 100
GSE90597	GEO	GPL10739	2018
n = 66
GSE6338	GEO	GPL570	2007
n = 40
GSE36172	GEO	GPL6480	2013
n = 38
E-TABM-783	ArrayExpress	GPL570	2009
n = 33
GSE65823	GEO	GPL570	2015
n = 31
GSE118238	GEO	GPL570	2018
n = 29
E-TABM-702	ArrayExpress	GPL570	2014
n = 23
GSE78513	GEO	GPL570	2016
n = 23
GSE51521	GEO	GPL17811	2018
n = 20
GSE14317	GEO	GPL571	2009
n= 19
GSE80631	GEO	GPL6883	2016
n = 19
GSE19067	GEO	GPL570	2010
n = 18
GSE20874	GEO	GPL10175	2011
n= 18
SRP049695	SRA	RNASeq
n = 17
SRP029591	SRA	RNASeq	2014
n= 10

In some embodiments, PTCL subtype may be determined using the techniques described herein for cells in a biological sample. PTCL subtype may include Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS), anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), Natural killer/T-cell lymphoma (NKTCL), Sezary syndrome, adult T-cell leukemia/lymphoma (ATLL), enteropathy-type T-cell lymphoma, nasal NK/T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell lymphomas of Follicular T-cell (TFH) origin, T-cell lymphomas of the gastrointestinal tract, and cutaneous T-cell lymphomas.
In some embodiments, a set of genes used to obtain a gene ranking may include genes associated with biological features, expression pathways, or otherwise associated with determining one or more PTCL subtypes. Examples of genes that may be included in such a gene set are listed in Table 10, below.

TABLE 10

Gene Set for PTCL Subtype Classifier

	NCBI
Gene	Gene ID	NCBI Accession Number(s)

EFNB2	1948	NM_004093
ROBO1	6091	NM_133631; NM_001145845; NM_002941; XM_006713277;
		XM_017006983
S1PR3	1903	NM_005226
ANK2	287	NM_001127493; NM_001148; NM_020977
LPAR1	1902	NM_001401; NM_057159; NM_001351414; NM_001351415;
		NM_001387481; NM_001387505; XM_005251782; NM_001351401;
		NM_001387470; NM_001387480; NM_001387486; NM_001387498;
		NM_001387502; NM_001387503; NM_001387509; NM_001387511;
		NM_001387521; NM_001351398; NM_001351399; NM_001351400;
		NM_001351419; NM_001387478; NM_001387493; NM_001387497;
		NM_001387506; NM_001387507; NM_001387508; NM_001387520;
		NM_001351416; NM_001387476; NM_001387491; NM_001387471;
		NM_001387472; NM_001387477; NM_001387485; NM_001387492;
		NM_001387494; NM_001387510; NM_001387517; NM_001351397;
		NM_001351405; NM_001351406; NM_001351407; NM_001351413;
		NM_001351418; NM_001387475; NM_001387483; NM_001387484;
		NM_001387488; NM_001387490; NM_001387496; NM_001387516;
		NM_001387519; NM_001351404; NM_001351408; NM_001351410;
		NM_001351420; NM_001387473; NM_001387474; NM_001387487;
		NM_001387489; NM_001387495; NM_001387514; NM_001387518;
		NM_001351402; NM_001351403; NM_001351409; NM_001351411;
		NM_001351412; NM_001351417; NM_001387479; NM_001387482;
		NM_001387501; NM_001387504; NM_001387512; NM_001387513;
		NM_001387515
SNAP91	9892	XM_011536269; XM_017011557; XM_017011558; XM_017011559;
		XM_017011560; XM_005248770; XM_017011564; XM_017011571;
		NM_001376687; NM_001376688; NM_001376698; NM_001376700;
		NM_001376709; NM_001376715; NM_001376716; NM_001376723;
		NM_001376728; NM_001376731; NM_001376734; XM_011536266;
		XM_017011575; XM_017011586; NM_001376676; NM_001376682;
		NM_001376685; NM_001376702; NM_001376711; NM_001376736;
		NM_001376738; NR_164844; XM_006715615; XM_011536265;
		XM_017011570; XM_017011582; XM_024446600; NM_001376691;
		NM_001376699; NM_001376720; NM_001376735; NM_001376740;
		NM_014841; NR_164843; NR_164845; XM_011536275; XM_017011562;
		XM_017011565; XM_017011569; XM_017011580; NR_026669;
		NM_001256717; NM_001376677; NM_001376680; NM_001376683;
		NM_001376708; NM_001376718; NM_001376726; NM_001376737;
		XM_011536273; XM_011536276; XM_017011567; XM_017011583;
		XM_017011584; NM_001242794; NM_001376679; NM_001376694;
		NM_001376695; NM_001376697; NM_001376710; NM_001376714;
		NM_001376717; NM_001376742; XM_017011572; XM_017011577;
		XM_017011581; XM_017011590; NM_001256718; NM_001363677;
		NM_001376678; NM_001376686; NM_001376690; NM_001376693
SOX8	30812	NM_014587
RAMP3	10268	NM_005856
TUBB2B	347733	NM_178012
ARHGEF10	9639	NM_001308152; NM_001308153; NM_014629; XM_017014003
NOTCH1	4851	NM_017617
ZBTB17	7709	NM_001242884; NM_001287603; NM_003443; XM_011542088
CCNE1	898	NM_001238; NM_001322262; NM_001322259; NM_001322261
FGF18	8817	NM_003862
MYCN	4613	NM_001293231; NM_001293228; NM_001293233; NM_005378
PTHLH	5744	NM_198965; NM_198966; XM_011520774; NM_002820; XM_017019675;
		NM_198964
SMARCA2	6595	NM_001289400; NM_001289399; NM_001289398; NM_001289397;
		NM_001289396; NM_003070; NM_139045
WNK1	65125	NM_014823; NM_018979; NM_001184985; NM_213655; XM_017019837;
		XM_017019838
NKX2-1	7080	NM_001079668; NM_003317
CYP26A1	1592	NM_000783; NM_057157
HPSE	10855	NM_001098540; NM_001166498; NM_001199830; NM_006665
CTLA4	1493	NM_001037631; NM_005214
PELI1	57162	NM_020651; XM_011532994; XM_017004520
PRKCB	5579	NM_002738; NM_212535
SPAST	6683	NM_014946; NM_199436
ALS2	57679	NM_001135745; NM_020919; XM_006712654
KIF3B	9371	NM_004798
ZFYVE27	118813	XM_005269509; XM_011539254; XR_945597; NM_001002262;
		NM_001174120; NM_001385878; NM_001385889; NM_001385895;
		NM_001385901; NM_001385918; NR_169800; XM_005269508;
		XR_945594; NM_001385877; NM_001385902; NM_001385903;
		NM_001385904; NR_169794; NR_169795; NR_169797; NR_169803;
		NR_169805; NR_169809; XM_011539253; XM_017015644;
		XR_002956957; NM_001174121; NM_001174122; NM_001385879;
		NM_001385881; NM_001385883; NM_001385900; XM_017015645;
		NM_001385875; NM_001385886; NM_001385896; NR_169796;
		XM_011539252; NM_001385876; NM_001385880; NM_001385887;
		NM_001385894; NM_001385898; NM_001385915; NM_001385919;
		XM_017015646; NM_001385882; NM_001385884; NM_001385890;
		NM_001385897; NM_001385899; NR_169804; NR_169810;
		NM_001002261; NM_001385871; NM_001385892; NM_001385905;
		NM_144588; NR_169802; NR_169806; NR_169808; NR_169811;
		XR_002956956; NM_001174119; NM_001385885; NM_001385888;
		NM_001385891; NM_001385893; NM_001385906; NM_001385908;
		NM_001385911; NM_001385916; NR_169798; NR_169799; NR_169801
FGF18	8817	NM_003862
FNTB	2342	NM_001202558; NM_002028
REL	5966	NM_001291746; NM_002908
DMRT1	1761	NM_021951
SLC19A2	10560	NM_001319667; NM_006996
STK3	6788	NM_001256313; NM_001256312; NM_006281
PERP	64065	NM_022121
TNFRSF8	943	NM_001243; NM_001281430
TMOD1	7111	NM_001166116; NM_003275
BATF3	55509	NM_018664
CDC14B	8555	NM_001077181; NM_003671; NM_033331; XM_011519153;
		XM_017015240; XM_017015247; XR_001746407; XR_001746409;
		NM_001351567; XM_017015248; XM_017015249; XR_929865;
		XM_011519147; XM_017015242; XM_017015244; XM_017015245;
		XR_929864; NM_001351568; XM_011519149; XM_011519152;
		XR_001746408; NM_001351570; NM_033332; XM_011519148;
		XM_011519151; XR_929868; NR_147239; XM_011519156;
		XR_002956814; XM_011519159; XM_017015241; XR_001746406;
		NM_001351569
WDFEY3	N/A	N/A
AGT	183	NM_000029
ALK	238	NM_004304
ANXA3	306	NM_005139
BTBD11	121551	NM_001017523; NM_001018072; NM_001347943
CCNA1	8900	NM_001111045; NM_001111046; NM_001111047; NM_003914;
		XM_011535294; XM_011535295; XM_011535296
DNER	92737	NM_139072
GAS1	2619	NM_002048
HS6ST2	90161	NM_001077188; NM_147175; XM_011531407; XM_017029945;
		XM_011531408; XM_017029946; XM_005262491; XM_011531406
IL1RAP	3556	NM_001167928; NM_001167929; NM_001167930; NM_001167931;
		NM_002182; NM_134470
PCOLCE2	26577	NM_013363
PDE4DIP	9659	XM_011510175; NM_001195261; NM_001002810; NM_001002811;
		NM_001002812; NM_001195260; NM_001198832; NM_001198834;
		NM_014644; NM_022359
SLC16A3	9123	NM_001042422; NM_001042423; NM_001206950; NM_001206951;
		NM_001206952; NM_004207; XM_024451023
TIAM2	26230	NM_001010927; NM_012454
TUBB6	84617	NM_001303527; NM_001303525; NM_001303524; NM_001303526;
		NM_001303528; NM_001303529; NM_001303530; NM_032525
WNT7B	7477	XM_011530366; NM_058238
SMOX	54498	NM_001270691; NM_175839; NM_175840; NM_175841; NM_175842;
		XM_011529261
TMEM158	25907	NM_015444
NLRP7	199713	NM_001127255; NM_139176; NM_206828; XM_006723075;
		XM_006723076; XM_011526599
ADRB2	154	NM_000024
GALNT2	2590	NM_004481; NM_001291866
HRASLS	57110	NM_020386; NM_001366112; XM_011513034; XM_011513035
CD244	51744	NM_001166663; NM_001166664; NM_016382; XM_011509622
FASLG	356	NM_000639; NM_001302746
KIR2DL4	3805	NM_001080772; NM_001080770; NM_002255
LOC100287534	100287534	HF584483.1
KLRD1	3824	NM_007334; XM_006719067; XR_001748697; XM_017019289;
		NM_001351062; NR_147038; XM_017019287; NM_001351063;
		XM_011520650; XM_017019286; XM_024448974; XR_001748696;
		NR_147040; XM_017019285; NM_001114396; NM_001351060;
		NR_147039; XM_011520651; XM_017019288; NM_002262
SH2D1B	117157	NM_053282
KLRC2	3822	NM_002260
NCAM1	4684	NM_001242607; NM_000615; NM_001076682; NM_001242608;
		NM_181351
CXCR5	643	NM_001716; NM_032966
IL6	3569	NM_001318095; NM_000600; XM_011515390
ICOS	29851	NM_012092
CD40LG	959	NM_000074
CD84	8832	NM_001184879; NM_001184881; NM_001184882; NM_001330742;
		NM_003874
IL21	59067	NM_021803; NM_001207006
BCL6	604	NM_001134738; NM_001130845; NM_001706; XM_005247694;
		XM_011513062
MAF	4094	XM_017023233; XM_017023234; XM_017023235; NM_001031804;
		NM_005360
SH2D1A	4068	NM_001114937; NM_002351
IL4	3565	NM_000589; NM_172348
PTPN1	5770	NM_002827; NM_001278618
PIM1	5292	NM_002648; NM_001243186
ENTPD1	953	NM_001098175; NM_001164178; NM_001164181; NM_001164182;
		NM_001164183; NM_001312654; NM_001320916; NM_001776;
		XM_011540371; XM_011540377; XM_017016959
IRF4	3662	NM_001195286; NM_002460
CCND2	894	NM_001759
IL16	3603	NM_001172128; NM_004513; NM_172217; NR_148035; NM_001352685;
		NM_001352686; NM_001352684
ETV6	2120	NM_001987
BLNK	29760	NM_001258441; NM_001258442; NM_001114094; NM_001258440;
		NM_013314
SH3BP5	9467	NM_001018009; XM_017007522; XM_017007523; XM_017007524;
		XM_017007525; NM_004844
FUT8	2530	NM_004480; NM_178155; NM_178156
CCR4	1233	NM_005508; XM_017005687
GATA3	2625	NM_001002295; NM_002051; XM_005252442; XM_005252443
IL5	3567	NM_000879; XM_011543373; XM_011543374
IL10	3586	NM_000572
IL13	3596	NM_002188
MMEITPKB	N/A	N/A
MYBL1	4603	NM_001080416; NM_001144755; NM_001294282
LRMP	16970	NM_001204126; NM_001204127; NM_006152; NM_001366543;
		NM_001366544; NM_001366546; NM_001366549; NM_001366545;
		NR_159367; NR_159368; NM_001366541; NR_159366; NM_001366540;
		NM_001366542; NM_001366547; NR_159369; NM_001366548
KIAA0870	22898	NM_014957
LMO2	4005	NM_001142315; NM_001142316; NM_005574; XM_005252921;
		XM_017017727; XM_017017728; XM_017017729; XM_017017730;
		XM_017017731; XM_017017732; XM_017017733
CR1	1378	NM_000651; NM_000573
LTBR	4055	NM_001270987; NM_002342
PDPN	10630	NM_001006624; NM_001006625; NM_006474; NM_198389;
		XM_006710295
TNFRSF1A	7132	NM_001346091; NM_001065; NM_001346092
FCER2	2208	NM_001207019; NM_001220500; NM_002002; XM_005272462
ICAM1	3383	NM_000201
FCGR2B	2213	NM_001002273; NM_001002274; NM_001002275; NM_001190828;
		NM_004001; XM_024454043; NM_001386004; NR_169827;
		NM_001386001; NM_001386002; NM_001386006; NM_001386003;
		XM_017000670; NM_001386000; NM_001386005; XM_024454047
IKZF2	22807	NM_001079526; NM_016260; XM_005246384; XM_005246385;
		XM_011510818; NM_001371277; XM_011510809; NM_001387220;
		XM_005246386; XM_011510810; XM_011510803; XM_011510804;
		XM_011510812; XM_011510815; XM_011510817; XM_017003592;
		NM_001371275; XM_011510808; NM_001371274; XM_011510802;
		XM_011510807; XM_011510819; NM_001371276; XM_011510805;
		XM_011510811; XM_017003591; XM_011510816
CCR8	1237	NM_005201
TNFRSF18	8784	XM_017002722; NM_004195; NM_148901; NM_148902
IKZF4	64375	NM_022465; XM_005269089; XM_017019813; XM_017019815;
		XM_024449128; XM_024449129; NM_001351090; XM_017019807;
		XM_017019812; XM_024449131; NM_001351089; XM_011538664;
		XM_011538669; XM_017019814; XM_017019808; XM_024449130;
		NM_001351092; XM_017019806; XM_017019809; XM_017019810;
		XM_005269086; XM_017019811; XM_017019816; NM_001351091
FOXP3	50943	XM_006724533; NM_001114377; NM_014009
IL2	3558	NM_000586
TBX21	30009	NM_013351
IFNG	3458	NM_000619
GZMH	2999	NM_001270781; NM_033423
GNLY	10578	NM_001302758; NM_006433; NM_012483
EOMES	8320	NM_001278182; NM_001278183; NM_005442
NCR1	9437	NM_004829; NM_001145458; NM_001145457; NM_001242356;
		NM_001242357
GZMB	3002	NM_001346011; NM_004131
NKG7	4818	NM_005601
FGFBP2	83888	NM_031950
KLRF1	51348	NM_001291822; NM_001291823; NM_016523
CD160	11126	NM_007053; XM_005272929; XM_011509104
KLRK1	22914	NM_001199805; NM_007360
CD226	10666	NM_001303619; XM_005266643; XM_017025525; NM_006566;
		XM_006722374; XM_017025526; NM_001303618; XM_005266642;
		XM_017025527
NCR3	259197	NM_001145466; NM_001145467; NM_147130; XM_006715049;
		XM_011514459
TNFRSF8	943	NM_001243; NM_001281430
BATF3	55509	NM_018664
TMOD1	7111	NM_001166116; NM_003275
TMEM158	25907	NM_015444
MSC	9242	NM_005098
POPDC3	64208	NM_022361; XM_011536067; XM_017011194; XM_017011195

Some embodiments involve using a gene set that includes genes associated with a molecular signature of one or more PTCL subtypes. Examples of genes that may be included in such a gene set are listed in Table 11, below, which shows different genes and their corresponding PTCL subtype. In some embodiments, one or more genes listed in Table 11 may be combined with one or more genes listed in Table 10 to form a gene set used for determining PTCL subtype according to the techniques described herein.

TABLE 11

Functional annotation of the representative genes in
the molecular signatures of the common PTCL subtypes.

PTCL
subgroups	Major functional category	Gene symbols

AITL	Cell morphology/Intracellular	EFNB2, ROBO1, S1PR3, ANK2, LPAR1,
	signaling	SNAP91,SOX8
	Cell migration/Vascularization	LPAR1, RAMP3, S1PR3, ROBO1, EFNB2,
		TUBB2B, SOX8
	Cell cycle	SOX8, ARHGEF10
ATLL	T-cell	NOTCH1, ZBTB17, CCNE1, FGF18, MYCN,
	homeostasis/activation/	PTHLH, SMARCA2, WNK1, NKX2-1, CYP26A1,
	differentiation	HPSE, CTLA4, MYCN, PELI1, PRKCB, SPAST,
		ALS2, KIF3B, ZFYVE27
	Cell cycle/Proliferation	FGF18, MYCN, NKX2-1, NOTCH1, PTHLH,
		SMARCA2, CCNE1, GF18, WNK1, CTLA4,
		PELI1, PRKCB, ZBTB17, HPSE, FNTB, REL-1
ALCL	Cell morphology/Intracellular	DMRT1, SLC19A21, STK3, PERP, TNFRSF8,
	signaling	TMOD1, BATF3, DNER, ADRB2, AGT, TIAM2,
	and interaction	HS6ST2, GAS1, IL1RAP, WNT7B, ARHGEF10,
		HRASLS
	P53-induced genes	CDC14B, PERP, WDFEY3, TMOD1
	Cell cycle/Proliferation	AGT, ALK, ANXA3, BTBD11, CCNA1, DNER,
		GAS1, HS6ST2, IL1RAP, PCOLCE2, PDE4DIP,
		SLC16A3, TIAM2, TUBB6, WNT7B, SMOX,
		TMEM158, NLRP7, ADRB2, GALNT2
NKTCL	NK-cell activation/survival	CD244, FASLG, KIR2DL4, KLRD1, SH2D1B
	NK cell markers	CD244, FASLG, KLRC2, KLRD1, NCAM1

Further examples of genes that may be included in a gene set used for determining PTCL subtype according to the techniques described herein are described and listed in Iqbal J, Wright G, Wang C, et al., Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma, Blood, 2014; 123(19):2915-2923 (doi:10.1182/blood-2013-11-536359), which is incorporated herein by reference in its entirety.
Some embodiments may involve using a gene set that includes genes that are up-regulated in angioimmunoblastic T-cell lymphoma (AITL) compared to normal T lymphocytes, which may be referred to herein as “up-regulated in AITL genes”. For example, one or more genes in the gene set PICCALUGA_ANGIOIMMUNOBLASTIC_LYMPHOMA_UP, with the systematic name M12225 in the Gene Set Enrichment Analysis (GSEA) database, may be used in determining PTCL subtype according to the techniques described herein. In some embodiments, the gene set may include one or more genes selected from the group consisting of: A2M, ABCC3, ABI3BP, ACKR1, ACTA2, ACVRL1, ADAMDEC1, ADAMTS1, ADAMTS9, ADGRF5, ADGRL4 ADRA2A, ANK2, ANKRD29, ANTXR1, APOC1, APOE, ARHGAP29, ARHGAP42, ARHGEF10, ASPM, ATOX1, C1QA, C1QB, C1QC, C1R, C1S, C2, C3, C4A, C7, CALD1, CARMN, CAV2, CAVIN1, CCDC102B, CCDC80, CCL14, CCL19, CCL2, CCL21, CCN4, CD63, CD93, CDH11, CDH5, CETP, CFB, CFH, CHI3L1, CLMP, CLU, CMKLR1, COL12A1, COL15A1, COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, COL6A1, COL8A2, COX7A1, CP, CSRP2, CTHRC1, CTSC, CTSL, CTTNBP2NL, CXCL10, CXCL12, CXCL9, CYBRD1, CYFIP1, CYPLB1, CYP26B1, CYP27A1, DAB2, DCLK1, DDR2, DEPP1, DHRS7B, DOCK4, DPYSL3, EMCN, EMILIN1, ENG, ENPP2, EPHX1, FAM107A, FAM114A1, FAM20A, FBN1, FCHO2, FERMT2, FLRT2, FN1, FSTL1, FUCA1, GABBR1, GASK1B, GJA1, GJC1, GPNMB, GPRC5B, GUCY1B1, HNMT, HSPB8, HSPG2, IDH1, IFI27, IGFBP5, IGFBP7, IL18, IL33, IRAK3, ITGA9, ITPRIPL2, KCNJ10, KCNMA1, KCTD12, LAMA4, LAMB1, LAMC1, LIFR, LOXL1, LPAR1, LUM, MARCKS, MFAP4, MIR1245A, MIR34AHG, MMP9, MXRA5, MYL9, MYLK, NAGK, NEXN, NFIB, NNMT, NPL, NR1H3, NR2F2, OSMR, P2RYl3, PAPSS2, PARVA, PCOLCE, PDGFRA, PDLIM5, PDPN, PGF, PLA2G2D, PLA2G4C, PLD1, PLPP3, PMP22, PPIC, PRRX1, PTGDS, RAB13, RA114, RARRES2, RASSF4, RBP5, RBPMS, RGL1, RGS5, RHOBTB3, RND3, RPE, RRAS, RSPO3, S1PR3, SAMD9L, SEPTIN10, SERPING1, SERPINH1, SLAMF8, SLC1A3, SLC40A1, SLCO2B1, SMOC2, SPARC, SPARCL1, SPRED1, SULF1, TAGLN, TANC1, TCIM, TDO2, TEAD2, THY1, TJP1, TLR4, TMEM163, TMEM176A, TMEM176B, TNC, TNS1, TNS3, TPM1, TRIM47, VCAM1, VWF, WDFY3, WLS, WWTR1, YAP1, and ZNF226.
Some embodiments may involve using a gene set that includes genes that are down-regulated in angioimmunoblastic lymphoma (AITL) compared to normal T lymphocytes, which may be referred to herein as “down-regulated in AITL genes”. For example, one or more genes in the gene set PICCALUGA_ANGIOIMMUNOBLASTIC_LYMPHOMA_DN, with the systematic name M4781 in the Gene Set Enrichment Analysis (GSEA) database, may be used in determining PTCL subtype according to the techniques described herein. In some embodiments, the gene set may include one or more genes selected from the group consisting of: AMD1, AREG, ATP2B1-AS1, B3GNT2, BOLA2, BTG1, C16orf72, CBX4, CCDC59, CCNL1, CD6, CD69, CHD1, CLK1, CNOT6L, CNST, COG3, CREM, CSGALNACT2, CSRNP1, DDX3X, DNAJB6, DUSP10, DUSP2, DUSP4, EIFI, EIF4E, EIF4G3, EIF5, EPC1, ETNK1, FBXO33, FBXW7, FOSB, FOSL2, FOXPI, G3BP2, GABARAPL1, GADD45A, GADD45B, GATA3, H2AC18, H3-3B, HAUS3, HECA, HIPK1, ID2, IDS, IER5, IFRD1, IKZF5, ING3, IRF2BP2, IRS2, JMJD1C, JMY, JUN, JUND, KDM3A, KDM6B, KLF10, KLF4, KLF6, LINC-PINT, LINC01578, LY9, MAP3K8, MCL1, MEX3C, MGAT4A, MOAP1, MPZL3, MXD1, MYLIP, NAMPT, NDUFA10, NR4A2, NR4A3, PCIF1, PDE4D, PELI1, PER1, PHF1, PIGA, PMAIP1, PNPLA8, PPPIR15A, PPPIR15B, PRNP, PTGER4, PTP4A1, PTP4A2, RAPGEF6, REL, RGCC, RGS1, RGS2, RNF103, RNF11, RNF139, RSRC2, SARAF, SBDS, SETD2, SIK1, SIK3, SLC2A3, SLC30A1, SMURF2, SNORD22, SNORD3B-1, SON, SRSF5, STK17B, SUCO, THAP2, TIPARP, TMX4, TNFAIP3, TOB1, TP53INP2, TRA2B, TSC22D2, TSC22D3, TSPYL2, TTC7A, TUBB2A, WIPF1, YPEL5, ZBTB10, ZBTB24, ZFAND2A, ZFAND5, ZFC3H1, ZFP36, and ZNF331.
Some embodiments may involve using a gene set that includes genes associated with a molecular functional (MF) profile of a subject, which may be referred to herein as “MF profile genes”. In some embodiments, genes associated with a MF profile may include genes in one or more modules of the MF profile. Examples of genes associated with a MF profile and modules of a MF profile are described and listed in U.S. Pat. No. 10,311,967, titled “SYSTEMS AND METHODS FOR GENERATING, VISUALIZING AND CLASSIFYING MOLECULAR FUNCTION PROFILES,” issued on Jun. 4, 2019, which is incorporated herein by reference in its entirety. In some embodiments, one or more of the genes associated with a MF profile and one or more of the genes listed in Table 10 may be used in combination as a gene set for determining a PTCL subtype.
Some embodiments may involve determining a PTCL subtype for cells in a biological sample by using a statistical model that outputs multiple PTCL subtype predictions corresponding to different PTCL subtypes, which are used to determine a PTCL subtype for the biological sample. FIG. 26 is a diagram of an illustrative processing pipeline 2600 for determining a PTCL subtype of a biological sample, which may include ranking genes based on their gene expression levels and using the ranking and statistical model to determine the PTCL subtype, in accordance with some embodiments of the technology described herein. Processing pipeline 2600 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, processing pipeline 2600 may be performed by a desktop computer, a laptop computer, a mobile computing device. In some embodiments, processing pipeline 2600 may be performed within one or more computing devices that are part of a cloud computing environment.
In some embodiments, gene expression data 102 and ranking process 108 are used to rank genes based on their expression levels in gene expression data 102 to obtain gene ranking 110. Gene ranking 110 may be input to statistical model 112. Statistical model 112 may be trained using training data indicating rankings of expression levels for some or all genes in the gene set.
In some embodiments, statistical model 112 may output predictions of the biological sample having particular PTCL subtypes. In some instances, a prediction output by a statistical model may include a probability of the biological sample having the PTCL subtype. As shown in FIG. 26 , statistical model 112 outputs PTCL Subtype Prediction 1 216 a, PTCL Subtype Prediction 2 216 b, PTCL Subtype Prediction 1 216 c, and PTCL Subtype Prediction 1 216 d. The predictions output by statistical model 112 may be analyzed using prediction analysis process 118 to determine PTCL subtype 214 for the biological sample. Prediction analysis process 118 may involve selecting a particular PTCL subtype for the biological sample from among the different PTCL subtype predictions. In some embodiments, a PTCL subtype prediction may include a probability that the biological sample has the particular PTCL subtype. In such embodiments, prediction analysis process 118 may involve selecting a PTCL subtype based on the probabilities. In some embodiments, selecting the PTCL subtype may involve selecting the PTCL subtype having the highest probability as being PTCL subtype 214.
In some embodiments, statistical model 112 may provide outputs, each corresponding to a different PTCL subtype. For example, PTCL Subtype Prediction 1 216 a may correspond to anaplastic large cell lymphoma (ALCL), PTCL Subtype Prediction 2 216 b may correspond to angioimmunoblastic T-cell lymphoma (AITL), PTCL Subtype Prediction 3 216 c may correspond to natural killer/T-cell lymphoma (NKTCL), and PTCL Subtype Prediction 4 216 d may correspond to adult T-cell leukemia/lymphoma (ATLL). In some embodiments, statistical model 112 may include a multi-class classifier. In some embodiments, a class weight may be implemented for one or more of the classes in the multi-class classifier. Examples of classifiers that statistical model 112 may include are a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier.
Although four outputs from statistical model 112 are shown in FIG. 26 , it should be appreciated that a statistical model having any suitable number of outputs for PTCL subtype predictions may be implemented using the techniques described above in determining a PTCL subtype of a biological sample. In some embodiments, the outputs may be in the range of 3 to 5, 3 to 10, 3 to 15, or 3 to 20.
Some embodiments may involve determining a PTCL subtype for cells in a biological sample by using multiple statistical models that correspond to different PTCL subtypes and output predictions for the PTCL subtypes, which are used to determine a PTCL subtype for the biological sample. FIG. 27 is a diagram of an illustrative processing pipeline 2700 for determining a PTCL subtype of a biological sample, which may include ranking genes based on their gene expression levels and using the ranking and statistical models to determine the PTCL subtype, in accordance with some embodiments of the technology described herein. Processing pipeline 2700 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, processing pipeline 2700 may be performed by a desktop computer, a laptop computer, a mobile computing device. In some embodiments, processing pipeline 2700 may be performed within one or more computing devices that are part of a cloud computing environment.
In some embodiments, gene expression data 102 and ranking process 108 are used to rank genes based on their expression levels in gene expression data 102 to obtain gene ranking 110. Gene ranking 110 may be input to statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d. Each of statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d may be trained using training data indicating rankings of expression levels for some or all genes in the gene set. Statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d may each correspond to a different PTCL subtype and output a prediction of the biological sample having its particular PTCL subtype. In some instances, the prediction output by a statistical model may include a probability of the biological sample having the PTCL subtype.
As shown in FIG. 27 , statistical model 1 112 a outputs PTCL Subtype Prediction 1 316 a, statistical model 2 112 b outputs PTCL Subtype Prediction 2 316 b, statistical model 3 112 c outputs PTCL Subtype Prediction 3 316 c, and statistical model 4 112 d outputs PTCL Subtype Prediction 4 316 d. Each of statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d may correspond to a different PTCL subtype. For example, statistical model 1 112 a and PTCL Subtype Prediction 1 316 a may correspond to anaplastic large cell lymphoma (ALCL) and statistical model 1 112 a may be trained using rankings of expression levels for one or more genes associated with ALCL, such as those listed in Table 11. As another example, statistical model 2 112 b and PTCL Subtype Prediction 2 316 b may correspond to angioimmunoblastic T-cell lymphoma (AITL) and statistical model 2 112 b may be trained using rankings of expression levels for one or more genes associated with AITL, such as those listed in Table 11. As yet another example, statistical model 3 112 c and PTCL Subtype Prediction 3 316 c may correspond to natural killer/T-cell lymphoma (NKTCL) and statistical model 3 112 c may be trained using rankings of expression levels for one or more genes associated with NKTCL, such as those listed in Table 11. As another example, statistical model 4 112 d and PTCL Subtype Prediction 4 316 d may correspond to adult T-cell leukemia/lymphoma (ATLL) and statistical model 4 112 d may be trained using rankings of expression levels for one or more genes associated with ATLL, such as those listed in Table 11.
The predictions output by statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d may be analyzed using prediction analysis process 118 to determine PTCL subtype 214 for the biological sample. Prediction analysis process 118 may involve selecting a particular PTCL subtype for the biological sample from among the different PTCL subtype predictions. In some embodiments, a PTCL subtype prediction may include a probability that the biological sample has the particular PTCL subtype. In such embodiments, prediction analysis process 118 may involve selecting a PTCL subtype based on the probabilities. In some embodiments, selecting the PTCL subtype may involve selecting the PTCL subtype having the highest probability as being PTCL subtype 214.
In some embodiments, one or more of statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d may include a binary classifier. In some embodiments, each of statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d includes a binary classifier. In such embodiments, if none of the binary classifiers used are not determinative as to which class the biological sample belongs, then the sample may be determined to be unclassified. In some embodiments, statistical model 1 112 a, statistical model 2 112 b, statistical model 3 112 c, and statistical model 4 112 d may have a hierarchical classifier configuration.
Some embodiments may involve a hierarchical configuration of four classifiers in the order of a first classifier for the NKTCL PTCL subtype, a second classifier for the ATLL PTCL subtype, a third classifier for the AITL PTCL subtype, a fourth classifier for the ALCL PTCL subtype. In some embodiments, each of the first, second, third, and fourth classifiers is a binary classifier.
Although four statistical models and corresponding outputs are shown in FIG. 27 , it should be appreciated that any number of statistical models may be implemented using the techniques described above in determining a PTCL subtype of a biological sample. In some embodiments, the number of statistical models may be in the range of 3 to 5, 3 to 10, 3 to 15, or 3 to 20.
Some embodiments may involve determining a PTCL subtype of a biological sample by using different gene sets and statistical models corresponding to the different gene sets to obtain PTCL subtype predictions, which are used to determine the PTCL subtype. FIG. 28 is a diagram of an illustrative processing pipeline 2800 for determining a PTCL subtype of a biological sample, which may include ranking genes based on their gene expression levels and using the rankings and statistical models to determine the PTCL subtype, in accordance with some embodiments of the technology described herein. Processing pipeline 2800 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, processing pipeline 2800 may be performed by a desktop computer, a laptop computer, a mobile computing device. In some embodiments, processing pipeline 2800 may be performed within one or more computing devices that are part of a cloud computing environment.
In some embodiments, gene expression data 102 is used to rank genes in different sets of genes based on their expression levels in gene expression data 102 to obtain multiple gene rankings. For example, a gene ranking may be obtained for each gene set and the gene ranking may be input to a statistical model trained using training data indicating rankings of expression levels for some or all genes in the gene set. As shown in FIG. 28 , ranking process 108 may involve using expression data 102 to rank genes in different gene sets, including Gene Set 1 106 a, Gene Set 2 106 b, Gene Set 3 106 c, and Gene Set 4 106 d, to obtain Gene Ranking 1 110 a, Gene Ranking 2 110 b, Gene Ranking 3 110 c, and Gene Ranking 4 110 d, respectively. Ranking process 108 may involve ranking genes in a set of genes based on numerical values of their expression levels. Different gene rankings may be obtained by ranking expression levels for different gene sets, and each gene ranking may be input to its respective statistical model to obtain a PTCL subtype prediction. As shown in FIG. 28 , Gene Ranking 1 10 a, Gene Ranking 2 110 b, Gene Ranking 3 110 c, and Gene Ranking 4 110 d is provided as input to Statistical Model 1 112 a, Statistical Model 2 112 b, Statistical Model 3 112 c, and Statistical Model 4 112 d, respectively.
In some embodiments, the different statistical models and their respective gene sets may correspond to a particular PTCL subtypes of the biological sample. In such embodiments, each of the statistical models may output a prediction of the biological sample having a particular PTCL subtype. In some instances, the prediction output by a statistical model may include a probability of the biological sample having the PTCL subtype.
As shown in FIG. 28 , Statistical Model 1 112 a outputs PTCL Subtype Prediction 1 416 a, Statistical Model 2 112 b outputs PTCL Subtype Prediction 2 416 b, Statistical Model 3 112 c outputs PTCL Subtype Prediction 3 416 c, and PTCL Subtype Prediction 4 116 d outputs PTCL Subtype Prediction 4 416 d. The predictions output by the different statistical models may be analyzed using prediction analysis process 118 to determine PTCL subtype 114 for the biological sample.
Although four gene sets and four statistical models are shown in FIG. 28 , it should be appreciated that any suitable number of gene sets and corresponding statistical models may be implemented using the techniques described above in determining PTCL subtype predictions to obtain a PTCL subtype of a biological sample. In some embodiments, the number of gene sets and corresponding statistical models may be in the range of 3 to 100, 3 to 70, 3 to 50, 3 to 40, 3 to 30, 5 to 50, 10 to 60, or 10 to 70.
In some embodiments, the number of gene sets and corresponding statistical models is equal to or less than the number of classes for the PTCL subtype. Such embodiments may involve a different gene set and corresponding statistical model for each PTCL subtype. For example, Gene Set 1 106 a and Statistical Model 1 112 a may be used for generating a prediction of the PTCL subtype being anaplastic large cell lymphoma (ALCL) (as PTCL Subtype Prediction 1 416 a), Gene Set 2 106 b and Statistical Model 2 112 b may be used for generating a prediction of the PTCL subtype being angioimmunoblastic T-cell lymphoma (AITL) (as PTCL Subtype Prediction 2 416 b), Gene Set 3 106 c and Statistical Model 3 112 c may be used for generating a prediction of the PTCL subtype being natural killer/T-cell lymphoma (NKTCL) (as PTCL Subtype Prediction 3 416 c), and Gene Set 4 106 d and Statistical Model 4 112 d may be used for generating a prediction of the PTCL subtype being adult T-cell leukemia/lymphoma (ATLL) (as PTCL Subtype Prediction 4 416 d). It should be appreciated that additional gene sets and their corresponding statistical models may be implemented for different tissue types.
FIG. 29 is a flow chart of an illustrative process 2900 for determining one or more characteristics of a biological sample using a gene ranking and a statistical model, in accordance with some embodiments of the technology described herein. Process 2900 may be performed on any suitable computing device(s) (e.g., a single computing device, multiple computing devices co-located in a single physical location or located in multiple physical locations remote from one another, one or more computing devices part of a cloud computing system, etc.), as aspects of the technology described herein are not limited in this respect. In some embodiments, ranking process 108 and statistical model 112 may perform some or all of process 2900 to determine PTCL subtype.
Process 2900 begins at act 2910, where expression data for a biological sample of a subject is obtained. In some embodiments, the expression data may be obtained using a gene expression microarray. In some embodiments, the expression data may be obtained by performing next generation sequencing. In some embodiments, the expression data may be obtained by using a hybridization-based expression assay. Some embodiments involve performing a sequencing process of the biological sample (e.g., a gene expression microarray, next generation sequencing) prior to obtaining expression data 102. In some embodiments, obtaining gene expression data 102 may involve obtaining gene expression data 102 in silico, such as by accessing, using a computing device, expression data (e.g., expression data that has been previously obtained from a biological sample) in one or more data stores, receiving the expression data from one or more other device, or any other way. In some embodiments, obtaining gene expression data 102 may involve analyzing a biological sample (in vitro) and accessing (e.g., by a computing device, processor) the expression data. Further aspects relating to obtaining expression data are provided in the section titled “Obtaining Expression Data”.
Next, process 2900 proceeds to act 2920, where genes in a set of genes are ranked based on their expression levels in the expression data to obtain a gene ranking, such as by using ranking process 108. The expression data may include values, each representing an expression level for a gene in the set of genes, and determining the gene ranking may involve determining a relative rank for each gene in the set of genes based on the values.
In some embodiments, the subject has, is suspected of having, or is at risk of having breast cancer. The set of genes may be selected from the group of genes listed in Table 10. The set of genes may include at least 3, 5, 10, or 20 genes selected from the group of genes listed in Table 10. In some embodiments, the set of genes may include all the genes listed in Table 10. In some embodiments, the set of genes may include 3-120 genes, 5-120 genes, 20-120 genes, 50-120 genes, 80-120 genes listed in Table 10. In some embodiments, the set of genes may include 120 or fewer genes, 100 or fewer genes, 80 or fewer genes, 50 or fewer genes, 20 or fewer genes listed in Table 10.
In some embodiments, the subject has, is suspected of having, or is at risk of having lymphoma. In some embodiments, the subject has, is suspected of having, or is at risk of having PTCL.
Next process 2900 proceeds to act 2920, where PTCL subtype of the biological sample is determined using the gene ranking and a statistical model, such as statistical model 112. The statistical model may be trained using rankings of expression levels for one or more genes in the set of genes. In some embodiments, the gene ranking may be used as an input to the statistical model to obtain an output indicating the PTCL subtype. In some embodiments, the statistical model comprises one or more classifiers selected from the group consisting of: a statistical model may include are a gradient boosted decision tree classifier, a decision tree classifier, a gradient boosted classifier, a random forest classifier, a clustering-based classifier, a Bayesian classifier, a Bayesian network classifier, a neural network classifier, a kernel-based classifier, and a support vector machine classifier. In some embodiments, a statistical model may involve using a machine learning algorithm that implements of a gradient boosting framework, such as a gradient boosting decision tree (GBDT) and a gradient boosted regression tree (GBRT). Examples of software packages that implement machine learning algorithms that may be used according to the techniques described herein include the LightGBM package, the XGBoost package, and the pGBRT package.
In some embodiments, the statistical model may include a multi-class classifier. The multi-class classifier may provide at least four outputs each corresponding to a different PTCL subtype. For example, a first output may correspond to anaplastic large cell lymphoma (ALCL), a second output may correspond to angioimmunoblastic T-cell lymphoma (AITL), a third output may correspond to natural killer/T-cell lymphoma (NKTCL), and a fourth output may correspond to adult T-cell leukemia/lymphoma (ATLL).
In some embodiments, the statistical model may include multiple classifiers corresponding to different PTCL subtypes. For example, a first classifier may correspond anaplastic large cell lymphoma (ALCL), a second classifier may correspond to angioimmunoblastic T-cell lymphoma (AITL), a third classifier may correspond to natural killer/T-cell lymphoma (NKTCL), and a fourth classifier may correspond to adult T-cell leukemia/lymphoma (ATLL). In some embodiments, the multiple classifiers may be binary classifiers. The binary classifiers may have a hierarchical classification. For example, a statistical mode may include four binary classifiers having a hierarchical configuration in the order of a first classifier for the NKTCL PTCL subtype, a second classifier for the ATLL PTCL subtype, a third classifier for the AITL PTCL subtype, a fourth classifier for the ALCL PTCL subtype.
In some embodiments, the subtype of PTCL is selected from the group consisting of: anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL). In some embodiments, the subtype of PTCL is selected from the group consisting of: Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS), anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), Natural killer/T-cell lymphoma (NKTCL), Sezary syndrome, adult T-cell leukemia/lymphoma (ATLL), enteropathy-type T-cell lymphoma, nasal NK/T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell lymphomas of Follicular T-cell (TFH) origin, T-cell lymphomas of the gastrointestinal tract, and cutaneous T-cell lymphomas.
In some embodiments, process 2900 may include outputting the PTCL subtype to a user (e.g., physician), such as by displaying the PTCL subtype to the user on a graphical user interface (GUI), including the PTCL subtype in a report, sending an email to the user, and in any other suitable way.
In some embodiments, process 2900 may include administering a treatment to the subject based on the determined PTCL subtype of the biological sample. For example, a physician may administer a treatment for the subject associated with treating lymphomas of the determined PTCL subtype. Further examples where PTCL subtype of a biological sample determined using the techniques described herein are used for administering a treatment are provided in the section titled “Methods of Treatment”.
In some embodiments, process 2900 may include identifying a treatment for the subject based on the determined PTCL subtype. For example, the determined PTCL subtype may be used to identify a treatment for the subject associated with treating lymphomas of the determined PTCL subtype.
In some embodiments, process 2900 may include determining a prognosis for the subject based on the determined PTCL subtype. For example, the determined PTCL subtype may be used to determine a prognosis for the subject associated with treating lymphomas of the determined PTCL subtype.
Further aspects relating to other applications where PTCL subtype of a biological sample determined using the techniques described herein are provided in the section titled “Applications”.
In some embodiments, a trained statistical model used for determining PTCL subtype may be evaluated using existing clinical data to determine its performance in identifying PTCL subtype. As an example, a gene set having the genes listed in Table 10 was used for rank process 108 and a multi-class classifier was used for determining whether samples belong to AITL, ATLL, ALCL, NKTCL, or PTCL NOS subtypes. The clinical data listed in Table 9 was used for this evaluation process and Table 12, below, shows the PTCL subtypes identified using this process. The statistical model used achieved a 0.84 f1 score. FIG. 30 is a plot of survival rates for the different PTCL subtypes (ATLL, ALCL, NKTCL, and PTCL NOS).

TABLE 12

PTCL Subtype Classification Performance.

Cohort	AITL	ATLL	ALCL	NKTCL	PTCL NOS

GSE58445

	15	0	9	7	160
n = 191
GSE45712	10	0	12	0	80
n = 101
GSE19069	29	11	24	0	36
n = 100
GSE90597	0	0	0	66	0
n = 66
GSE6338	6	0	6	0	28
n = 40
GSE36172	0	0	0	0	38
n = 38
E-TABM-783	17	0	0	0	16
n = 33
GSE65823	0	0	31	0	0
n = 31
GSE118238	0	0	29	0	0
n = 29
E-TABM-702	0	0	0	7	16
n = 23
GSE78513	0	0	23	0	0
n = 23
GSE51521	20	0	0	0	0
n = 20
GSE14317	0	19	0	0	0
n = 19
GSE80631	0	0	0	19	0
n = 19
GSE19067	0	0	0	18	0
n = 18
GSE20874	8	0	0	4	6
n = 18
SRP049695	0	0	0	17	0
n = 17
SRP029591	10	0	0	0	0
n = 10

In some aspects, methods for characterization of cancers as described herein may be applied to any lymphoma. “Lymphoma” generally refers to a cancer (e.g., neoplasm) that originates from lymph node and lymphoid cells. Lymphomas are typically classified according to the normal cell type from which the tumor cells originate, for example T-cell lymphomas, B-cell lymphomas, Hodgkin (lymphocyte) lymphomas, and histiocytic and dendritic cell neoplasms. Classification of lymphomas is described, for example, by Jiang et al. Expert Rev. Hematol. 2017 March; 10(3):239-249. Classification of PTCL lymphomas is described, for example, by Iqbal J, Wright G, Wang C, et al., Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma, Blood, 2014; 123(19):2915-2923 (doi:10.1 182/blood-2013-11-536359), which is incorporated herein by reference in its entirety.
In some embodiments, a lymphoma is a B-cell lymphoma. In some embodiments, a B-cell lymphoma is a diffuse large B-cell lymphoma (DLBCL). Classification of DLBCL is described, for example, Alizadeh et al., Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling, Nature 403, 503-511 (2000) (doi: 10.1038/35000501). Examples of DLBCLs include but are not limited to germinal center B-cell (GCB) subtype and activated B-cell (ABC) subtype.
In some embodiments, a lymphoma is a T-cell lymphoma. In some embodiments, a T-cell lymphoma is a mature T-cell lymphoma, such as a peripheral T-cell lymphoma (PTCL). Over 25 mature T-cell lymphomas have been identified. Examples of PTCLs include but are not limited to Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS), anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), Natural killer/T-cell lymphoma (NKTCL), Sezary syndrome, adult T-cell leukemia/lymphoma (ATLL), enteropathy-type T-cell lymphoma, nasal NK/T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell lymphomas of Follicular T-cell (TFH) origin, T-cell lymphomas of the gastrointestinal tract (e.g., EATL, MEITL), cutaneous T-cell lymphomas, etc.
In some embodiments, the lymphoma is an anaplastic large cell lymphoma (ALCL). In some embodiments, the ALCL is systemic ALCL. In some embodiments, the ALCL is cutaneous ALCL (e.g., ALCL affecting the skin). In some embodiments, the ALCL is ALK-positive ALCL. In some embodiments, the ALCL is ALK-negative ALCL.
In some embodiments, the lymphoma is an angioimmunoblastic T-cell lymphoma (AITL). In some embodiments, AITL tumor cells express one or more follicular T cell markers, for example CD10 and CD279 (PD-1, PDCD1), CXCL13, BCL6, CD40L, or NFATC1.
In some embodiments, the lymphoma is an adult T-cell leukemia/lymphoma (ATLL). In some embodiments, ATLL results from infection with HTLV-1 virus.
In some embodiments, the lymphoma is a Natural killer/T-cell lymphoma (NKTCL). In some embodiments, NKTCL tumors are located in the palate and/or sinuses of a subject. In some embodiments, NKTCL tumors are located in the nasal cavity of a subject.
Obtaining Expression Data
Expression data (e.g., microarray data, next-generation sequencing (NGS) data) as described herein may be obtained from a variety of sources. In some embodiments, expression data may be obtained by analyzing a biological sample of a subject. The biological sample may be analyzed prior to performance of the techniques described herein, including the techniques for ranking genes based on their expression levels and using the ranking(s) to determine one or more characteristics of the biological sample. In some such embodiments, data obtained from the biological sample may be stored (e.g., in a database) and accessed during performance of the techniques described herein. Thus, “obtaining expression data” as described herein may involve obtaining gene expression data in silico, such as by accessing, using a computing device, expression data (e.g., expression data that has been previously obtained from a biological sample) in one or more data stores, receiving the expression data from one or more other device, or any other way, analyzing a biological sample (in vitro), or a combination thereof. Examples of additional techniques relating to how expression data is obtained are described in U.S. Pat. No. 10,311,967, titled “SYSTEMS AND METHODS FOR GENERATING, VISUALIZING AND CLASSIFYING MOLECULAR FUNCTION PROFILES,” issued on Jun. 4, 2019, which is incorporated herein by reference in its entirety.
In some embodiments, expression data may include expression levels for the entire cellular RNA, for all mRNAs in a cell, or a subset of RNAs in a cell (e.g., for a subset of RNAs expressed from a group of genes comprising or consisting of one or more gene sets described in this application, or at least some of the genes in those gene sets). RNA levels can be obtained using any appropriate technique including sequencing and/or hybridization based techniques (e.g., whole exome sequencing data, target specific sequencing data for a subset of RNAs, microarray data, etc.).
Biological Samples
Any of the methods, systems, assays, or other suitable techniques may be used to analyze any biological sample from a subject (e.g., a patient). In some embodiments, the biological sample may be any sample from a subject known or suspected of having cancer, including cancerous cells or pre-cancerous cells.
The biological sample may be any type of sample including, for example, a sample of a bodily fluid, one or more cells, a piece of tissue, or some or all of an organ. In some embodiments, the sample may be from a cancerous tissue or organ or a tissue or organ suspected of having one or more cancerous cells. In some embodiments, the sample may be from a healthy (e.g., non-cancerous) tissue or organ. In some embodiments, a sample from a subject (e.g., a biopsy from a subject) may include both healthy and cancerous cells and/or tissue. In certain embodiments, one sample will be taken from a subject for analysis.
Any of the biological samples described herein may be obtained from the subject using any known technique. In some embodiments, the biological sample may be obtained from a surgical procedure (e.g., laparoscopic surgery, microscopically controlled surgery, or endoscopy), bone marrow biopsy, punch biopsy, endoscopic biopsy, or needle biopsy (e.g., a fine-needle aspiration, core needle biopsy, vacuum-assisted biopsy, or image-guided biopsy). In some embodiments, each of the biological samples is a bodily fluid sample, a cell sample, or a tissue biopsy. In some embodiments, one or more than one cell (a cell sample) is obtained from a subject using a scrape or brush method. The cell sample may be obtained from any area in or from the body of a subject including, for example, from one or more of the following areas: the cervix, esophagus, stomach, bronchus, or oral cavity. In some embodiments, one or more than one piece of tissue (e.g., a tissue biopsy) from a subject may be used. In certain embodiments, the tissue biopsy may comprise one or more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) samples from one or more tumors or tissues known or suspected of having cancerous cells.
Sample Analysis
Methods described herein are based, at least in part, on the identification and characterization of certain biological processes and/or molecular and cellular compositions that are present within and/or surrounding the cancer (e.g., the tumor).
Biological processes within and/or surrounding cancer (e.g., a tumor) include, but are not limited to, angiogenesis, metastasis, proliferation, cell activation (e.g., T cell activation), tumor invasion, immune response, cell signaling (e.g., HER2 signaling), and apoptosis.
Molecular and cellular compositions within and/or surrounding cancer (e.g., a tumor) include, but are not limited to, nucleic acids (e.g., DNA and/or RNA), molecules (e.g., hormones), proteins (e.g., wild-type and/or mutant proteins), and cells (e.g., malignant and/or non-malignant cells). The cancer microenvironment, as used herein, refers to the molecular and cellular environment in which the cancer (e.g., a tumor) exists including, but not limited to, blood vessels that surround and/or are internal to a tumor, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM).
The molecular and cellular composition and biological processes present within and/or surrounding the tumor may be directed toward promoting cancer (e.g., tumor) growth and survival (e.g., pro-tumor) and/or inhibiting cancer (e.g., tumor) growth and survival (e.g., anti-tumor).
The cancer (e.g., tumor) microenvironment may comprise cellular compositions and biological processes directed toward promoting cancer (e.g., tumor) growth and survival (e.g., pro-tumor microenvironment) and/or inhibiting cancer (e.g., tumor) growth and survival (e.g., anti-tumor microenvironment). In some embodiments, the cancer (e.g., tumor) microenvironment comprises a pro-cancer (e.g., tumor) microenvironment. In some embodiments, the cancer (e.g., tumor) microenvironment comprises an anti-cancer (e.g., tumor) microenvironment. In some embodiments, the cancer (e.g., tumor) microenvironment comprises a pro-cancer (e.g., tumor) microenvironment and an anti-cancer (e.g., tumor) microenvironment.
Any information relating to molecular and cellular compositions, and biological processes that are present within and/or surrounding cancer (e.g., a tumor) may be used in methods for characterization of cancers (e.g., tumors) as described herein. In some embodiments, cancer (e.g., a tumor) may be characterized based on gene group expression level (e.g., on gene group RNA expression level). In some embodiments, cancer (e.g., a tumor) is characterized based on protein expression.
Methods for characterization of cancers as described herein may be applied to any cancer (e.g., any tumor). Exemplary cancers include, but are not limited to, adrenocortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, colon adenocarcinoma, esophageal carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, skin cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, uterine corpus endometrial carcinoma, and cholangiocarcinoma.
Expression Data
Expression data (e.g., indicating expression levels) for a plurality of genes may be used for any of the methods described herein. The number of genes which may be examined may be up to and inclusive of all the genes of the subject.
Any method may be used on a sample from a subject in order to acquire expression data (e.g., indicating expression levels) for the plurality of genes. As a set of non-limiting examples, the expression data may be RNA expression data, DNA expression data, or protein expression data.
DNA expression data, in some embodiments, refers to a level of DNA in a sample from a subject. The level of DNA in a sample from a subject having cancer may be elevated compared to the level of DNA in a sample from a subject not having cancer, e.g., a gene duplication in a cancer patient's sample. The level of DNA in a sample from a subject having cancer may be reduced compared to the level of DNA in a sample from a subject not having cancer, e.g., a gene deletion in a cancer patient's sample.
DNA expression data, in some embodiments, refers to data for DNA (or gene) expressed in a sample, for example, sequencing data for a gene that is expressed in a patient's sample. Such data may be useful, in some embodiments, to determine whether the patient has one or more mutations associated with a particular cancer.
RNA expression data may be acquired using any method known in the art including, but not limited to: whole transcriptome sequencing, total RNA sequencing, mRNA sequencing, targeted RNA sequencing, small RNA sequencing, ribosome profiling, RNA exome capture sequencing, and/or deep RNA sequencing. DNA expression data may be acquired using any method known in the art including any known method of DNA sequencing. For example, DNA sequencing may be used to identify one or more mutations in the DNA of a subject. Any technique used in the art to sequence DNA may be used with the methods described herein. As a set of non-limiting examples, the DNA may be sequenced through single-molecule real-time sequencing, ion torrent sequencing, pyrosequencing, sequencing by synthesis, sequencing by ligation (SOLiD sequencing), nanopore sequencing, or Sanger sequencing (chain termination sequencing). Protein expression data may be acquired using any method known in the art including, but not limited to: N-terminal amino acid analysis, C-terminal amino acid analysis, Edman degradation (including though use of a machine such as a protein sequenator), or mass spectrometry.
In some embodiments, the expression data comprises next-generation sequencing (NGS) data. In some embodiments, the expression data comprises microarray data. In some embodiments, the expression data comprises whole exome sequencing (WES) data. In some embodiments, the expression data comprises whole genome sequencing (WGS) data. In some embodiments, expression data comprises RNA Seq data (e.g., by performing RNA sequencing). In some embodiments, expression data comprises a combination of RNA Seq data and WGS data. In some embodiments, expression data comprises a combination of RNA Seq data and WES data.
Assays
Any of the biological samples described herein can be used for obtaining expression data using conventional assays or those described herein. Expression data, in some embodiments, includes gene expression levels. Gene expression levels may be detected by detecting a product of gene expression such as mRNA and/or protein.
In some embodiments, gene expression levels are determined by detecting a level of a protein in a sample and/or by detecting a level of activity of a protein in a sample. As used herein, the terms “determining” or “detecting” may include assessing the presence, absence, quantity and/or amount (which can be an effective amount) of a substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values and/or categorization of such substances in a sample from a subject.
The level of a protein may be measured using an immunoassay. Examples of immunoassays include any known assay (without limitation), and may include any of the following: immunoblotting assay (e.g., Western blot), immunohistochemical analysis, flow cytometry assay, immunofluorescence assay (IF), enzyme linked immunosorbent assays (ELISAs) (e.g., sandwich ELISAs), radioimmunoassays, electrochemiluminescence-based detection assays, magnetic immunoassays, lateral flow assays, and related techniques. Additional suitable immunoassays for detecting a level of a protein provided herein will be apparent to those of skill in the art.
Such immunoassays may involve the use of an agent (e.g., an antibody) specific to the target protein. An agent such as an antibody that “specifically binds” to a target protein is a term well understood in the art, and methods to determine such specific binding are also well known in the art. An antibody is said to exhibit “specific binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target protein than it does with alternative proteins. It is also understood by reading this definition that, for example, an antibody that specifically binds to a first target peptide may or may not specifically or preferentially bind to a second target peptide. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. In some examples, an antibody that “specifically binds” to a target peptide or an epitope thereof may not bind to other peptides or other epitopes in the same antigen. In some embodiments, a sample may be contacted, simultaneously or sequentially, with more than one binding agent that binds different proteins (e.g., multiplexed analysis).
It will be apparent to those of skill in the art that this disclosure is not limited to immunoassays. Detection assays that are not based on an antibody, such as mass spectrometry, are also useful for the detection and/or quantification of a protein and/or a level of protein as provided herein. Assays that rely on a chromogenic substrate can also be useful for the detection and/or quantification of a protein and/or a level of protein as provided herein.
Alternatively, the level of nucleic acids encoding a gene in a sample can be measured via a conventional method. In some embodiments, measuring the expression level of nucleic acid encoding the gene comprises measuring mRNA. In some embodiments, the expression level of mRNA encoding a gene can be measured using real-time reverse transcriptase (RT) Q-PCR or a nucleic acid microarray. Methods to detect nucleic acid sequences include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), in situ PCR, quantitative PCR (Q-PCR), real-time quantitative PCR (RT Q-PCR), in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, detection of a reporter gene, or other DNA/RNA hybridization platforms.
In some embodiments, the level of nucleic acids encoding a gene in a sample can be measured via a hybridization assay. In some embodiments, the hybridization assay comprises at least one binding partner. In some embodiments, the hybridization assay comprises at least one oligonucleotide binding partner. In some embodiments, the hybridization assay comprises at least one labeled oligonucleotide binding partner. In some embodiments, the hybridization assay comprises at least one pair of oligonucleotide binding partners. In some embodiments, the hybridization assay comprises at least one pair of labeled oligonucleotide binding partners.
Any binding agent that specifically binds to a desired nucleic acid or protein may be used in the methods and kits described herein to measure an expression level in a sample. In some embodiments, the binding agent is an antibody or an aptamer that specifically binds to a desired protein. In other embodiments, the binding agent may be one or more oligonucleotides complementary to a nucleic acid or a portion thereof. In some embodiments, a sample may be contacted, simultaneously or sequentially, with more than one binding agent that binds different proteins or different nucleic acids (e.g., multiplexed analysis).
To measure an expression level of a protein or nucleic acid, a sample can be in contact with a binding agent under suitable conditions. In general, the term “contact” refers to an exposure of the binding agent with the sample or cells collected therefrom for suitable period sufficient for the formation of complexes between the binding agent and the target protein or target nucleic acid in the sample, if any. In some embodiments, the contacting is performed by capillary action in which a sample is moved across a surface of the support membrane.
In some embodiments, an assay may be performed in a low-throughput platform, including single assay format. In some embodiments, an assay may be performed in a high-throughput platform. Such high-throughput assays may comprise using a binding agent immobilized to a solid support (e.g., one or more chips). Methods for immobilizing a binding agent will depend on factors such as the nature of the binding agent and the material of the solid support and may require particular buffers. Such methods will be evident to one of ordinary skill in the art.
Genes
The various genes recited herein are, in general, named using human gene naming conventions. The various genes, in some embodiments, are described in publicly available resources such as published journal articles. The gene names may be correlated with additional information (including sequence information) through use of, for example, the NCBI GenBank® databases; the HUGO (Human Genome Organization) Gene Nomination Committee (HGNC) databases; the DAVID Bioinformatics Resource. The gene names may also be correlated with additional information through printed publications from the foregoing organizations, which are incorporated by reference herein for this purpose. It should be appreciated that a gene may encompass all variants of that gene. For organisms or subjects other than human subjects, corresponding specific-specific genes may be used. Synonyms, equivalents, and closely related genes (including genes from other organisms) may be identified using similar databases including the NCBI GenBank® databases described above.
Some embodiments involve using a gene set for predicting breast cancer grade, including the genes listed in Table 1. Some embodiments involve using a gene set for predicting kidney clear cell cancer grade, including the genes listed in Table 2. Some embodiments involve using a gene set for predicting tissue of origin for Diffuse Large B-Cell Lymphoma (DLBCL), such as germinal center B-cell (GCB) and activated B-cell (ABC), including the genes listed in Table 3. Some embodiments involve using a gene set for predicting PTCL subtype, including the genes listed in Table 10.
Applications
Methods for biological sample characterization, which may include tumor type characterization, as described herein may be used for various clinical purposes including, but not limited to, monitoring the progress of cancer in a subject, assessing the efficacy of a treatment for cancer, identifying patients suitable for a particular treatment, evaluating suitability of a patient for participating in a clinical trial and/or predicting relapse in a subject. Accordingly, described herein are diagnostic and prognostic methods for cancer treatment based on tumor type described herein.
Methods described herein can be used to evaluate the efficacy of a cancer treatment, such as those described herein, given the correlation between cancer type (e.g., tumor types) and cancer prognosis. For example, multiple biological samples, such as those described herein, can be collected from a subject to whom a treatment is performed either before and after the treatment or during the course of the treatment. The cancer type (e.g., the tumor type) in the biological sample from the subject can be determined using any of the methods described herein. For example, if the cancer type indicates that the subject has a poor prognosis and the cancer type changes to a cancer type indicative of a favorable prognosis after the treatment or over the course of treatment, it indicates that the treatment is effective.
In some embodiments, cancer types can also be used to identify a cancer that may be treatable using a specific anti-cancer therapeutic agent (e.g., a chemotherapy). To practice this method, the cancer type in a sample (e.g., a tumor biopsy) collected from a subject having cancer can be determined using methods described herein. If the cancer type is identified as being susceptible to treatment with an anti-cancer therapeutic agent, the method may further comprise administering to the subject having the cancer an effective amount of the anti-cancer therapeutic agent.
In some embodiments, the methods for cancer type characterization as described herein may be relied on in the development of new therapeutics for cancer. In some embodiments, the cancer type may indicate or predict the efficacy of a new therapeutic or the progression of cancer in a subject prior to, during, or after the administration of the new therapy.
In some embodiments, methods for cancer type characterization as described herein may be used to evaluate suitability of a patient for participating in a clinical trial. In some embodiments, the cancer type may be used to include patients in a clinical trial. In some embodiments, patients having a specified cancer grade (e.g., Grade 1) are included in a clinical trial. In some embodiments, patients having a specified tissue of origin for the cancer are included in a clinical trial. In some embodiments, the cancer type may be used to exclude patients in a clinical trial. In some embodiments, patients having a specified cancer grade (e.g., Grade 3) are excluded from a clinical trial. In some embodiments, patients having a specified tissue of origin are excluded from a clinical trial. In some embodiments, patients having a specified PTCL subtype are excluded from a clinical trial.
In some embodiments, the methods described herein may be used in monitoring progression of a patient's disease and identifying one or more treatments based on a stage of disease determined using the techniques described herein. In some embodiments, the monitoring occurs over a period of time where a first disease stage is identified for the patient at a first time and a second disease stage is identified for the patient at a second time. The second disease stage may be used to identify a different type of treatment. For example, in the context of using the techniques described herein for predicting cancer grade, monitoring a patient's disease and identifying different treatments based on stage of disease may involve obtaining first expression data obtained by sequencing a first biological sample of a subject (e.g., a subject having kidney cancer), determining a first cancer grade using the first expression data and a statistical model described herein, identifying or recommending a first treatment for the subject based on the first cancer grade, and optionally, administering the first treatment. Monitoring the patient's disease may further involve obtaining second expression data obtained by sequencing a second biological sample of the subject (e.g., a biological sample obtained from the subject at a different time than the first biological sample), determining a second cancer grade using the second expression data, identifying or recommending a second treatment for the subject based on the second cancer grade, and optionally, administering the second treatment. In some embodiments, the first cancer grade is different from the first cancer grade and the first treatment is different from the second treatment. In some embodiments, monitoring may be performed multiple times (e.g., along with multiple medical visits) to evaluate progress of a treatment, determine how a patient is responding to a particular treatment, or a combination thereof.
In some embodiments, the methods described herein may be used in assessing how a subject has responded to a treatment. For example, these techniques described herein may be used in determining whether a subject is responding to a line of treatment or not, whether a subject is in remission, and whether there is a recurrence of a disease.
In some embodiments, characteristic(s) for cells of a biological sample of a subject determined using the techniques described herein may be used in identifying a diagnosis for the subject. In some embodiments, the characteristic(s) may provide information for a physician or other user to determine a diagnosis for the subject. For example, the characteristic(s) alone may be sufficient to allow a physician to determine the diagnosis. In some embodiments, a combination of the characteristic(s) and other patient medical data may be used by a physician or other user in determining a diagnosis for the subject.
In some embodiments, characteristic(s) for cells of a biological sample of a subject determined using the techniques described herein may be used in identifying a prognosis for the subject. In some embodiments, the characteristic(s) may provide information for a physician or other user to determine a prognosis for the subject. For example, the characteristic(s) alone may be sufficient to allow a physician to determine the prognosis. In some embodiments, a combination of the characteristic(s) and other patient medical data may be used by a physician or other user in determining a prognosis for the subject.
In some embodiments, a diagnosis or prognosis determined using the techniques described herein may be used in recommending a treatment or therapy for the subject. The therapy may be a drug treatment, radiation, surgery, diet or lifestyle change, or other therapy. A treatment may be chemotherapy, immunotherapy, hormone therapy, or other treatment. In some embodiments, recommending a treatment or therapy may include a change in treatment (e.g., a different treatment, an additional treatment, or a different frequency or dose).
In some embodiments, a diagnosis or prognosis determined using the techniques described herein may be used in generating a recommendation for further analysis of the patient. For example, a recommendation for further diagnostic intervention (e.g., more extensive CAT scan, MRI, more extensive or invasive biopsies, more detailed genetic, proteomic, or histological analysis of one or more tissue samples, etc.).
In some embodiments, a diagnosis or prognosis determined using the techniques described herein may be used in generating a recommendation to change the frequency of follow up medical checks. For example, a recommendation to have more frequent medical checks if the analysis suggests a higher risk, or less frequent medical checks if the analysis suggests a lower risk or that the subject is in remission.
In some embodiments, characteristic(s) for cells of a biological sample of a subject determined using the techniques described herein may be using in generating a report specific to the subject. For example, the report may be a patient-specific cancer characteristics report. Generating the report may involve generating a file comprising information indicative of disease characteristics determined using the techniques described herein (e.g., cancer grade, tissue of origin, tissue subtype).
In the context of providing a recommendation or other information to a physician or other user, providing such information may involve transmitting electronic information to the physician or other user. In some embodiments, the electronic information may be transmitted to a medical center or to a computer system that hosts the patient medical information, and the physician or other user may access the information using a computing device.
Examples of additional applications for how characteristics of a biological sample, as determined using the techniques described herein, may be used are described in in U.S. Pat. No. 10,311,967, titled “SYSTEMS AND METHODS FOR GENERATING, VISUALIZING AND CLASSIFYING MOLECULAR FUNCTION PROFILES,” issued on Jun. 4, 2019, which is incorporated herein by reference in its entirety.
Methods of Treatment
In certain methods described herein, an effective amount of anti-cancer therapy described herein may be administered or recommended for administration to a subject (e.g., a human) in need of the treatment via a suitable route (e.g., intravenous administration).
The subject to be treated by the methods described herein may be a human patient having, suspected of having, or at risk for a cancer. A subject having, suspected of having, or at risk of having cancer may be a subject exhibiting one or more signs or symptoms of cancer, subject that is diagnosed as having cancer, a subject that has a family history and/or a genetic predisposition to having cancer, and/or a subject that has one or more other risk factors for cancer (e.g., age, exposure to carcinogens, environmental exposure, exposure to a virus associated with a higher likelihood of developing cancer, etc.). Examples of a cancer include, but are not limited to, melanoma, lung cancer, brain cancer, breast cancer, colorectal cancer, pancreatic cancer, liver cancer, prostate cancer, skin cancer, kidney cancer, bladder cancer, or prostate cancer. The subject to be treated by the methods described herein may be a mammal (e.g., may be a human). Mammals include, but are not limited to: farm animals (e.g., livestock), sport animals, laboratory animals, pets, primates, horses, dogs, cats, mice, and rats.
“An effective amount” as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons, or for virtually any other reasons.
Examples of additional methods of treatment are described in U.S. Pat. No. 10,311,967, titled “SYSTEMS AND METHODS FOR GENERATING, VISUALIZING AND CLASSIFYING MOLECULAR FUNCTION PROFILES,” issued on Jun. 4, 2019, which is incorporated herein by reference in its entirety.
Quality Control Analysis
In some embodiments, the techniques described herein may be used in performing quality control. One application is quality control analysis in a laboratory setting. For example, a sequencing laboratory may receive a biological sample together with information about the biological sample. Aside from an identifier and/or tracking number, such information may include information about the characteristics of the biological sample (e.g., the tissue source, cancer type, cancer grade, etc.). However, due to laboratory errors, it is possible that the biological sample provided does not actually have these characteristics (e.g., due to an error where patient samples are switched, mislabeled, wrong information is provided, etc.).
Another application is to quality control analysis in data analysis setting. For example, a patient's sequencing data (e.g., reads, aligned reads, expression levels, etc.) may be provided as input to a data processing pipeline. However, if that sequencing data does not correspond to the alleged source (e.g., it comes from a different patient due to an error), the results of the analysis are likely meaningless.
In some embodiments, quality control may be performed by comparing an asserted characteristic of a biological sample to a predicted characteristic determined using the techniques described herein. When the asserted characteristic and the predicted characteristic match (e.g., are the same or are within a tolerated difference), then it may be determined that a quality control check has been satisfied. On the other hand, if the predicted and asserted characteristics do not match, then further action may need to be taken. For example, further analysis of the biological sample may be performed, the biological sample may be rejected, a data processing pipeline may be stopped or not executed (thereby saving valuable and costly computational resources), a laboratory operator and/or other party (e.g., clinician, staff, etc.) may be notified of a potential discrepancy (e.g., by an e-mail alert, a message, a report, an entry in a log-file, etc.).
For example, a classifier for determining cancer grade may be used to predict cancer grade from gene expression data of a sample, and the predicted cancer grade may be compared to an asserted cancer grade for the sample. If the predicted and asserted cancer grades match, then it may be determined that the sample analysis has met quality control standards. However, if the predicted and asserted cancer grades do not match, then further analysis may be performed. As another example, a classifier for determining tissue of origin may be used to predict a type of tissue for a sample and the predicted tissue type may be compared to an asserted tissue type for the sample. If the predicted and asserted tissue types do not match, then further analysis of the biological sample may be performed to identify the tissue type for the sample. Any of the classification techniques described herein may be used in this manner, either alone or in combination with one another to provide multiple quality control checkpoints.
Examples of additional quality control analysis are described in U.S. patent application Ser. No. 16/920,636, titled “TECHNIQUES FOR BIAS CORRECTION IN SEQUENCE DATA,” filed Jul. 3, 2020, which is incorporated herein by reference in its entirety.
Computational System
An illustrative implementation of a computer system 1000 that may be used in connection with any of the embodiments of the technology described herein is shown in FIG. 10 . The computer system 1000 includes one or more processors 1010 and one or more articles of manufacture that comprise non-transitory computer-readable storage media (e.g., memory 1020 and one or more non-volatile storage media 1030). The processor 1010 may control writing data to and reading data from the memory 1020 and the non-volatile storage device 1030 in any suitable manner, as the aspects of the technology described herein are not limited in this respect. To perform any of the functionality described herein, the processor 1010 may execute one or more processor-executable instructions stored in one or more non-transitory computer-readable storage media (e.g., the memory 1020), which may serve as non-transitory computer-readable storage media storing processor-executable instructions for execution by the processor 1010.
Computing device 1000 may also include a network input/output (I/O) interface 1040 via which the computing device may communicate with other computing devices (e.g., over a network), and may also include one or more user I/O interfaces 1050, via which the computing device may provide output to and receive input from a user. The user I/O interfaces may include devices such as a keyboard, a mouse, a microphone, a display device (e.g., a monitor or touch screen), speakers, a camera, and/or various other types of I/O devices.
The above-described embodiments can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor (e.g., a microprocessor) or collection of processors, whether provided in a single computing device or distributed among multiple computing devices. It should be appreciated that any component or collection of components that perform the functions described above can be generically considered as one or more controllers that control the above-discussed functions. The one or more controllers can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above.
In this respect, it should be appreciated that one implementation of the embodiments described herein comprises at least one computer-readable storage medium (e.g., RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or other tangible, non-transitory computer-readable storage medium) encoded with a computer program (e.g., a plurality of executable instructions) that, when executed on one or more processors, performs the above-discussed functions of one or more embodiments. The computer-readable medium may be transportable such that the program stored thereon can be loaded onto any computing device to implement aspects of the techniques discussed herein. In addition, it should be appreciated that the reference to a computer program which, when executed, performs any of the above-discussed functions, is not limited to an application program running on a host computer. Rather, the terms computer program and software are used herein in a generic sense to reference any type of computer code (e.g., application software, firmware, microcode, or any other form of computer instruction) that can be employed to program one or more processors to implement aspects of the techniques discussed herein.
The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of processor-executable instructions that can be employed to program a computer or other processor to implement various aspects of embodiments as discussed above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the disclosure provided herein need not reside on a single computer or processor, but may be distributed in a modular fashion among different computers or processors to implement various aspects of the disclosure provided herein.
Processor-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.
Also, data structures may be stored in one or more non-transitory computer-readable storage media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a non-transitory computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish relationships among information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationships among data elements.
Also, various inventive concepts may be embodied as one or more processes, of which examples have been provided. The acts performed as part of each process may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
Aspects of the technology described herein provide computer implemented methods for generating, visualizing and classifying biological characteristic(s) (e.g., cancer grade, tissue of origin) of cancer patients.
In some embodiments, a software program may provide a user with a visual representation of a patient's characteristic(s) and/or other information related to a patient's cancer using an interactive graphical user interface (GUI). Such a software program may execute in any suitable computing environment including, but not limited to, a cloud-computing environment, a device co-located with a user (e.g., the user's laptop, desktop, smartphone, etc.), one or more devices remote from the user (e.g., one or more servers), etc.
For example, in some embodiments, the techniques described herein may be implemented in the illustrative environment 1100 shown in FIG. 11 . As shown in FIG. 11 , within illustrative environment 1100, one or more biological samples of a patient 1102 may be provided to a laboratory 1104. Laboratory 1104 may process the biological sample(s) to obtain expression data (e.g., DNA, RNA, and/or protein expression data) and provide it, via network 1108, to at least one database 1106 that stores information about patient 1102.
Network 1108 may be a wide area network (e.g., the Internet), a local area network (e.g., a corporate Intranet), and/or any other suitable type of network. Any of the devices shown in FIG. 11 may connect to the network 1108 using one or more wired links, one or more wireless links, and/or any suitable combination thereof.
In the illustrated embodiment of FIG. 11 , the at least one database 1106 may store expression data for the patient, medical history data for the patient, test result data for the patient, and/or any other suitable information about the patient 1102. Examples of stored test result data for the patient include biopsy test results, imaging test results (e.g., MRI results), and blood test results. The information stored in at least one database 1106 may be stored in any suitable format and/or using any suitable data structure(s), as aspects of the technology described herein are not limited in this respect. The at least one database 1106 may store data in any suitable way (e.g., one or more databases, one or more files). The at least one database 1106 may be a single database or multiple databases.
As shown in FIG. 11 , illustrative environment 1100 includes one or more external databases 1116, which may store information for patients other than patient 1102. For example, external databases 1116 may store expression data (of any suitable type) for one or more patients, medical history data for one or more patients, test result data (e.g., imaging results, biopsy results, blood test results) for one or more patients, demographic and/or biographic information for one or more patients, and/or any other suitable type of information. In some embodiments, external database(s) 1116 may store information available in one or more publicly accessible databases such as TCGA (The Cancer Genome Atlas), one or more databases of clinical trial information, and/or one or more databases maintained by commercial sequencing suppliers. The external database(s) 1116 may store such information in any suitable way using any suitable hardware, as aspects of the technology described herein are not limited in this respect.
In some embodiments, the at least one database 1106 and the external database(s) 1116 may be the same database, may be part of the same database system, or may be physically co-located, as aspects of the technology described herein are not limited in this respect.
For example, in some embodiments, server(s) 1110 may access information stored in database(s) 1106 and/or 1116 and use this information to perform process 300, described with reference to FIG. 3 , for determining one or more characteristics of a biological sample.
As another example, in some embodiments, server(s) 1110 may access information stored in database(s) 1106 and/or 1116 and use this information to perform process 400, described with reference to FIG. 4 , for determining tissue of origin for some or all cells in a biological sample.
As another example, in some embodiments, server(s) 1110 may access information stored in database(s) 1106 and/or 1116 and use this information to perform process 500, described with reference to FIG. 5 , for determining cancer grade for some or all cells in a biological sample.
As another example, in some embodiments, server(s) 1110 may access information stored in database(s) 1106 and/or 1116 and use this information to perform process 800, described with reference to FIG. 8 , for selecting a gene set.
As another example, in some embodiments, server(s) 1110 may access information stored in database(s) 1106 and/or 1116 and use this information to perform process 2900, described with reference to FIG. 29 , for determining PTCL subtype of a biological sample. In some embodiments, server(s) 1110 may include one or multiple computing devices. When server(s) 1110 include multiple computing devices, the device(s) may be physically co-located (e.g., in a single room) or distributed across multi-physical locations. In some embodiments, server(s) 1110 may be part of a cloud computing infrastructure. In some embodiments, one or more server(s) 1110 may be co-located in a facility operated by an entity (e.g., a hospital, research institution) with which doctor 1114 is affiliated. In such embodiments, it may be easier to allow server(s) 1110 to access private medical data for the patient 1102.
As shown in FIG. 11 , in some embodiments, the results of the analysis performed by server(s) 1110 may be provided to doctor 1114 through a computing device 1112 (which may be a portable computing device, such as a laptop or smartphone, or a fixed computing device such as a desktop computer). The results may be provided in a written report, an e-mail, a graphical user interface, and/or any other suitable way. It should be appreciated that although in the embodiment of FIG. 11 , the results are provided to a doctor, in other embodiments, the results of the analysis may be provided to patient 1102 or a caretaker of patient 1102, a healthcare provider such as a nurse, or a person involved with a clinical trial.
In some embodiments, the results may be part of a graphical user interface (GUI) presented to the doctor 1114 via the computing device 1112. In some embodiments, the GUI may be presented to the user as part of a webpage displayed by a web browser executing on the computing device 1112. In some embodiments, the GUI may be presented to the user using an application program (different from a web-browser) executing on the computing device 1112. For example, in some embodiments, the computing device 1112 may be a mobile device (e.g., a smartphone) and the GUI may be presented to the user via an application program (e.g., “an app”) executing on the mobile device.
The GUI presented on computing device 1112 may provide a wide range of oncological data relating to both the patient and the patient's cancer in a new way that is compact and highly informative. Previously, oncological data was obtained from multiple sources of data and at multiple times making the process of obtaining such information costly from both a time and financial perspective. Using the techniques and graphical user interfaces illustrated herein, a user can access the same amount of information at once with less demand on the user and with less demand on the computing resources needed to provide such information. Low demand on the user serves to reduce clinician errors associated with searching various sources of information. Low demand on the computing resources serves to reduce processor power, network bandwidth, and memory needed to provide a wide range of oncological data, which is an improvement in computing technology. All definitions, as defined and used herein, should be understood to control over dictionary definitions, and/or ordinary meanings of the defined terms.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed. Such terms are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term).
The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing”, “involving”, and variations thereof, is meant to encompass the items listed thereafter and additional items.
Having described several embodiments of the techniques described herein in detail, various modifications, and improvements will readily occur to those skilled in the art. Such modifications and improvements are intended to be within the spirit and scope of the disclosure. Accordingly, the foregoing description is by way of example only, and is not intended as limiting. The techniques are limited only as defined by the following claims and the equivalents thereto.

Claims

What is claimed is:

1-30. (canceled)

31. A system, comprising:

at least one computer hardware processor; and

at least one non-transitory computer-readable storage medium storing processor-executable instructions configured to perform a method comprising:

accessing a trained statistical model comprising parameters estimated using training data, the training data indicating a plurality of gene rankings of at least some genes in a set of genes, the parameters being estimated after the at least some genes in the set of genes were selected;

obtaining expression data for a biological sample of a subject, the expression data previously obtained at least in part by sequencing the biological sample and comprising first expression levels for the set of genes;

ranking the at least some genes in the set of genes, based on their first expression levels in the expression data to obtain a gene ranking; and

determining at least one characteristic of the biological sample by providing the gene ranking as an input to the trained statistical model and processing the input with the trained statistical model, using the parameters, to obtain an output indicating the at least one characteristic.

32. The system of claim 31, wherein the at least one characteristic is selected from the group consisting of cancer grade for cells in the biological sample, tissue of origin for cells in the biological sample, tissue type for cells in the biological sample, and cancer subtype for cells in the biological sample.

33. The system of claim 31, wherein the at least one characteristic includes cancer grade for cells in the biological sample, and the cancer grade is selected from the group consisting of Grade 1, Grade 2, Grade 3, Grade 4, and Grade 5.

34. The system of claim 31, wherein the at least one characteristic includes tissue of origin for cells in the biological sample, and the tissue of origin is selected from the group consisting of lung tissue, pancreas tissue, stomach tissue, colon tissue, liver tissue, bladder tissue, kidney tissue, thyroid tissue, lymph node tissue, adrenal gland tissue, skin tissue, breast tissue, ovary tissue, prostate tissue, urothelial tissue, cervical tissue, esophagus tissue, brain tissue, soft tissue, connective tissue, head tissue, and neck tissue.

35. The system of claim 31, wherein the at least one characteristic includes tissue type for cells in the biological sample, and the tissue type is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, carcinoma, cystadenocarcinoma, sarcoma, and glioma.

36. The system of claim 31, wherein the at least one characteristic includes human papillomavirus (HPV) status for cells in the biological sample, and wherein the set of genes includes at least 5 genes selected from the group of genes listed in Table 8.

37. The system of claim 31, wherein the at least one characteristic includes a subtype of peripheral T-cell lymphoma (PTCL) for cells in the biological sample, and wherein the set of genes includes at least 5 genes selected from the group of genes listed in Table 10.

38. The system of claim 37, wherein the subtype of PTCL is selected from the group consisting of: anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma (NKTCL), and adult T-cell leukemia/lymphoma (ATLL).

39. The system of claim 31, wherein the method further comprises: presenting, to a user, an indication of the at least one characteristic via a graphical user interface (GUI).

40. A method, comprising:

using at least one computer hardware processor to perform:

accessing a trained statistical model comprising parameters estimated using training data, the training data indicating a plurality of gene rankings of some genes in a set of genes, the parameters being estimated after the some genes in the set of genes were selected;

ranking the some genes in the set of genes, based on their first expression levels in the expression data to obtain a gene ranking; and

41. The method of claim 40, wherein the at least one characteristic includes cancer grade for cells in the biological sample.

42. The method of claim 41, wherein the subject has, is suspected of having, or is at risk of having breast cancer, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 1.

43. The method of claim 42, wherein the set of genes comprises at least 10 genes selected from the group of genes listed in Table 1.

44. The method of claim 41, wherein the subject has, is suspected of having, or is at risk of having kidney cancer, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 2.

45. The method of claim 41, wherein the subject has, is suspected of having, or is at risk of having lymphoma, and wherein the set of genes comprises at least 5 genes selected from the group of genes listed in Table 3.

46. The method of claim 45, wherein the subject has, is suspected of having, or is at risk of having Diffuse Large B-Cell Lymphoma (DLBCL), the set of genes comprises at least 10 genes selected from the group of genes listed in Table 3, and the at least one characteristic is a cell of origin selected from the group consisting of germinal center B-cell (GCB) and activated B-cell (ABC).

47. The method of claim 40, wherein the at least one characteristic is selected from the group consisting of cancer grade for cells in the biological sample, tissue of origin for cells in the biological sample, tissue type for cells in the biological sample, and cancer subtype for cells in the biological sample.

48. At least one non-transitory computer-readable storage medium storing processor-executable instructions that are configured to cause, when executed by at least one computer hardware processor, the at least one computer hardware processor to perform:

accessing a trained statistical model previously trained and comprising parameters estimated using training data after at least some genes in a set of genes were selected, the training data indicating a plurality of gene rankings of the at least some genes in the set of genes;

49. The at least one non-transitory computer-readable storage medium of claim 48, wherein the gene ranking includes a value identifying a relative rank for each gene of the at least some genes in the set of genes.

50. The at least one non-transitory computer-readable storage medium of claim 48, wherein the gene ranking includes values identifying relative ranks of the at least some genes in the gene ranking, wherein the values are different from the first expression levels.