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WO2021092299A1 - Systèmes et essais pour évaluer l'instabilité de microsatellites - Google Patents

Systèmes et essais pour évaluer l'instabilité de microsatellites Download PDF

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Publication number
WO2021092299A1
WO2021092299A1 PCT/US2020/059295 US2020059295W WO2021092299A1 WO 2021092299 A1 WO2021092299 A1 WO 2021092299A1 US 2020059295 W US2020059295 W US 2020059295W WO 2021092299 A1 WO2021092299 A1 WO 2021092299A1
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Prior art keywords
seq
polynucleotide sequence
microsatellite
abi
loci
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English (en)
Inventor
Charles HIGDON
Harrison Leong
Charles BAUDO
Carole Bornarth
Edgar SCHREIBER
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Life Technologies Corp
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Life Technologies Corp
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Priority to CN202080083538.7A priority Critical patent/CN114761580A/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This disclosure relates generally to DNA fragment analysis.
  • a causal factor in cancer is thought to be the breakdown of biomolecular machinery to repair DNA.
  • DNA repair mechanisms are critical to the integrity of the replicate cells. When these mechanisms break down, mistakes can accumulate in the DNA carried by the resulting cells.
  • One way of detecting the circumstances in which the drugs would be most effective is to examine the degree to which DNA deviates from normal at loci where the DNA consists of many repeated subsequences.
  • Microsatellites also known as short tandem repeats (STRs) are polymorphic
  • DNA loci consisting of short nucleotide sequences, usually 1-6 base repeats. These motifs comprise approximately 3% of the human genome. During DNA replication, these sequences are susceptible to errors that can result in deletions and insertions. When deficiencies in the DNA MMR system are present, microsatellite replication errors accumulate in the genome. This phenomenon is commonly referred to as microsatellite instability (MSI).
  • MSI microsatellite instability
  • microsatellite loci are amplified by polymerase chain reaction (PCR) using fluorescently labeled forward primers and unlabeled reverse primers. The PCR amplicons are separated by size using electrophoresis.
  • Applications include linkage mapping; animal breeding; human, animal, and plant typing; pathogen sub-typing; genetic diversity; microsatellite instability; Loss of Heterozygosity (LOH); Inter-simple sequence repeat (ISSR); Multilocus Variant Analysis (MLVA); and companion diagnostics for cancer treatments.
  • LH Loss of Heterozygosity
  • ISSR Inter-simple sequence repeat
  • MLVA Multilocus Variant Analysis
  • the instant technology generally relates to methods, systems, compositions, and kits for detecting microsatellite instability (MSI) in a DNA sample.
  • MSI microsatellite instability
  • a method for detecting microsatellite instability (MSI) in a DNA sample including: a) co-amplifying a plurality of microsatellite loci of the DNA sample to produce amplified fragments comprising nucleic acid sequences (e.g., DNA fragments) from each locus; b) determining the size of the amplified fragments from each locus; and c) comparing the size of the amplified fragments from each locus to the size of corresponding amplified fragments from a control.
  • MSI microsatellite instability
  • the plurality of loci includes at least one locus selected from BAT25, BAT 26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • a difference in size between one or more amplified fragments between the sample and the control indicates the presence of MSI at the corresponding locus in the DNA sample.
  • a method for analyzing a DNA sample to determine microsatellite instability (MSI) in the DNA sample including: a) co amplifying a plurality of microsatellite loci of the DNA sample to produce amplified fragments comprising nucleic acid sequences (e.g., DNA fragments) from each locus; b) determining the size of the amplified fragments from each locus; c) comparing the size of the amplified fragments from each locus to the size of corresponding amplified fragments from a control, a difference in size between one or more amplified fragments between the sample and the control indicating the presence of MSI in the DNA sample; and d) assigning a degree of MSI to the DNA sample, thereby determining the MSI status of the DNA sample.
  • MSI microsatellite instability
  • the plurality of loci includes at least one locus selected from BAT25, BAT 26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • a method for diagnosing the presence of cancerous tissue in a biological sample including: a) co-amplifying a plurality of microsatellite loci of the DNA sample to produce amplified fragments comprising nucleic acid sequences (e.g., DNA fragments) from each locus; b) determining the size of the amplified fragments from each locus; and c) comparing the size of the amplified fragments from each locus to the size of corresponding amplified fragments from a control.
  • the plurality of loci includes at least one locus selected from BAT25, BAT 26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • a difference in size between one or more amplified fragments between the sample and the control indicates the presence of microsatellite instability (MSI) at the corresponding locus in the DNA sample, wherein the presence of MSI in the DNA sample indicates that the biological sample contains cancerous tissue.
  • MSI microsatellite instability
  • the method further includes assigning a degree of MSI to the
  • the DNA sample is assigned a high degree of MSI (microsatellite instability high) if more than about 30% of the loci in the DNA sample are determined to have MSI. In embodiments, the DNA sample is assigned a low degree of MSI ((microsatellite instability low) if less than about 30% but more than about 1% of the loci in the DNA sample are determined to have MSI. In embodiments, the DNA sample is assigned a stable degree (microsatellite stable) if none of the loci in the DNA sample are determined to have MSI.
  • a method for diagnosing cancer in a subject having or suspected of having cancer including: a) co-amplifying a plurality of microsatellite loci of the DNA sample to produce amplified fragments comprising nucleic acid sequences (e.g., DNA fragments) from each locus; b) determining the size of the amplified fragments from each locus; and c) comparing the size of the amplified fragments from each locus to the size of corresponding amplified fragments from a control, a difference in size between one or more amplified fragments between the sample and the control indicating the presence of microsatellite instability (MSI) at the corresponding locus in the DNA sample.
  • MSI microsatellite instability
  • the presence of MSI in the DNA sample indicates that the subject has cancer.
  • the plurality of loci includes at least one locus selected from BAT25, BAT 26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI- 19.
  • a method for treating cancer in a subject having or suspected of having cancer including: a) co-amplifying a plurality of microsatellite loci of the DNA sample to produce amplified fragments comprising nucleic acid sequences (e.g., DNA fragments) from each locus; b) determining the size of the amplified fragments from each locus; c) comparing the size of the amplified fragments from each locus to the size of corresponding amplified fragments from a control; and d) administering to the subject having cancer an anti-cancer agent.
  • nucleic acid sequences e.g., DNA fragments
  • a difference in size between one or more amplified fragments between the samples indicates the presence of microsatellite instability (MSI) in the DNA sample.
  • MSI microsatellite instability
  • the presence of MSI in the DNA sample indicates that the subject has cancer.
  • the plurality of loci includes at least one locus selected from BAT25, BAT 26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • a method for analyzing amplified fragments comprising nucleic acid sequences (e.g., DNA fragments) to determine microsatellite instability (MSI) including: a) providing amplified fragments comprising nucleic acid sequences (e.g., DNA fragments) amplified from a plurality of microsatellite loci in a DNA sample; b) determining the size of the amplified fragments; c) comparing the size of the amplified fragments of step b) to the size of corresponding amplified fragments from a paired normal DNA sample, a difference in size between one or more fragments between the samples indicating the presence of MSI in the DNA sample; and d) assigning a degree of MSI to the DNA sample, thereby determining the MSI status of the DNA sample.
  • MSI microsatellite instability
  • the amplified fragments include a modification.
  • the modification includes a detectable marker.
  • the plurality of loci includes at least one locus selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-
  • the method further includes co- amplifying one or more identification markers in step a).
  • the one or more identification markers include PENTAD and/or TH01.
  • the plurality of loci include at least two, three, four, five, six, or seven loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19. In embodiments, the plurality of loci include at least eight, nine, ten, eleven, or twelve loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • the plurality of loci includes each of the following loci: BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI- 19.
  • the DNA sample is from tumor cells, cells suspected of being cancerous, or other biological material suspected of being cancerous.
  • the control is a paired normal DNA sample, a DNA sample from a non-cancerous tissue, DNA from a blood sample, an average based on a normal (non-cancerous) population, or a median based on a normal (non-cancerous) population.
  • the microsatellite loci are co-amplified using one or more primers selected from SEQ ID NOS.: 1-26.
  • the microsatellite loci are co amplified using a primer pair including a first primer and a second primer, wherein polynucleotide sequences of the first primer and the second primer include one of the following pairs: the polynucleotide sequence of SEQ ID NO.: 1 and the polynucleotide sequence of SEQ ID NO.: 2; the polynucleotide sequence of SEQ ID NO.: 3 and the polynucleotide sequence of SEQ ID NO.: 4; the polynucleotide sequence of SEQ ID NO.: 5 and the polynucleotide sequence of SEQ ID NO.: 6; the polynucleotide sequence of SEQ ID NO.: 7 and the polynucleotide sequence of SEQ ID NO.: 8; the polynucleotide sequence of SEQ
  • the plurality of microsatellite loci is amplified using thermal cycling, and analyzed via fragment analysis, Sanger sequencing, ion semiconductor sequencing, or high-resolution melt curve analysis.
  • the Sanger sequencing is capillary electrophoresis Sanger sequencing.
  • the DNA sample and the paired normal DNA sample are from the same individual.
  • the paired normal DNA sample is a control DNA from a non-cancerous tissue.
  • the DNA sample and the paired normal DNA sample are from the same type of tissue.
  • the cancer is colorectal cancer, gastric cancer, adrenocortical carcinoma, cervical cancer, mesothelioma, or endometrial cancer.
  • each locus is amplified using a primer pair, and further wherein at least one primer of the primer pair includes a modification.
  • the modification includes a detectable marker.
  • a primer set including one or more primer pairs, each primer pair including a first primer and a second primer, wherein polynucleotide sequences of the first primer and the second primer include one of the following pairs: the polynucleotide sequence of SEQ ID NO.: 1 and the polynucleotide sequence of SEQ ID NO.: 2; the polynucleotide sequence of SEQ ID NO.: 3 and the polynucleotide sequence of SEQ ID NO.: 4; the polynucleotide sequence of SEQ ID NO.: 5 and the polynucleotide sequence of SEQ ID NO.: 6; the polynucleotide sequence of SEQ ID NO.: 7 and the polynucleotide sequence of SEQ ID NO.: 8; the polynucleotide sequence of SEQ ID NO.: 9 and the polynucleotide sequence of SEQ ID NO.: 10; the polynucleotide sequence of SEQ ID NO.:
  • a composition including a primer set of any one of the previously described primer sets, including embodiments.
  • the primer set includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 primer pairs.
  • the composition includes 13 primer pairs including the polynucleotide sequences of SEQ ID NOs.: 1-26.
  • at least one primer is modified.
  • the modification includes a detectable label.
  • the composition further includes a polymerase.
  • the composition further includes a plurality of deoxyribonucleotide triphosphates.
  • the composition further includes a DNA sample.
  • the composition further includes one or more salts.
  • a system including the composition as previously described, including embodiments, and a first device configured to perform DNA amplification.
  • the first device is configured to perform Sanger sequencing, ion semiconductor sequencing, capillary electrophoresis, or high-resolution melt analysis.
  • the system further includes a second device configured to compare and/or analyze nucleic acid fragments resulting from amplification of DNA with the primers.
  • kits including a buffer and at least one primer pair for amplification of a microsatellite locus, the primer pair including a first primer and a second primer.
  • the microsatellite locus includes BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and/or ABI-19.
  • the at least one primer pair includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12, or at least 13 primer pairs.
  • the at least one primer pair includes at least one primer having the polynucleotide sequence of any one of SEQ ID NOs.: 1-26. In embodiments, the at least one primer pair includes 13 primer pairs, the primers having the polynucleotide sequence of each of SEQ ID NOs.: 1-26. In embodiments, the kit further includes a primer pair for amplification of an identification marker. In embodiments, the identification marker includes PENTAD and/or TH01.
  • polynucleotide sequences of the first primer and the second primer include one of the following pairs: the polynucleotide sequence of SEQ ID NO.: 1 and the polynucleotide sequence of SEQ ID NO.: 2; the polynucleotide sequence of SEQ ID NO.: 3 and the polynucleotide sequence of SEQ ID NO.: 4; the polynucleotide sequence of SEQ ID NO.: 5 and the polynucleotide sequence of SEQ ID NO.: 6; the polynucleotide sequence of SEQ ID NO.: 7 and the polynucleotide sequence of SEQ ID NO.: 8; the polynucleotide sequence of SEQ ID NO.: 9 and the polynucleotide sequence of SEQ ID NO.: 10; the polynucleotide sequence of SEQ ID NO.: 11 and the polynucleotide sequence of SEQ ID NO.: 12; the polynucleot
  • the kit includes primers including the polynucleotide sequence of each of SEQ ID NO.: 1-26.
  • the kit further includes a polymerase and/or a plurality of deoxynucleotide triphosphates.
  • the buffer is a PCR buffer.
  • at least one primer from the primer set includes a modification.
  • the modification is a detectable label.
  • the kit further includes a computer program for identification of microsatellite instability in a biological sample.
  • Embodiments of the invention used to detect microsatellite instability in a biological sample are disclosed.
  • Signal data is received from a capillary electrophoresis genetic analysis instrument, wherein the signal data is measured from fluorescence of fragments comprising nucleic acid sequences amplified from the biological sample via polymerase chain reaction.
  • the nucleic acid sequences correspond to a plurality of different microsatellite loci. Different loci can exhibit different signal characteristics. At a particular locus, a hierarchy of analysis methods can be applied that may also be peculiar to the characteristics of the signal data at that locus.
  • a three- level hierarchy could be described as follows: A first processing algorithm is implemented to obtain a first determination, based on the signal data, regarding instability of one or more first microsatellite loci of the plurality of different microsatellite loci. A second processing algorithm is implemented to obtain a second determination, based on the signal data, regarding instability of one or more second microsatellite loci of the plurality of different microsatellite loci. A third processing algorithm is then implemented to measure microsatellite instability of the biological sample based on at least the first determination and the second determination ⁇
  • Embodiments of the invention describe a collection of ways to analyze the CE data to determine whether a given DNA locus is abnormal and to determine whether the overall genetic profile, combining results from all loci, can be considered MSI high, MSI low, or MSS.
  • the methods described herein provide a means to automatically make the calls.
  • the methods described can also be used to assign a confidence metric to the calls, for example, by reporting the proximity of calculated results to decision thresholds, which, in turn, can be used to focus human review efforts on those cases where the automated MSI assessment is less confident.
  • Embodiments of the present invention disclosed herein describe a heterogeneous approach to the analysis ranging from simple thresholds up to utilizing deep learning technologies.
  • the reason for this is that assigning the overall genetic profile to MSI high, MSI low or MSI stable can involve one locus of microsatellites in the DNA up to many loci of microsatellites in the DNA.
  • the complexity of analysis algorithms depends on the nature of DNA replication patterns at the loci chosen. Different loci might be chosen for different cancers since some may be more sensitive to a given cancer type compared to other cancer types and/or, in combination with other loci, may yield a more sensitive and/or specific test for MSI status, and/or the DNA may be more reliably amplified.
  • FIG. 1 illustrates a system in accordance with an embodiment of the present invention
  • FIG. 2 illustrates an exemplary capillary electrophoresis process used in some embodiments of the present invention
  • FIG. 3 illustrates an exemplary genetic analyzer instrument used in some embodiments of the present invention
  • FIG. 4 illustrates an exemplary all-in-one cartridge used in the exemplary genetic analyzer instrument of FIG. 3;
  • FIG. 5 illustrates four exemplary screenshots of user interface displays used in some embodiments of the present invention
  • FIG. 6 illustrates a flow diagram depicting a cloud integration process of the exemplary genetic analyzer instrument of FIG. 3;
  • FIG. 7 illustrates a flow diagram of a method according to some embodiments of the present invention.
  • FIG. 8 illustrates a flow diagram of alternate methods according to some embodiments of the present invention.
  • FIG. 9 illustrates a block diagram of a distributed computer system that can implement one or more aspects of an embodiment of the invention.
  • FIG. 10 illustrates a block diagram of an electronic device that can implement one or more aspects of an embodiment of the invention.
  • FIG. 11 shows a representative electropherogram of the 15-plex assay on DNA samples from colorectal carcinoma (left) or normal tissue (right), with the markers being spaced across the channels.
  • Top row shows results from amplification of BAT25, NR24 and NR21 loci.
  • Second row shows results from amplification of TH01, BAT40, and CAT25 loci.
  • Third row shows results from amplification of NR22, NR27, ABI19, and ABI20B loci.
  • Fourth row shows results from amplification of PentaD locus.
  • Bottom row shows results from amplification of ABI17, ABI16, BAT26, and ABI20A loci. Loci are indicated by the gray bars over each trace, with divisions between each locus’ trace indicated by red triangles on the X axis.
  • FIG. 12A is a table comparing the results of an MSI assay as described herein with a competitor (ProMega) MSI assay when ran on endometrial carcinoma samples.
  • FIG. 12B shows trace profiles showing the clear shifts across the different types of markers: 20bp (ABI-20A & ABI-20B) vs 40bp (BAT-40) homopolymers.
  • FIG. 13 shows synthetic constructs revealing detection complexities.
  • FIG. 14 is a graph of tumor-only analysis with >98% specificity and >90% sensitivity at 5 bpd.
  • FIG. 15 is a graph of tumor-normal analysis with >95% specificity and sensitivity at > 3bdp.
  • FIG. 16 is an ABI MSI software illustration of automatic calls and reports comparing normal versus tumor colon samples at BAT-25, NR-24, and NR-21 (top) and all 15 loci (bottom).
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas.
  • exemplary cancers that may be treated with a compound or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and Non-Hodgkin's Lymphomas.
  • Exemplary cancers that may be treated with a compound or method provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus.
  • Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract
  • leukemia refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood- leukemic or aleukemic (subleukemic).
  • Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy- cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia,
  • lymphoma refers to a group of cancers affecting hematopoietic and lymphoid tissues. It begins in lymphocytes, the blood cells that are found primarily in lymph nodes, spleen, thymus, and bone marrow. Two main types of lymphoma are non-Hodgkin lymphoma and Hodgkin’s disease. Hodgkin’s disease represents approximately 15% of all diagnosed lymphomas. This is a cancer associated with Reed- Stemberg malignant B lymphocytes. Non-Hodgkin’s lymphomas (NHL) can be classified based on the rate at which cancer grows and the type of cells involved.
  • B-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, small lymphocytic lymphoma, Mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large cell B-lymphoma, Burkitt’s lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B-lymphoblastic lymphoma.
  • T-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, cunateous T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sar
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding- Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid
  • the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. “Metastatic cancer” is also called “Stage IV cancer.” Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body.
  • a second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor.
  • the metastatic tumor and its cells are presumed to be similar to those of the original tumor.
  • the secondary tumor in the breast is referred to a metastatic lung cancer.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
  • non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors.
  • metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.
  • treating refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation.
  • the term "treating" and conjugations thereof, may include prevention of an injury, pathology, condition, or disease.
  • treating is preventing.
  • treating does not include preventing.
  • Treating” or “treatment” as used herein also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (/. ⁇ ? ., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
  • treatment includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.
  • “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
  • a patient is human.
  • An “effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition).
  • an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • a “reduction” of a symptom or symptoms means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • a “prophylactic ally effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.
  • An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist.
  • a “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). [0066]
  • the term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above.
  • a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as “-fold” increase or decrease.
  • a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5- fold, or more effect over a control.
  • Dosages may be varied depending upon the requirements of the patient and the compound being employed.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • administering means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • the administering does not include administration of any active agent other than the recited active agent.
  • nucleic acid As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,”
  • nucleic acid oligomer oligonucleotide
  • nucleic acid sequence oligonucleotide
  • polynucleotide polynucleotide
  • nucleotides covalently linked together may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof.
  • Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer.
  • Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
  • a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • T thymine
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleo
  • amplified DNA fragments or “amplified fragments comprising nucleic acid sequences” refers to polynucleotide sequences that were produced by an in vitro amplification method, for example Polymerase Chain Reaction (PCR), isothermal amplification, strand displacement amplification, or any other DNA amplification method.
  • PCR Polymerase Chain Reaction
  • microsatellite locus or “microsatellite loci” refers to loci in the genome of an organism that contain a microsatellite.
  • a “microsatellite” is a tract of repetitive DNA in which certain DNA motifs (generally from one to six or more base pairs) are repeated, typically 5-50 times. Microsatellites occur at thousands of locations within an organism's genome.
  • identification marker refers to a locus that can be used to identify a DNA sample.
  • Identification markers can be any locus that has allelic variability in a population, such that the allelic variability can be differentiated by the amplification used.
  • identification markers are short tandem repeat (STR) polymorphisms, and these polymorphisms can be differentiated based on length of the STR.
  • an identification marker may be any locus used in forensic DNA profiling, parentage determination, or similar methods.
  • identification markers include, without limitation, Amelogenin, CSF1PO, D13S317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA, PentaD, PentaE, TH01, TPOX, and vWA.
  • the identification marker is a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • the length of fragments comprising nucleic acid sequences amplified from the identification marker does not change or shows a lesser degree of change in cancers exhibiting MSI. Detection of MSI
  • MSI neoplastic inherited syndrome
  • MSI status is used as a predictive biomarker for cancer immunotherapy.
  • Microsatellite instability due to inherited germline mutations of mismatch repair genes or epigenetic inactivation of these genes, is found in many cancer types at varying frequencies.
  • the tumors with the highest rates of MSI include uterine corpus endometrial carcinoma, colorectal adenocarcinoma, and stomach adenocarcinoma (Cortes-Ciriano et al, 2017).
  • Lynch syndrome is the most common inherited colorectal cancer (CRC) susceptibility syndrome. It accounts for approximately 3-5% of newly diagnosed causes of CRCs and 2-3% of endometrial cancers.
  • CRC colorectal cancer
  • the American Society of Clinical Oncology recommends that tumor testing for Lynch syndrome be performed in all people diagnosed with colorectal cancer. Recent guidelines recommend tumor testing for all endometrial cancers as well. Screening tests can be performed on tumor tissue to determine if Lynch syndrome is likely.
  • One way to test is analyzing MSI. The result of MSI testing can indicate whether more specific genetic testing should be considered.
  • Fragment analysis has frequently been used to assess MSI status. Fragment analysis applications are those in which fluorescent fragments of DNA are produced by PCR using dye-labelled primers designed for a specific interrogation task. These fragments are then separated using capillary electrophoresis and sized by comparison to a size standard. MSI analysis via fragment analysis follows this same paradigm; PCR amplification of the microsatellite loci of interest using fluorescently labeled primers. The labeled PCR products are then analyzed by capillary electrophoresis (CE) or electrophoresis to separate the alleles by size.
  • CE capillary electrophoresis
  • CE fragment analysis There are possible alternatives to using CE fragment analysis.
  • sequencing technologies can be used to sequence the DNA loci of interest and, through sequence analysis (e.g., counting the number of microsatellites in the sequence), assign MSI status.
  • sequence analysis e.g., counting the number of microsatellites in the sequence
  • assign MSI status e.g., assign MSI status.
  • using sequencing technologies or similar approaches other than CE fragment analysis may be disadvantageous.
  • DNA sequence analysis has a limited ability to multiplex data.
  • the process of DNA sequence analysis takes longer, and the analysis may be more error prone.
  • a method for detecting microsatellite instability (MSI) in a DNA sample is provided.
  • a method for analyzing a DNA sample to determine microsatellite instability (MSI) in the DNA sample is provided.
  • a method for diagnosing the presence of cancerous tissue in a biological sample is provided.
  • a method for treating cancer in a subject having or suspected of having cancer is provided.
  • a method for diagnosing cancer in a subject having or suspected of having cancer is provided
  • the method includes: a) co-amplifying a plurality of microsatellite loci of the DNA sample to produce amplified fragments comprising nucleic acid sequences from each locus; b) determining the size of the amplified fragments from each locus; and c) comparing the size of the amplified fragments from each locus to the size of corresponding amplified fragments from a control.
  • the plurality of loci includes at least one locus selected from BAT25, BAT 26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • a difference in size between one or more amplified fragments between the sample and the control indicates the presence of MSI at the corresponding locus in the DNA sample or biological sample.
  • a method for analyzing amplified fragments comprising nucleic acid sequences to determine microsatellite instability is provided, the method including: a) providing amplified fragments amplified from a plurality of microsatellite loci in a DNA sample or biological sample; b) determining the size of the amplified fragments; c) comparing the size of the amplified fragments of step b) to the size of corresponding amplified fragments from a paired normal DNA sample or paired normal biological sample, a difference in size between one or more amplified fragments between the samples indicating the presence of MSI in the DNA sample; and d) assigning a degree of MSI to the DNA sample, thereby determining the MSI status of the DNA sample.
  • a difference in size between one or more amplified fragments between the sample and the control indicates the presence of microsatellite instability (MSI) at the corresponding locus in the DNA sample.
  • MSI microsatellite instability
  • the presence of MSI in the DNA sample indicates that the biological sample from which the DNA sample was derived contains cancerous tissue.
  • the method further includes assigning a degree of MSI to the
  • the DNA sample is assigned a high degree of MSI (microsatellite instability high) if more than about 30% of the loci in the DNA sample are determined to have MSI. In embodiments, the DNA sample is assigned a low degree of MSI (microsatellite instability low) if less than about 30% but more than about 1% of the loci in the DNA sample are determined to have MSI. In embodiments, the DNA sample is assigned a stable degree (microsatellite stable) if none of the loci in the DNA sample are determined to have MSI.
  • a DNA sample where at least 4 (or at least 3) loci are determined to have MSI is assigned a high degree of MSI (i.e., is MSI-high); a DNA sample where at least 1 but less than 4 (or less than 3) loci are determined to have MSI is assigned a low degree of MSI (i.e., is MSI-low); and a DNA sample where 0 loci are determined to have MSI is assigned a stable degree of MSI (i.e., is MSI-stable).
  • the presence of MSI in the DNA sample indicates that the subject has cancer.
  • the method includes administering to the subject having cancer an anti-cancer agent.
  • a primer used to amplify a locus includes a modification.
  • each locus is amplified using a primer pair, and at least one primer of the primer pair includes a modification.
  • the modification may be migration modifiers such as poly ethylene glycol (PEG), locked nucleic acids (LNA), 3'-minor groove binders or the addition of non-specific nucleic acids to one end of the primer sequence.
  • the amplified fragments include a modification.
  • the modification includes a detectable marker. Detectable markers include, without limitation, fluorescent markers/dyes and radioactive markers. Fluorescent markers/dyes are well known in the art, and include, without limitation, fluorescein, FAM (carboxyfluorescein) (e.g., 5-FAM, 6-FAM), TET, VIC
  • LC Red 640 LC Red 705, SID, TAZ, YY, rhodamine (or derivatives), coumarin (or derivatives), and cyanine (or derivatives, e.g. Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7), or derivatives of any of these molecules.
  • cyanine or derivatives, e.g. Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, or derivatives of any of these molecules.
  • the method further includes co- amplifying one or more identification markers in step a).
  • the one or more identification markers include Amelogenin, CSF1PO, D13S317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA, PentaD, PentaE, TH01, TPOX, and/or vWA.
  • the one or more identification markers include PENTAD.
  • the one or more identification markers include TH01.
  • the one or more identification markers include PENTAD and TH01.
  • the term “comparing the size of’ refers to comparison of the size or size distribution of nucleic acid fragments. Comparison can be performed by any method, such as gel electrophoresis, capillary electrophoresis, DNA sequencing, microscopic visualization (e.g., adsorption grid electron microscopic visualization or Kleinschmidt Electron Microscopic visualization), and the like. Comparison may be performed, for example, by visualization of the fragment size, and/or by a computer-implemented program.
  • the plurality of loci include at least 2 loci selected from
  • the plurality of loci include at least 3 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • the plurality of loci include at least 4 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19. In embodiments, the plurality of loci include at least 5 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • the plurality of loci include at least 6 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19. In embodiments, the plurality of loci include at least 7 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • the plurality of loci include at least 8 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19. In embodiments, the plurality of loci include at least 9 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI-19.
  • the plurality of loci include at least 10 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI- 16, ABI-20B and ABI-19. In embodiments, the plurality of loci include at least 11 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI- 16, ABI-20B and ABI-19.
  • the plurality of loci include at least 12 loci selected from BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI- 16, ABI-20B and ABI-19.
  • the plurality of loci includes BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and ABI- 19.
  • the plurality of loci includes BAT25. In embodiments, the plurality of loci includes BAT26. In embodiments, the plurality of loci includes BAT40. In embodiments, the plurality of loci includes CAT25. In embodiments, the plurality of loci includes NR21. In embodiments, the plurality of loci includes NR22. In embodiments, the plurality of loci includes NR24. In embodiments, the plurality of loci includes NR27. In embodiments, the plurality of loci includes ABI-20A. In embodiments, the plurality of loci includes ABI-17. In embodiments, the plurality of loci includes ABI-16. In embodiments, the plurality of loci includes ABI-20B.
  • the plurality of loci includes ABI-19.
  • the DNA sample is from a biological sample.
  • the biological sample includes tumor cells, cells suspected of being cancerous, or other biological material suspected of being cancerous or from a cancer cell.
  • control is a paired normal DNA sample, a DNA sample from a non-cancerous tissue, DNA from a blood sample, an average based on a normal population, or a median based on a normal population.
  • normal refers to a DNA sample from non-cancerous tissue.
  • normal refers to a DNA sample from non-cancerous tissue adjacent to cancerous tissue.
  • the microsatellite loci are co-amplified using one or more primers selected from SEQ ID NOS.: 1-26. In embodiments, the microsatellite loci are co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 1. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 2. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 3.
  • the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 4. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 5. In embodiments, the microsatellite loci are co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 6. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 7.
  • the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 8. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 9. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 10. In embodiments, the microsatellite loci are co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 11.
  • the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 12. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 13. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 14. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 15.
  • the microsatellite loci are co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 16. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 17. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 18. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 19.
  • the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 20. In embodiments, the microsatellite loci are co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 21. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 22. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 23.
  • the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 24. In embodiments, the microsatellite loci are co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 25. In embodiments, the microsatellite loci are co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 26.
  • the microsatellite loci are co-amplified using a primer pair including a first primer and a second primer.
  • the polynucleotide sequences of the first primer and the second primer include one of the following pairs (respectively): the polynucleotide sequence of SEQ ID NO.: 1 and the polynucleotide sequence of SEQ ID NO.: 2; the polynucleotide sequence of SEQ ID NO.: 3 and the polynucleotide sequence of SEQ ID NO.: 4; the polynucleotide sequence of SEQ ID NO.: 5 and the polynucleotide sequence of SEQ ID NO.: 6; the polynucleotide sequence of SEQ ID NO.: 7 and the polynucleotide sequence of SEQ ID NO.: 8; the polynucleotide sequence of SEQ ID NO.: 9 and the polynucleotide sequence of SEQ ID NO.: 10; the polyn
  • the identification marker(s) is co-amplified using one or more primers selected from SEQ ID NOS.: 27-31. In embodiments, the identification marker(s) is co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 27. In embodiments, the identification marker(s) is co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 28. In embodiments, the identification marker(s) is co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 29. In embodiments, the identification marker(s) is co-amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 30. In embodiments, the identification marker(s) is co amplified using a primer containing the nucleotide sequence of SEQ ID NO.: 31.
  • the plurality of microsatellite loci is amplified using thermal cycling.
  • the resulting nucleic acid fragments are analyzed via fragment analysis, Sanger sequencing, ion semiconductor sequencing, or high-resolution melt curve analysis.
  • the Sanger sequencing is capillary electrophoresis Sanger sequencing.
  • the DNA sample and the paired normal DNA sample are from the same individual.
  • the paired normal DNA sample is a control DNA from a non-cancerous tissue.
  • the DNA sample and the paired normal DNA sample are from the same type of tissue.
  • the cancer can be any type of cancer.
  • the cancer can be any cancer that is associated with MSI.
  • the cancer is colorectal cancer, gastric cancer, adrenocortical carcinoma, cervical cancer, mesothelioma, or endometrial cancer.
  • capillary electrophoresis can be used to measure the number of microsatellites by using fragment analysis.
  • Automated CE uses fluorescent dyes and separates with higher resolution and higher accuracy than other methods such as agarose or polyacrylamide gel electrophoresis.
  • probes and primers can be designed to flank a region of interest. This can be done by attaching fluorescent dyes to primers or probes used with the polymerase chain reaction (PCR) to amplify a DNA locus of interest before the electrophoresis and submitting the amplicons to CE.
  • PCR polymerase chain reaction
  • There is also a sizing standard a collection of fragments of known sizes labelled with a color that is different than the colors of the test fragments. The labelled PCR products and the sizing standard are then electrokinetically injected into the capillaries. During electrophoresis, the negatively charged DNA fragments moves from the cathode, through the polymer-filled capillary towards the positively charged anode when high voltage is applied between the electrodes.
  • DNA fragment analysis using CE can be multiplexed, meaning there are multiple fragments in a reaction well going through the same capillary. The smaller fragments usually run faster, and the bigger ones run slower. Shortly before reaching the positive electrode, the fluorescently labelled DNA fragments, separated by size, move through the path of a laser beam. The laser beam causes the dyes on the fragments to fluoresce at different emission wavelengths. A CCD camera detects the fluorescence, and the fluorescence intensities are digitalized, color-coded and displayed as peaks in the electropherogram. Longer fragments will occur later in the data relative to shorter fragments.
  • a primer set may include one or more primer pairs, each primer pair including a first primer and a second primer (e.g., forward and reverse primers).
  • the polynucleotide sequences of the first primer and the second primer include one of the following pairs: the polynucleotide sequence of SEQ ID NO.: 1 and the polynucleotide sequence of SEQ ID NO.: 2; the polynucleotide sequence of SEQ ID NO.: 3 and the polynucleotide sequence of SEQ ID NO.: 4; the polynucleotide sequence of SEQ ID NO.: 5 and the polynucleotide sequence of SEQ ID NO.: 6; the polynucleotide sequence of SEQ ID NO.: 7 and the polynucleotide sequence of SEQ ID NO.: 8; the polynucleotide sequence of SEQ ID NO.: 9 and the polynucleotide sequence of SEQ ID NO.
  • a composition including a primer set of any one of the previously described primer sets, including embodiments.
  • the primer set includes at least 2 primer pairs.
  • the primer set includes at least 3 primer pairs.
  • the primer set includes at least 4 primer pairs.
  • the primer set includes at least 5 primer pairs.
  • the primer set includes at least 6 primer pairs.
  • the primer set includes at least 7 primer pairs.
  • the primer set includes at least 8 primer pairs.
  • the primer set includes at least 9 primer pairs.
  • the primer set includes at least 10 primer pairs.
  • the primer set includes at least 11 primer pairs.
  • the primer set includes at least 12 primer pairs.
  • the composition includes at least 13 primer pairs.
  • the primer pairs include the polynucleotide sequence of one or more of SEQ ID NOs.: 1-26. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 1. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 2. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 3. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 4. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 5. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 6.
  • the primer pairs include the polynucleotide sequence of SEQ ID NO.: 7. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 8. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 9. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 10. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 11. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 12. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 13.
  • the primer pairs include the polynucleotide sequence of SEQ ID NO.: 14. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 15. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 16. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 17. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 18. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 19. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 20.
  • the primer pairs include the polynucleotide sequence of SEQ ID NO.: 21. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 22. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 23. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 24. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 25. In embodiments, the primer pairs include the polynucleotide sequence of SEQ ID NO.: 26.
  • the primer set further includes a primer pair for amplification of an identification marker.
  • the identification marker includes PENTAD and/or TH01.
  • the identification marker includes TH01.
  • the identification marker includes PENTAD and TH01.
  • the primer pair for amplification of PENTAD comprises primers including the polynucleotide sequence of one or more of SEQ ID NO.: 27-29.
  • the primer pair for amplification of TH01 comprises primers including the polynucleotide sequence of one or more of SEQ ID NO.: 30-
  • the primers include a primer containing the nucleotide sequence of SEQ ID NO.: 27. In embodiments, the primers include a primer containing the nucleotide sequence of SEQ ID NO.: 28. In embodiments, the primers include a primer containing the nucleotide sequence of SEQ ID NO.: 29. In embodiments, the primers include a primer containing the nucleotide sequence of SEQ ID NO.: 30. In embodiments, the primers include using a primer containing the nucleotide sequence of SEQ ID NO.: 31.
  • At least one primer is modified.
  • the modification includes a detectable label.
  • the composition further includes a polymerase.
  • the composition further includes a plurality of deoxyribonucleotide triphosphates.
  • the composition further includes a DNA sample.
  • the composition further includes one or more salts.
  • kits including a buffer and a primer set including at least one primer pair for amplification of a microsatellite locus, the primer pair including a first primer and a second primer.
  • the microsatellite locus includes BAT25, BAT26, BAT40, CAT25, NR21, NR22, NR24, NR27, ABI-20A, ABI-17, ABI-16, ABI-20B and/or ABI-19.
  • the primer set includes at least 2 primer pairs. In embodiments, the primer set includes at least 3 primer pairs. In embodiments, the primer set includes at least 4 primer pairs. In embodiments, the primer set includes at least 5 primer pairs. In embodiments, the primer set includes at least 6 primer pairs. In embodiments, the primer set includes at least 7 primer pairs. In embodiments, the primer set includes at least 8 primer pairs. In embodiments, the primer set includes at least 9 primer pairs. In embodiments, the primer set includes at least 10 primer pairs. In embodiments, the primer set includes at least 11 primer pairs. In embodiments, the primer set includes at least 12 primer pairs. In embodiments, the primer set includes at least 13 primer pairs. In embodiments, the primer set includes at least 14 primer pairs.
  • the primer set includes at least 14 primer pairs. In embodiments, the primer set includes at least one primer having the polynucleotide sequence of any one of SEQ ID NOs.: 1-26. In embodiments, the primer set includes 13 primer pairs, the primers having the polynucleotide sequence of each of SEQ ID NOs.: 1-26. In embodiments, the primer set is a primer set as described above.
  • the primer set further includes a primer pair for amplification of an identification marker.
  • the identification marker includes PENTAD.
  • the identification marker includes TH01.
  • the identification marker includes PENTAD and TH01.
  • the primer set includes primers having the polynucleotide sequence of any one or more of SEQ ID NOs.: 27-31.
  • polynucleotide sequences of the first primer and the second primer include one of the following pairs: the polynucleotide sequence of SEQ ID NO.: 1 and the polynucleotide sequence of SEQ ID NO.: 2; the polynucleotide sequence of SEQ ID NO.: 3 and the polynucleotide sequence of SEQ ID NO.: 4; the polynucleotide sequence of SEQ ID NO.: 5 and the polynucleotide sequence of SEQ ID NO.: 6; the polynucleotide sequence of SEQ ID NO.: 7 and the polynucleotide sequence of SEQ ID NO.: 8; the polynucleotide sequence of SEQ ID NO.: 9 and the polynucleotide sequence of SEQ ID NO.: 10; the polynucleotide sequence of SEQ ID NO.: 11 and the polynucleotide sequence of SEQ ID NO.: 12; the polynucleot
  • all primers of the primer set are present in the same container.
  • the kit further includes a polymerase. In embodiments, the kit further includes a plurality of deoxy nucleotide triphosphates. In embodiments, the buffer is a PCR buffer. In embodiments, the kit further includes a computer program for identification of microsatellite instability in a biological sample.
  • a system including a composition as previously described, including embodiments, and a first device configured to perform DNA amplification.
  • the first device is configured to perform Sanger sequencing, ion semiconductor sequencing, capillary electrophoresis, or high-resolution melt analysis.
  • the system further includes a second device configured to compare and/or analyze nucleic acid fragments resulting from amplification of DNA with the primers.
  • a system including a composition as previously described, including embodiments, and a device configured to compare and/or analyze nucleic acid fragments resulting from amplification of DNA with the primers.
  • FIG. 1 illustrates system 1000 in accordance with an exemplary embodiment of the present invention.
  • the DNA fragment analysis processes set forth in embodiments of the present invention start with extracting the DNA from a tissue sample.
  • a plurality of microsatellite DNA loci of interest in one or more nucleic acid samples 111 under investigation may be amplified in a PCR reaction performed in amplification instrument 112 such as a thermal cycler.
  • amplification instrument 112 such as a thermal cycler.
  • Exemplary thermal cycle instruments used in some embodiments of the present invention include the Applied Biosystems ProFlex PCR System, the SimpliAmp Thermal Cycler, and other thermal cycler systems manufactured by Thermo Fisher Scientific.
  • System 1000 comprises capillary electrophoresis based genetic analyzer instrument (e.g. a sequencing instrument) 101, one or more computers 103, and user device 108.
  • Exemplary genetic analyzer instruments used in some embodiments of the present invention include the Applied Biosystems SeqStudio Genetic Analyzer by Thermo Fisher Scientific, Models 3500, 3720, and 3130 and similar capillary electrophoresis-based genetic analyzers manufactured by Thermo Fisher Scientific and others.
  • capillary electrophoresis is a process 200 used to separate ionic fragments by size.
  • an electrokinetic injection is used to inject DNA fragments from solution and into each capillary of a capillary array 201 comprising one or more capillaries.
  • the extension products of the PCR reaction and any other negatively charged molecules such as salt or unincorporated primers and nucleotides
  • a high voltage charge applied to the sample forces the negatively charged fragments into the capillaries.
  • the extension products are separated by size based on their total charge.
  • the electrophoretic mobility of the sample can be affected by the ran conditions: the buffer type, concentration, and pH; the ran temperature; the amount of voltage applied; and the type of polymer used.
  • the fluorescently labeled DNA fragments move across the path of a laser beam 202.
  • the laser beam causes the dyes attached to the fragments to fluoresce.
  • the dye signals are separated by a diffraction system 203, and a CCD camera detects the fluorescence as shown in 204. Because each dye emits light at a different wavelength when excited by the laser, all colors, and therefore loci, can be detected and distinguished in one capillary injection.
  • the fluorescence signal is converted into digital data, then the data is stored in a file format compatible with an analysis software application.
  • the data coming out of the CE instrumentation is a series of fluorescent peaks instead of a single peak at the exact size of the amplicon expected for a given number of microsatellites. This is caused by nuances in the amplification of the DNA of interest; “stutter” of the biomolecular machinery involved can result in generating amplicons with a few more or a few less microsatellites in the amplicons than the true number of microsatellites. As a result of this “stutter”, there can be some uncertainty in determining the number of microsatellites and/or in determining whether the number of microsatellites differs from the number expected in normal, non-cancerous tissue.
  • a single dye may be used with several different PCR primers that target different DNA loci. This is done because the instrumentation imposes limitations on the number of different dyes that can be used, and the number of DNA loci of interest may exceed the maximum number of dyes that can be used. If the amplicon sizes are sufficiently different between a group of loci for which the same dye is used on their respective PCR primers, the fluorescent peaks associated with each of the loci would be well separated in the data generated by the CE instrument.
  • FIG. 1 reside in computer program product 104 which is stored in storage 105 and those instructions are executable by processor 106.
  • processor 106 When processor 106 is executing the instructions of computer program product 104, the instructions, or a portion thereof, are typically loaded into working memory 109 from which the instructions are readily accessed by processor 106.
  • computer program product 104 is stored in storage 105 or other non-transitory computer readable medium (which may include being distributed across media on different devices and different locations). In alternative embodiments, the storage medium is transitory.
  • processor 106 in fact comprises multiple processors which may comprise additional working memories (additional processors and memories not individually illustrated) including a graphics processing unit (GPU) comprising at least thousands of arithmetic logic units supporting parallel computations on a large scale. GPUs are often utilized in deep learning applications because they can perform the relevant processing tasks more efficiently than can typical general-purpose processors (CPUs). Other embodiments comprise one or more specialized processing units comprising systolic arrays and/or other hardware arrangements that support efficient parallel processing. In some embodiments, such specialized hardware works in conjunction with a CPU and/or GPU to carry out the various processing described herein.
  • graphics processing unit GPU
  • CPUs general-purpose processors
  • Other embodiments comprise one or more specialized processing units comprising systolic arrays and/or other hardware arrangements that support efficient parallel processing. In some embodiments, such specialized hardware works in conjunction with a CPU and/or GPU to carry out the various processing described herein.
  • such specialized hardware comprises application specific integrated circuits and the like (which may refer to a portion of an integrated circuit that is application specific), field programmable gate arrays and the like, and combinations thereof.
  • a processor such as processor 106 may be implemented as one or more general purpose processors (preferably having multiple cores) without necessarily departing from the spirit and scope of the present invention.
  • FIG. 3 shows an exemplary genetic analyzer instrument system 300 that may be used in the system of FIG. 1.
  • exemplary genetic analyzer instrument system 300 comprises an Applied BiosystemsTM SeqStudioTM Genetic Analyzer instrument system, manufactured by ThermoFisher Scientific Inc., although other genetic analyzer instrument systems similarly capable of performing capillary electrophoresis may be used.
  • System 300 comprises genetic analyzer instrument 310, all-in-one cartridge
  • Genetic analyzer instrument 310 Built into genetic analyzer instrument 310 is touchscreen display 340 and USB port 350.
  • Genetic analyzer system 300 used in some embodiments of the present invention allows multiple fragment analysis and/or sequencing runs on the same plate. Genetic analyzer system 300 is easy to use with integrated cartridge-based system 320 and allows researchers to access and monitor experimental runs as well as view data on the integrated touchscreen display 340, or remotely. The fully connected genetic analyzer, along with the simple cartridge design, can be easily shared by multiple researchers in a lab or facility.
  • an easy-to-use functional core of the instrument includes a cartridge design that helps maximize efficiency and convenience.
  • the SeqStudio Genetic Analyzer mentioned above utilizes an all-in-one cartridge 320, shown in more detail in FIG. 4, that contains the capillary array 440, polymer reservoir 410, polymer delivery system 420, and anode buffer 430.
  • a laser detection window may also be provided in some embodiments of cartridge 320.
  • Cartridge 320 is removable and can be stored on the instrument for up to four months.
  • each cartridge contains a new polymer unique to the SeqStudio system that allows Sanger sequencing and fragment analysis to be performed with no reconfiguration.
  • the cartridge has four capillaries, and can process samples from either standard 96- well plates or 8- well strip tubes.
  • the cartridge and cathode buffer container include radio-frequency identification (RFID) tags that track the number of injections (cartridge) and length of time on the instrument (cathode buffer container). This allows scientists, using the same instrument, to maintain custody of their own cartridges.
  • RFID radio-frequency identification
  • Genetic analyzer instrument system 300 allows real-time monitoring of runs on the SeqStudio Genetic Analyzer. As shown in FIG. 5, Instrument 300 displays results 510 for each capillary in real time. Once an injection is finished, several quality checks are calculated and displayed in exemplary screen display 520. If an injection produces poor traces or poor QC values, those samples can be re-injected, with altered injection parameters, if desired.
  • Exemplary screenshot 530 from an off-site computer monitoring shows the progress of a run.
  • exemplary screenshot 540 as used in some embodiments of the present invention, runs set up in a PlateManager user interface can be uploaded directly to the instrument.
  • PlateManager allows investigators to assign multiple sequencing and fragment analysis runs on the same plate, taking advantage of the universal polymer in the cartridge and use them only when needed, providing another level of flexibility.
  • maintenance of the SeqStudio Genetic Analyzer used in some embodiments of the present invention is simple and straightforward for the user, and instrument calibrations used in the genetic analyzer instrument used in exemplary embodiments of the invention described herein may be handled automatically by leveraging advancements in imaging and algorithm tools.
  • FIG. 6 shows how the SeqStudio genetic analyzer instrument may be accessible to the user in several different ways: via the onboard interface, a remote computer, or a mobile device app.
  • assays or experimental runs can be set up using either the onboard computer or by using PlateManager, the stand-alone software that operates within Thermo Fisher Connect or on a separate computer, as shown in 610.
  • PlateManager the stand-alone software that operates within Thermo Fisher Connect or on a separate computer, as shown in 610.
  • web browser- based software access to run setup, plate maps, run conditions, and analysis settings are all immediately available from anywhere you have Internet access.
  • Injection conditions, reinjections, and reordering of injections can all be monitored and modified during the run as shown in 620, maximizing the ability to collect quality data from each plate.
  • the web browser-based suite of applications including applications to measure microsatellite instability, allows accessible analysis in some embodiments of the present invention. Determination of DNA sequence variants, alignments, and fragment analysis are all available immediately upon completion of a run, in analysis step 630.
  • the cloud connectivity 650 enables collaborators in different locations to monitor, access, share data information, and rapidly analyze the same data sets anytime post-run in sharing and collaboration step 640.
  • the SeqStudio Genetic Analyzer provides touchscreen usability via the instrument itself or via smartphone, tablet or other user device, allowing researches to collaborate and analyze data remotely as well as onsite with equal effectiveness.
  • the exemplary genetic analyzer system discussed herein as used in some embodiments of the present invention is designed for both new and experienced users who need simple and affordable Sanger sequencing and fragment analysis, without compromising performance or quality.
  • FIG. 7 shows a flow diagram of a method 700 used in some embodiments of the present invention to determine the MSI status of a biological sample.
  • step 710 one or more DNA loci of the biological sample are selected to investigate for tumors which exhibit a high mutation rate and hence a high microsatellite instability.
  • Microsatellite instability is a form of genomic instability due to reduced fidelity during the replication of DNA; this is thought to be caused by defects in DNA repair mechanisms. Defects in this biomolecular machinery is most easily observed by examining places in the DNA where there is a single nucleotide (one of the four possible nucleotides) repeated many times (a homopolymer); e.g., GGGGGGGGGGG (SEQ ID NO: 32) is an 11- base repeat of Guanine. Extending this example, with damaged DNA repair mechanisms that often manifest in tumor cells, the section of DNA with the 11 -base repeat of Guanine may be replicated as, for example, 10 bases or 5 bases or 13 bases instead of the normal 11 bases.
  • Microsatellite instability analysis involves chemistries designed to examine several different regions in DNA at which there are homopolymers. These chemistries select out and amplify sections of DNA (an amplified fragment of DNA at specific DNA loci) that include each of the homopolymers of interest. Hence, again building on our 11 -base Guanine example, normally, the amplified DNA at this locus would have a fragment size of, say, 20 bases (some number larger than 11 selected out by the chemistry). However, if DNA replication repair mechanisms are damaged, the replicated DNA may only have 10, for example, instead of the usual 11 Guanines so the amplified fragments will be of size 19 instead of 20.
  • step 710 particular DNA loci may be selected for the sensitivity of the loci to the cancer type under investigation as compared to other cancer types.
  • a particular DNA locus (also referred to as a marker) may also be selected for the reliability of DNA amplification at that particular locus.
  • each DNA locus is e amined and one or more algorithms may be selected for each locus to determine whether that given locus is microsatellite unstable, MSU, or microsatellite stable, MSS.
  • Embodiments of the present invention utilize a number of algorithms for determining whether or not a given DNA locus is MSU or MSS, including algorithms 1 through 11 below.
  • the selected algorithm(s) is executed for each selected DNA locus.
  • the overall MSI status is determined for the biological sample by combining the MSI results for each selected DNA locus.
  • FIG. 8 shows another embodiment of a method for assessing the microsatellite instability of a biological sample, which is first described above and in FIG. 7.
  • the embodiment shown in FIG. 8 provides additional detail and alternate data analysis pathways elaborating on the embodiment of the microsatellite instability assessment method shown in FIG. 7.
  • the CE fragment analysis data across all DNA loci for the biological sample is obtained.
  • Each DNA locus, or marker may be analyzed separately, as shown in step 805.
  • the markers may be analyzed together as shown in step 815 of FIG. 8.
  • one or more signal features can be extracted for each marker in step 820, and one or more classification functions such as the exemplary classification functions described below can be applied to the extracted signal features to determine the MSI status of each marker in step 830.
  • a simple size threshold Any fluorescence peaks appearing below the fragment size threshold (or above depending on the location for an MSS situation) is considered MSU. This is appropriate if there is only one DNA locus covered by a given dye and the number of nucleotides differs significantly between MSU and MSS DNA molecule situations.
  • Fragment size interval If there are any fluorescence peaks appearing within a given fragment size interval (an interval on the DNA fragment size axis of the data), the DNA locus is considered MSU. This presumes that there is no overlap between the size intervals covering all the DNA loci using the same dye and the fluorescent peaks associated with MSU and MSS situations are also well separated.
  • Peak count within a given size interval If the number of significant fluorescent peaks (significance determined by peak size) is above a threshold, the locus is considered MSU. This presumes that there is no overlap between the size intervals covering all the DNA loci using the same dye.
  • Peak envelope peaks within a given size interval If the number of envelope peaks is two or more, the locus is considered MSI-high. This presumes that there is no overlap between the size intervals covering all the DNA loci using the same dye.
  • Peak patterns can consist of two or more values among the following: peak amplitudes and/or locations along the fragment size axis; peak amplitudes and/or locations relative to the largest peak; peak envelope peak amplitudes, locations, and/or widths; peak metrics relative to peak envelope metrics;
  • Peak pattern deviations from normal Peak patterns of (7) above relative to these patterns from normal tissue samples;
  • Peak pattern deviations from normal (non-cancer) population peak patterns Peak patterns of (7) above relative to these patterns from nominal values for these patterns, such as the mean, median, z-score, etc., across a population of people without cancer.
  • Peak pattern deviations from normal relative to population deviations A combination of (8) and (9) above where the metrics of (8) are compared to nominal values of these patterns across a population of people without cancer.
  • Difference signal patterns In the case that data from a given person is available from both normal and tumor tissue, signal patterns described above can be computed on the difference between normalized data from tumor and normal tissue. In addition, other metrics derived from the difference signal can be used to characterize the difference signal at each locus. For example, asymmetry of the difference signal can be characterized by the difference between the center of mass of the positive peaks of the difference signal compared to the negative peaks. Other examples include the relative position of the difference signal maximum and minimum, the root-mean-square (RMS) values of positive compared to negative peaks, overall RMS value for the difference signal, etc.
  • RMS root-mean-square
  • the algorithm for determining whether a DNA locus is MSS or MSU would consist of a suitable classification function that can process multi-dimensional vectors.
  • discriminant functions, multi-layer artificial neural networks, vector machines, etc. are examples of typical machine learning methods that can be applied.
  • deep learning methods can be applied to automatically learn the best signal features to distinguish MSU from MSS by using a large number of samples of CE fragment analysis data localized to the fragment size intervals of interest.
  • the MSI results for each DNA locus can be combined across all of the markers in the nucleic acid sample under investigation to assign an overall MSI status call in step 840.
  • Weighted sum In one embodiment of the invention, a weighted sum across DNA loci can be calculated after assigning MSU loci an exemplary value of 1 and MSS loci an exemplary value of 0; the overall assessment can be assigned MSI-high if the weighted sum across loci exceeds a threshold. Linear discriminant functions are an exemplary way to determine the weightings.
  • Non-linear classification As shown in FIG. 8 at steps 820 and 830, the
  • MSI results across DNA loci can alternatively be combined in a non-linear fashion to determine an overall assessment of MSI status in step 840.
  • An exemplary way to determine the non-linear classification function is to train a multi-layer artificial neural network to make the assignment.
  • Standard 3-layer artificial neural networks trained with customary backpropagation techniques known in the art to minimize cross entropy, have been found to provide adequate accuracy in distinguishing MSU from MSS cases.
  • the markers may be analyzed together in step 815 and the signal features expressed in items (7) to (11) can be combined across DNA loci as shown in step 860 and used to generate one or more classification functions that directly assigns the overall MSI status as shown in step 870 of FIG. 8. For example, suppose marker A used 3 signal features to determine whether it is MSU or MSS and marker B used 4 features for this.
  • the features from both markers can be combined into a 7 -dimensional feature vector to feed into an artificial intelligence generated classifier, such as an artificial neural network trained to map this vector to an overall MSI status call.
  • artificial intelligence generated classifiers may be implemented via deep learning methods, which can be used as described above except that data across DNA loci are combined in the analysis either after the markers are analyzed separately in step 805 or together, as shown in step 815.
  • the signal features are fed directly into a deep learning neural network for mapping directly into an MSI status call, as shown in step 850 of FIG. 8.
  • signals from each locus can be concatenated into one large signal vector and fed into a deep learning network to map them into an overall MSI status call at step 880.
  • Some embodiments of the present invention comprise methods for using one or more anti-tumor drugs to treat tumor patients.
  • one or more of the methods, computer program products, systems, or kits disclosed herein are used to determine microsatellite instability of tumor cells in a biological sample obtained from a patient. Then, if microsatellite instability is determined to be high, the one or more anti tumor drugs are administered to the patient to treat the tumor.
  • Systems, apparatus, and methods described herein may be implemented using a computer program product tangibly embodied in an information carrier, e.g., in a non- transitory machine-readable storage device, for execution by a programmable processor; and the method steps described herein, including one or more of the steps of the methods in FIG. 6, FIG. 7 and FIG. 8 and alternative embodiments may be implemented using one or more computer programs that are executable by such a processor.
  • a computer program is a set of computer program instructions that can be used, directly or indirectly, in a computer to perform a certain activity or bring about a certain result.
  • a computer program can be written in any form of programming language, including compiled or interpreted languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment.
  • FIG. 9 illustrates components of one embodiment of an environment 900 in which the invention may be practiced. Not all the components may be required to practice the invention, and variations in the arrangement and type of the components may be made without departing from the spirit or scope of the invention.
  • the system 900 includes one or more Local Area Networks ("LANs”) / Wide Area Networks ("WANs") 912, one or more wireless networks 910, one or more wired or wireless client devices 906, mobile or other wireless client devices 902-906, servers 907-909, and may include or communicate with one or more data stores or databases.
  • LANs Local Area Networks
  • WANs Wide Area Networks
  • client devices 902-906 may include, for example, desktop computers, laptop computers, set top boxes, tablets, monitors, cell phones, smart phones, devices for interfacing with, or viewing dashboards or analytics relating to, genetic analysis related systems or entities, etc.
  • the servers 907-909 can include, for example, one or more application servers, content servers, search servers, database servers, database management or SQL servers, other servers relating to genetic analysis related systems, etc.
  • FIG. 10 illustrates a block diagram of an electronic device 1100 that can implement one or more aspects of genetic analysis related systems and methods according to embodiments of the invention.
  • Instances of the electronic device 1100 may include servers, e.g., servers 907-909, and client devices, e.g., client devices 902-906.
  • FIG. 11 shows an example of a computer system 1100, one or more of which may provide one or more of the components of, or alternatives to computer 103 of FIG. 1.
  • Computer system 1100 executes instruction code contained in a computer program product 1122 comprising genetic analyzer program 1123 (which may, for example, comprise CE data analyzer program 104 of the computer program product 102 of the embodiment of FIG. 1.)
  • Computer program product 1122 comprises executable code in an electronically readable medium that may instruct one or more computers such as computer system 1100 to perform processing that accomplishes the exemplary method steps performed by the embodiments referenced herein.
  • the electronically readable medium may be any non-transitory medium that stores information electronically and may be accessed locally or remotely, for example via a network connection.
  • the medium may be transitory.
  • the medium may include a plurality of geographically dispersed media each configured to store different parts of the executable code at different locations and/or at different times.
  • the executable instruction code in an electronically readable medium directs the illustrated computer system 1100 to carry out various exemplary tasks described herein.
  • the executable code for directing the carrying out of tasks described herein would be typically realized in software.
  • computers or other electronic devices might utilize code realized in hardware to perform many or all the identified tasks without departing from the present invention.
  • Those skilled in the art will understand that many variations on executable code may be found that implement exemplary methods within the spirit and the scope of the present invention.
  • the code or a copy of the code contained in computer program product 1100 may reside in one or more storage persistent media (not separately shown) communicatively coupled to system 1100 for loading and storage in persistent storage device.
  • the electronic device 1100 can include a processor/CPU 1102, memory 1130, a power supply 1106, and input/output (I/O) components/devices 1140, e.g., microphones, speakers, displays, touchscreens, keyboards, mice, keypads, microscopes, GPS components, etc., which may be operable, for example, to provide graphical user interfaces, dashboards, etc.
  • a user may provide input via a touchscreen of an electronic device 1100.
  • a touchscreen may determine whether a user is providing input by, for example, determining whether the user is touching the touchscreen with a part of the user's body such as his or her fingers.
  • the electronic device 1100 can also include a communications bus 1104 that connects the aforementioned elements of the electronic device 1100.
  • Network interfaces 1114 can include a receiver and a transmitter (or transceiver), and one or more antennas for wireless communications.
  • the processor 1102 can include one or more of any type of processing device, e.g., a Central Processing Unit (CPU), and a Graphics Processing Unit (GPU).
  • the processor can be central processing logic, or other logic, may include hardware, firmware, software, or combinations thereof, to perform one or more functions or actions, or to cause one or more functions or actions from one or more other components.
  • central processing logic, or other logic may include, for example, a software-controlled microprocessor, discrete logic, e.g., an Application Specific Integrated Circuit (ASIC), a programmable/programmed logic device, memory device containing instructions, etc., or combinatorial logic embodied in hardware.
  • ASIC Application Specific Integrated Circuit
  • logic may also be fully embodied as software.
  • the memory 1130 which can include Random Access Memory (RAM) 1112 and Read Only Memory (ROM) 1132, can be enabled by one or more of any type of memory device, e.g., a primary (directly accessible by the CPU) or secondary (indirectly accessible by the CPU) storage device (e.g., flash memory, magnetic disk, optical disk, and the like).
  • RAM Random Access Memory
  • ROM Read Only Memory
  • the ROM 1132 can also include Basic Input / Output System (BIOS) 1120 of the electronic device.
  • BIOS Basic Input / Output System
  • the RAM can include an operating system 1121, data storage 1124, which may include one or more databases, and programs and/or applications 1122 and a genetic analyzer program 1123.
  • the genetic analyzer program 1123 is intended to broadly include all programming, applications, algorithms, software and other and tools necessary to implement or facilitate methods and systems according to embodiments of the invention. Elements of the genetic analyzer program 1123 program may exist on a single server computer or be distributed among multiple computers, servers, devices or entities, or sites. Moreover, those skilled in the art will appreciate that in addition to storing computer program product 1122 for carrying out processing described herein, memory 1130 may be configured to store the various data elements referenced and illustrated herein.
  • the power supply 1106 contains one or more power components and facilitates supply and management of power to the electronic device 1100.
  • the input/output components can include, for example, any interfaces for facilitating communication between any components of the electronic device 1100, components of external devices (e.g., components of other devices of the network or system 1100), and end users.
  • components can include a network card that may be an integration of a receiver, a transmitter, a transceiver, and one or more input/output interfaces.
  • a network card for example, can facilitate wired or wireless communication with other devices of a network. In cases of wireless communication, an antenna can facilitate such communication.
  • some of the input/output interfaces 1140 and the bus 1104 can facilitate communication between components of the electronic device 1100, and in an example can ease processing performed by the processor 1102.
  • the electronic device 1100 can include a computing device that can be capable of sending or receiving signals, e.g., via a wired or wireless network, or may be capable of processing or storing signals, e.g., in memory as physical memory states.
  • the server may be an application server that includes a configuration to provide one or more applications.
  • Any computing device capable of sending, receiving, and processing data over a wired and/or a wireless network may act as a server, such as in facilitating aspects of implementations of genetic analyzer related systems and methods according to embodiments of the invention.
  • Devices acting as a server may include devices such as dedicated rack mounted servers, desktop computers, laptop computers, set top boxes, integrated devices combining one or more of the preceding devices, etc.
  • Servers may vary widely in configuration and capabilities, but they generally include one or more central processing units, memory, mass data storage, a power supply, wired or wireless network interfaces, input/output interfaces, and an operating system such as
  • a server may include, for example, a device that is configured, or includes a configuration, to provide data or content via one or more networks to another device, such as in facilitating aspects of systems and methods according to embodiments of the invention.
  • One or more servers may, for example, be used in hosting a Web site utilized in embodiments of the present invention.
  • One or more servers may host a variety of sites, such as, for example, business sites, informational sites, social networking sites, educational sites, wikis, financial sites, government sites, personal sites, and the like.
  • Servers may also, for example, provide a variety of services, such as Web services, third-party services, audio services, video services, email services, HTTP or HTTPS services, Instant Messaging (IM) services, Short Message Service (SMS) services, Multimedia Messaging Service (MMS) services, File Transfer Protocol (FTP) services, Voice Over IP (VOIP) services, calendaring services, phone services, and the like, all of which may work in conjunction with example aspects of systems and methods according to embodiments of the invention.
  • Content may include, for example, text, images, audio, video, and the like.
  • client devices may include, for example, any computing device capable of sending and receiving data over a wired and/or a wireless network.
  • client devices may include desktop computers as well as portable devices such as cellular telephones, smart phones, display pagers, Radio Frequency (RF) devices, Infrared (IR) devices, Personal Digital Assistants (PDAs), handheld computers, GPS-enabled devices tablet computers, monitors, sensor-equipped devices, laptop computers, set top boxes, wearable computers, integrated devices combining one or more of the preceding devices, and the like.
  • RF Radio Frequency
  • IR Infrared
  • PDAs Personal Digital Assistants
  • handheld computers GPS-enabled devices tablet computers, monitors, sensor-equipped devices, laptop computers, set top boxes, wearable computers, integrated devices combining one or more of the preceding devices, and the like.
  • Client devices may range widely in terms of capabilities and features.
  • a cell phone, smart phone or tablet may have a numeric keypad and a few lines of monochrome Liquid-Crystal Display (LCD) display on which only text may be displayed.
  • LCD liquid-Crystal Display
  • a Web-enabled client device may have a physical or virtual keyboard, data storage (such as flash memory or SD cards), accelerometers, gyroscopes, GPS or other location-aware capability, and a 2D or 3D touch-sensitive color screen on which both text and graphics may be displayed.
  • Client devices such as client devices 1002-1006, for example, as may be used in example systems and methods according to embodiments of the invention, may ran a variety of operating systems, including personal computer operating systems such as Windows, iOS or Linux, and mobile operating systems such as iOS, Android, Windows Mobile, and the like. Client devices may be used to run one or more applications that are configured to send or receive data from another computing device. Client applications may provide and receive textual content, multimedia information, and the like.
  • Client applications may perform actions such as viewing or interacting with analytics or dashboards, interacting with genetic analyzer instruments, methods or systems used in embodiments of the present invention, browsing webpages, using a web search engine, interacting with various apps stored on a smart phone, sending and receiving messages via email, SMS, or MMS, playing games, receiving advertising, watching locally stored or streamed video, or participating in social networks.
  • one or more networks such as networks 1010 or 1012, for example, may couple servers and client devices with other computing devices, including through wireless network to client devices.
  • a network may be enabled to employ any form of computer readable media for communicating information from one electronic device to another.
  • a network may include the Internet in addition to Local Area Networks (LANs), Wide Area Networks (WANs), direct connections, such as through a Universal Serial Bus (USB) port, other forms of computer-readable media, or any combination thereof.
  • LANs Local Area Networks
  • WANs Wide Area Networks
  • USB Universal Serial Bus
  • a network may include the Internet in addition to Local Area Networks (LANs), Wide Area Networks (WANs), direct connections, such as through a Universal Serial Bus (USB) port, other forms of computer-readable media, or any combination thereof.
  • LANs Local Area Networks
  • WANs Wide Area Networks
  • USB Universal Serial Bus
  • Communication links within LANs may include twisted wire pair or coaxial cable, while communication links between networks may utilize analog telephone lines, cable lines, optical lines, full or fractional dedicated digital lines including Tl, T2, T3, and T4, Integrated Services Digital Networks (ISDNs), Digital Subscriber Lines (DSLs), wireless links including satellite links, optic fiber links, or other communications links known to those skilled in the art.
  • ISDNs Integrated Services Digital Networks
  • DSLs Digital Subscriber Lines
  • wireless links including satellite links, optic fiber links, or other communications links known to those skilled in the art.
  • remote computers and other related electronic devices could be remotely connected to either LANs or WANs via a modem and a telephone link.
  • a wireless network such as wireless network 1010, as in example genetic analysis related systems and methods according to embodiments of the invention, may couple devices with a network.
  • a wireless network may employ stand-alone ad-hoc networks, mesh networks, Wireless LAN (WLAN) networks, cellular networks, and the like.
  • WLAN Wireless LAN
  • a wireless network may further include an autonomous system of terminals, gateways, routers, or the like connected by wireless radio links, or the like. These connectors may be configured to move freely and randomly and organize themselves arbitrarily, such that the topology of wireless network may change rapidly.
  • a wireless network may further employ a plurality of access technologies including 2nd (2G), 3rd (3G), 4th (4G) generation, Long Term Evolution (LTE) radio access for cellular systems, WLAN, Wireless Router (WR) mesh, and the like.
  • Access technologies such as 2G, 2.5G, 3G, 4G, 5G and future access networks may enable wide area coverage for client devices, such as client devices with various degrees of mobility.
  • a wireless network may enable a radio connection through a radio network access technology such as Global System for Mobile communication (GSM), Universal Mobile Telecommunications System (UMTS), General Packet Radio Services (GPRS), Enhanced Data GSM Environment (EDGE), 3GPP Long Term Evolution (LTE), LTE Advanced, Wideband Code Division Multiple Access (WCDMA), Bluetooth,
  • GSM Global System for Mobile communication
  • UMTS Universal Mobile Telecommunications System
  • GPRS General Packet Radio Services
  • EDGE Enhanced Data GSM Environment
  • LTE Long Term Evolution
  • LTE Advanced Long Term Evolution
  • WCDMA Wideband Code Division Multiple Access
  • a wireless network may include virtually any wireless communication mechanism by which information may travel between client devices and another computing device, network, and the like.
  • IP Internet Protocol
  • the Internet includes local area networks (LANs), Wide Area Networks (WANs), wireless networks, and long-haul public networks that may allow packets to be communicated between the local area networks.
  • the packets may be transmitted between nodes in the network to sites each of which has a unique local network address.
  • a data communication packet may be sent through the Internet from a user site via an access node connected to the Internet.
  • the packet may be forwarded through the network nodes to any target site connected to the network provided that the site address of the target site is included in a header of the packet.
  • Each packet communicated over the Internet may be routed via a path determined by gateways and servers that switch the packet according to the target address and the availability of a network path to connect to the target site.
  • the header of the packet may include, for example, the source port (16 bits), destination port (16 bits), sequence number (32 bits), acknowledgement number (32 bits), data offset (4 bits), reserved (6 bits), checksum (16 bits), urgent pointer (16 bits), options (variable number of bits in multiple of 8 bits in size), padding (may be composed of all zeros and includes a number of bits such that the header ends on a 32 bit boundary).
  • the number of bits for each of the above may also be higher or lower.
  • Such services may make use of ancillary technologies including, but not limited to, "cloud computing,” distributed storage, DNS request handling, provisioning, data monitoring and reporting, content targeting, personalization, and business intelligence.
  • a CDN may also enable an entity to operate and/or manage a third party's Web site infrastructure, in whole or in part, on the third party's behalf.
  • a Peer-to-Peer (or P2P) computer network relies primarily on the computing power and bandwidth of the participants in the network rather than concentrating it in a given set of dedicated servers.
  • P2P networks are typically used for connecting nodes via largely ad hoc connections.
  • a pure peer-to-peer network does not have a notion of clients or servers, but only equal peer nodes that simultaneously function as both "clients" and “servers” to the other nodes on the network.
  • One embodiment of the present invention includes systems, methods, and a non-transitory computer readable storage medium or media tangibly storing computer program logic capable of being executed by a computer processor.
  • computer system 1100 illustrates just one example of a system in which a computer program product in accordance with an embodiment of the present invention may be implemented.
  • execution of instructions contained in a computer program product in accordance with an embodiment of the present invention may be distributed over multiple computers, such as, for example, over the computers of a distributed computing network.
  • FFPE paraffin-embedded
  • the ABI MSI amplification mix consists of 4 pi of Multiplex PCR MasterMix, 4 pi of IX low- EDTA TE buffer, and 2 pi of DNA at 1 ng/pl.
  • the PCR was carried out in an Applied Biosystems ProFlex PCR system in GeneAmpTM PCR System 9700 simulation mode under the following conditions: 95°C for 11 minutes, 29 cycles of denaturation at 94°C for 20 seconds and annealing at 59°C for 2 minutes; and a final extension at 60°C for 25 minutes.
  • Amplified PCR products were denatured for 95 °C for 3 minutes in a 20 pi reaction volume consisting of 17 pi of Hi-DiTM Formamide, 1 pi of GeneScanTM 600 LIZTM dye Size Standard v2.0, and 2 pi of PCR product. Denatured amplified PCR products were then analyzed by fragment analysis on an ABI 3500 (xL) or SeqStudioTM Genetic Analyzer.
  • PCR primers including associated labeled, are shown in Table 1. PCR primers were labeled with the indicated dye at the 5' end of the forward primer in each set, with the exception of ABI- 16, which was labeled at the 5' end of the reverse primer. The label was attached via a C3 linker.
  • Example 2 Assessment of MSI in Colorectal Cancer versus Normal Tissue
  • DNA was isolated from FFPE sections of colorectal carcinoma (“Tumor”) or normal tissue (“Normal”) and PCR was performed as described in Example 1. Normal tissue is generally a matched normal control (tissue from the same patient). The signals in the electropherograms of FIG. 11 show clear differences between the signals of tumor versus normal samples. Signals are clear and easily interpretable with consistent signal strength.
  • Example 3 Comparison of ABI-MSI with Other MSI Assays
  • Endometrial carcinomas are notoriously difficult to assay, due to small deletions that are hard to resolve by standard MSI assays.
  • DNA samples from endometrial carcinomas were amplified and analyzed as described in Example 1 (“ABI-MSI”), or using the PROMEGATM MSI Analysis System using the protocol provided by the manufacturer.
  • the PROMEGATM system amplifies DNA from 5 loci (NR21, BAT26, BAT25, NR24, and M0N027), and includes two controls (PentaC and PentaD). As shown in FIG.
  • the ABI- MSI assay identified one sample as MSI-high (MSI-H) that was identified as MSI-low (MSI- L) by the PROMEGATM system, and identified five samples as MSI-low that were identified as MSI-stable (MSS) by the PROMEGATM system.
  • FIG. 12B shows clear shifts for several loci between normal (blue) and tumor (green) samples.
  • MSI assay has been developed that has a fast, simple workflow, low sample input (2 ng FFPE DNA), expanded content, automated analysis and interpretable results, and tumor-only analysis.
  • the assay takes only 3.5 hours from DNA to answer: 15 minutes of PCR sample preparation, 2 hours of fluorescent PCR, 15 minutes of fragment analysis sample preparation, 55 minutes of CE-based fragment analysis, and 5 minutes of data analysis.
  • FIG. 13 shows how synthetic constructs reveal detection complexities.
  • Detection of instability is a complex interplay between deletion size and mutant allele fraction present in a sample. Synthetic constructs were generated to: 1) understand the peak morphology of difficult to assess MSI samples, and 2) train the algorithm at various allele frequencies and with variable deletion sizes for each homopolymer.
  • FIG. 14 shows tumor-only analysis at >98% specificity and >90% sensitivity at
  • FIG. 15 shows tumor- normal analysis at >95% specificity and sensitivity at >
  • Tunable algorithm parameters allow for maximization of sensitivity and specificity on both the Applied BiosystemsTM 3500 and SeqStudioTM Genetic Analyzer Systems.
  • FIG. 16 illustrates the ABI MSI software (top) and example MSI report
  • the software provides an automated genotyping solution for streamlined analysis and reporting, saving customers time and effort required by current manual analysis.
  • the ABI MSI Assay achieves robust identification of microsatellite instability in multiple cancer types, with low sample input.
  • MSI analysis software has fast analysis and can include automated calling at sensitivity and specificity.

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Abstract

L'invention concerne des systèmes, des amorces, des kits et des procédés de détection de l'instabilité des microsatellites dans un échantillon biologique. Des données de signal sont reçues d'un instrument d'analyse génétique d'électrophorèse capillaire, les données de signal étant mesurées à partir de la fluorescence de fragments comprenant des séquences d'acide nucléique amplifiées à partir de l'échantillon biologique par l'intermédiaire d'une réaction d'amplification en chaîne par polymérase (PCR). Les séquences d'acides nucléiques correspondent à une pluralité de loci microsatellites différents et sont obtenues à l'aide d'une pluralité d'amorces PCR conçues pour flanquer une pluralité de loci microsatellites d'un échantillon biologique. Lorsque les amorces de PCR et l'échantillon biologique sont combinés et sont soumis à une amplification par PCR, des fragments d'ADN marqués par fluorescence sont générés comprenant la pluralité de loci microsatellites. Des données de fluorescence obtenues à partir de la pluralité de loci microsatellites marqués par fluorescence sont utilisées pour classifier l'instabilité des microsatellites de l'échantillon biologique.
PCT/US2020/059295 2019-11-08 2020-11-06 Systèmes et essais pour évaluer l'instabilité de microsatellites Ceased WO2021092299A1 (fr)

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