Detection of microsatellite instability

Abstract

To provide: methods and systems for detecting MSI; methods for enriching human genomic DNA for microsatellite loci; and an oligonucleotide array for detecting MSI.SOLUTION: A method for detecting microsatellite instability (MSI) includes: detecting sequencing reads from human genomic DNA for a plurality of microsatellite loci; determining a metric for repeat length distribution (RLD) for each microsatellite locus; comparing the metric with a threshold value for the microsatellite locus, wherein each microsatellite has an independent threshold value; quantifying the number of microsatellite loci having an RLD metric exceeding the threshold value; and comparing the number of microsatellite loci having an RLD metric exceeding the threshold value with a microsatellite instability (MSI) proportion threshold, wherein if the number exceeds the MSI proportion threshold, the human has MSI.SELECTED DRAWING: None

Description

[Background technology]

[0001] Microsatellite loci are regions of genomic DNA that have short tandem repeats (STRs), which are repeating units of 1-6 base pairs in length. Hundreds of thousands of microsatellite loci are distributed throughout the human genome. Microsatellite loci are classified based on the length of the minimal repeating unit. For example, microsatellite loci with repeating units of 1-6 base pairs in length are referred to as "mononucleotide", "dinucleotide", "trinucleotide", "tetranucleotide", "pentanucleotide", and "hexanucleotide" repeat loci, respectively. Generally, microsatellite loci are classified into two types: single repeat type (e.g., (GT) n ) and can have up to 100 repeats, but microsatellites with multiple repeat types are called compound microsatellites (e.g., (GT) n (CT) m Microsatellite loci are known as nucleotide substitutions (incomplete microsatellites, e.g., (GT) n A(GT) m ) or insertions (translated microsatellites, e.g., (GT) n CCC(GT) m ).

[0002] Each microsatellite locus in normal genomic DNA of most diploid species, such as genomic DNA from mammalian species, consists of two alleles at each locus. The two alleles may be the same or different in length from one individual to another, and may vary from one individual to another. Microsatellite alleles are usually maintained at a constant length in a given individual and its descendants, but microsatellite length instability has been identified in several tumor types (Aaltonen et al., 1993, Science 260:812-815; Thibodeau et al., 1993 Science 260:816-819; Peltomaki et al., 1993 Cancer Research 53:5853-5855; Ionov et al., 1993 Nature 363:558-561).

[0003] This type of genomic instability in tumors, termed "microsatellite instability" (MSI), is a molecular hallmark of the inherited cancer syndrome hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Causes of MSI are thought to include failure of the DNA mismatch repair (MMR) system, which fails to reverse errors that occur during DNA replication, for example, by polymerase slippage (Fishel et al., 1993 Cell 75:1027-38; Leach et al., 1993 Cell 75:215-25; Bronner et al., 1994 Nature 368:258-61; Nicolaides et al., 1994 Nature 371:75-80; Miyaki et al., 1997 Nat Genetics 17:271-2). Insertions or deletions of one or more repeat units at a microsatellite locus (e.g., due to defects in MMR) can be detected, for example, by comparing the allele sizes found at microsatellite loci amplified from normal and tumour DNA samples (Thibodeau et al., 1993, supra).

[0004] MSI has been found in over 90% of HNPCC and 10-20% of sporadic colorectal tumors (Liu et al., 1996 Nature Med 2:169-174; Thibodeau et al., 1993, supra; Ionov et al., 1993 Nature 363:558-561; Aaltonen et al., 1993 Science 260:812-816; Lothe et al., 1993 Cancer Res.53:5849-5852; and Boland and Goel, 2010 Gastroenterology 138:2073-2087). However, MSI is not exclusive to colorectal tumors. MSI has been detected in pancreatic cancer (Han et al., 1993 Cancer Res 53:5087-5089), gastric cancer (Peltomaki et al., 1993 Cancer Res 53:5853-5855; Mironov et al., 1994 Cancer Res 54:41-44), prostate cancer (Gao et al., 1994 Oncogene 9:2999-3003), endometrial cancer (Risinger et al., 1993 Cancer Res 53:5100-5103), and breast cancer (Patel et al., 1994 Oncogene 9:3695-3700).

[0005] Colorectal tumors with MSI-high (MSI-H) status have a unique molecular etiology resulting in tumor hallmarks, including aberrant phenotypic features and hypermutations in the tumor genome. Samples are typically classified as MSI-H when MSI is detected in more than 40% of tiled MS loci. Several clinical studies have shown that MSI-high (MSI-H) colorectal cancer (CRC) patients have distinct responses to certain treatments, including both chemotherapy and immunotherapy, and have a different prognosis than CRC patients without MSI (e.g., microsatellite stable (MSS) patients) (e.g., Popat et al., J Clin Oncol. 2005;23:609-618; Kim et al. JJ Clin Oneal. 2007;25:767-772; Elsaleh et al. Lancet. 2000;355:1745-1750; Sturojeva et al. Oneal Rep. 2005;14:241-249; Jover et al. Gut. 2006;55:848-855; Des Guetz et al. Eur J Cancer. 2009;45:1890-1896; Bertagnolli et al. al. J Clin Oncol. 2009;27:1814-1821; Ribic et al. N Engl J Med. 2003;349:247-257; and Benatti et al. Clin Cancer Res. 2005;11:8332-8340). In 2017, the FDA approved pembrolizumab for patients with unresectable or metastatic, MSI-H or mismatch repair deficient (dMMR) solid tumors that have progressed on prior therapy. This is believed to be the first time that a cancer drug has been approved based on tumor genetics rather than histology or tumor site. Thus, detection of the molecular alterations underlying MSI-H features will impact the development of new diagnostic and therapeutic approaches in CRC and other oncology indications.

[0006] Existing criteria for determining MSI-H status include the Bethesda Panel (Berg et al., J Mol Diagn. 2000;2:20-28), which interrogates five microsatellite loci (three dinucleotide and two mononucleotide repeats) recommended by the 1997 National Cancer Institute-sponsored MSI Workshop, and the Promega MSI Analysis System (Bacher et al., J Mol Diagn. 2000;2:20-28), which includes seven microsatellite loci (five mononucleotide and two pentanucleotide repeats). al. Dis. Markers 2004.20(4-5):237-50). Within these criteria, instability at two or more MS loci defines MSI-H status. These existing criteria are limited in that they are polymerase chain reaction (PCR) based and require manual analysis of electropherogram output to determine MSI or MSS status. Furthermore, the criteria are for tissue samples rather than cell-free DNA (cfDNA) samples, and the mutant allele fraction of MSI loci in tissue samples is significantly higher than that seen in cfDNA samples.

[0007] Moreover, the state of the art relies on a small set of known MSI loci and either the presence of matched normal samples or manual visual analysis of the electropherogram output to determine whether a sample is MSI. Furthermore, the state of the art cannot identify the MSI status of circulating tumor DNA (ctDNA) samples due to the low abundance of mutant alleles present in cfDNA samples. Summary of the Invention

[0008] In one aspect, the present disclosure generally relates to a method for detecting microsatellite instability (MSI). In some embodiments, the method for detecting MSI comprises: detecting sequencing reads for a plurality of microsatellite loci from a sample of human genomic DNA; determining a repeat length distribution (RLD) reference value for each microsatellite locus; comparing the reference value with a threshold value for the microsatellite locus (each microsatellite has an independent threshold); quantifying the number of microsatellite loci that have a RLD reference value that exceeds the threshold; and comparing the number of microsatellite loci that have a RLD reference value that exceeds the threshold value with a microsatellite instability (MSI) ratio threshold. If the number of microsatellite loci exceeds the MSI ratio threshold, the human genomic DNA from the sample has MSI. In some embodiments, the plurality of microsatellite loci are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or more microsatellite loci selected from the group consisting of microsatellite loci: 1-170. In another embodiment, the plurality of microsatellite loci are about 5 to about 160, about 10 to about 150, about 20 to about 100, about 25 to about 80, or about 30 to about 50 microsatellite loci selected from the group consisting of microsatellite loci: 1-170 (Table 1). In yet another embodiment, the plurality of microsatellite loci correspond to loci: 1-170 (Table 1). In yet another embodiment, the plurality of microsatellite loci correspond to the loci of Table 2. In one embodiment, the plurality of microsatellite loci are at least 10, 20, 30, 40, 50, or more microsatellite loci selected from the group consisting of the microsatellite loci in Table 2. In one embodiment, the plurality of microsatellite loci are about 10 to about 50, more preferably about 20 to about 50, and most preferably about 20 to about 40 microsatellite loci selected from the group consisting of the microsatellite loci corresponding to the loci in Table 2.In some embodiments, the plurality of microsatellite loci corresponds to the microsatellite loci in Table 2.

[0009] In some embodiments, the reference value of repeat length distribution (RLD) is a t-statistic. In some embodiments, the reference value of RLD can comprise a t-statistic based on the average read length of repeat units and the variance of RLD at the microsatellite locus of interest. In some embodiments, the t-statistic is determined based on the RLD of the test sample and the control RLD.

[0010] In some embodiments, detecting the sequencing reads comprises generating nucleotide sequencing reads from human genomic DNA, and identifying the sequencing reads that contain microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences. In some embodiments, detecting further comprises counting the number of repeat lengths of each sequencing read that is determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus.

[0011] In some embodiments, prior to generating nucleotide sequencing reads, human genomic DNA is enriched for microsatellite loci by hybridizing the human genomic DNA to an oligonucleotide probe that hybridizes to the microsatellite locus-containing DNA. In some embodiments, the oligonucleotide probe is in solution and the oligonucleotide probe hybridizes to the microsatellite locus-containing DNA to form a hybridization complex in solution. In another embodiment, the oligonucleotide probe is bound or attached to a solid support and the oligonucleotide probe is hybridized ... The nucleotide probe hybridizes to the microsatellite locus-containing DNA to form a hybridization complex on the solid support. In some embodiments, one or more of the oligonucleotide probes are labeled with a heterologous agent. In another embodiment, the oligonucleotide probe comprises one or more modified nucleotides. In one embodiment, the heterologous agent is a biotin or streptavidin moiety. In another embodiment, the heterologous agent is a fluorescent or chemiluminescent moiety. In some embodiments, the heterologous agent comprises at least one label moiety and / or at least one quencher moiety.

[0012] In some embodiments, the method includes providing a sample having human genomic DNA. In some embodiments, the sample includes human genomic DNA fragments. In one embodiment, the fragments of human genomic DNA are prepared by mechanical, chemical, or enzymatic means. In some embodiments, the human genomic DNA fragments include microsatellite loci. In some embodiments, the human genomic DNA fragments are contacted with a pool of oligonucleotide probes. In some embodiments, a portion of the human genomic DNA fragments contacted with the pool of oligonucleotide probes hybridize to the oligonucleotide probes to form hybridization complexes, thereby generating captured DNA. In some embodiments, the captured DNA is isolated from other cellular, polypeptide, or polynucleotide components.

[0013] In some embodiments, the method further comprises providing a sample containing genomic DNA fragments, contacting the sample with a pool of oligonucleotide probes, capturing DNA fragments containing microsatellite loci in hybridization complexes with the pooled oligonucleotide probes, thereby generating captured DNA, separating the hybridization complexes from unbound bound nucleic acids, eluting the captured DNA from the hybridization complexes, sequencing the captured DNA eluted from the hybridization complexes, identifying sequencing reads containing microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences, and counting the number of repeat lengths of each sequencing read determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus. In some embodiments, the genomic DNA fragments are ligated to an adaptor oligonucleotide prior to contacting with the pool of oligonucleotide probes. In some embodiments, the adaptor oligonucleotide comprises one or more barcodes, a universal primer binding site, or comprises one or more barcodes and a universal primer binding site. In some embodiments, prior to sequencing, the captured DNA can be amplified with a universal primer that hybridizes to a universal priming binding site.

[0014] In some embodiments, the sequencing comprises nanopore-based sequencing. In other embodiments, the sequencing comprises ion-based sequencing. In yet other embodiments, the sequencing comprises semiconductor-based sequencing.

[0015] In one embodiment, a method includes providing a sample comprising human genomic DNA fragments, amplifying DNA fragments comprising microsatellite loci using oligonucleotide primers specific for the microsatellite loci to generate amplicons, sequencing the amplicons, identifying sequencing reads comprising microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences, and counting the number of repeat lengths for each sequencing read determined to comprise a microsatellite locus, thereby generating an RLD for each microsatellite locus.

[0016] In some embodiments, the sample is a tissue sample or a blood sample. In some embodiments, the sample is a blood sample, such as plasma, serum and / or mononuclear peripheral cells. In some embodiments, the sample is cell-free DNA (cfDNA). In another embodiment, the sample comprises circulating tumor DNA (ctDNA). In some embodiments, the tissue sample is a tumor sample. In another embodiment, the sample is obtained from a human having or suspected of having cancer. In some embodiments, the tumor sample is obtained from a human having colorectal cancer. In some embodiments, the human has or is suspected of having HNPCC.

[0017] In some embodiments, the method further comprises treating the human with an antibody, small molecule, or other compound if the human has MSI. In some embodiments, the antibody is pembrolizumab.

[0018] In another aspect, the disclosure generally relates to an array of oligonucleotides linked to a solid support or heterologous agent. In some embodiments, the array comprises a set of oligonucleotides complementary to microsatellite loci selected from the group consisting of microsatellite loci corresponding to loci: 1-170 over a contiguous sequence of at least 12 (or 14, 16, 18, 20) nucleotides, where the set of oligonucleotides comprises at least 10, 20, 30, 40, or 50 oligonucleotides each complementary to a different microsatellite locus corresponding to loci: 1-170. In some embodiments, each oligonucleotide in the array is linked to a separate heterologous agent. In one embodiment, the heterologous agent is biotin. In some embodiments, the set of oligonucleotides in the array are linked to the same solid support. In yet another embodiment, each oligonucleotide in the array is linked to a separate solid support.

[0019] In another aspect, the disclosure generally relates to methods of enriching microsatellite loci in a human genomic DNA sample. In some embodiments, the method comprises hybridizing human genomic DNA to an array of oligonucleotides linked to a solid support or heterologous agent, where the array comprises a set of oligonucleotides complementary to microsatellite loci selected from the group consisting of microsatellite loci: 1-170 over a contiguous sequence of at least 12 (or 14, 16, 18, 20) nucleotides, where the set of oligonucleotides comprises at least 10, 20, 30, 40, or 50 oligonucleotides each complementary to a different microsatellite locus corresponding to microsatellite loci: 1-170. In some embodiments, the method further comprises removing unhybridized genomic DNA, eluting hybridized human genomic DNA from the array of oligonucleotides, thereby enriching the microsatellite loci.

[0020] In another aspect, the disclosure generally relates to a method for detecting MSI, comprising a processor and a non-transitory computer readable medium coupled to the processor, wherein the non-transitory computer readable medium comprises code executable by the processor to perform the MSI detection method. In some embodiments, the method comprises a processor; a non-transitory computer readable medium coupled to the processor, the non-transitory computer readable medium comprising code executable by the processor to perform the method, the method comprising receiving sequencing reads from human genomic DNA for a plurality of microsatellite loci; determining a repeat length distribution (RLD) for each microsatellite locus; generating a reference value for the RLD for each microsatellite locus; comparing the reference value for each microsatellite locus with an independent threshold to a threshold value for the microsatellite locus; and quantifying the number of detected microsatellite loci with an RLD reference value above the threshold. (i) comparing the number of microsatellite loci with an RLD reference value above the threshold with (ii) a locus set proportion threshold, where if the number exceeds the locus set proportion threshold, the human has microsatellite instability. In some embodiments, the method further comprises instructions for identifying sequencing reads that include microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences, and for counting the number of repeat lengths of each sequencing read determined to include a microsatellite locus, thereby generating the RLD of each microsatellite locus. In some embodiments, the RLD reference value is a t-statistic. In some embodiments, the RLD reference value can include a t-statistic based on the average read length of the repeat unit and the variance of the RLD at the microsatellite locus of interest. In some embodiments, the t-statistic is determined based on the RLD of the test sample and the control RLD. In some embodiments, the method further comprises at least one device configured to assay a plurality of MSI loci in a patient sample to determine the RLD of the plurality of MSI loci.

[0021] In some embodiments, the computer readable medium comprises computer readable program code comprising: a database comprising a list of available therapeutic agents according to MSI status; instructions for inputting a number of microsatellite loci with RLD reference values ​​above a threshold; and comparing the number of microsatellite loci with RLD reference values ​​above the threshold to a proportional threshold for the set of loci; and instructions for generating a report comprising the list of therapeutic agents, wherein the comparison to the proportional threshold for the set of loci indicates a likelihood that at least one therapeutic agent in the database is effective. [Brief description of the drawings]

[0022] [Figure 1A] FIG. 1 provides exemplary electropherogram results of MSI or MSS cell lines using the Promega MSI Analysis System. FIG. 1A shows the distribution of five different MS loci (NR-21, NR-24, BAT-25, BAT-26 and MONO-27) using an MSS cell line (CCL-228). The distribution of MS loci is consistent between the test sample (MSS) and the control sample. FIG. 1B shows the distribution of three MS loci (NR-21, BAT-25 and MONO-27) using an MSI cell line (CCL-253). The distribution of MS loci is shifted to the right in the MSI cell line compared to the control sample, indicating smaller MS repeat lengths in the MSI cell line. [Figure 1B]FIG. 1 provides exemplary electropherogram results of MSI or MSS cell lines using the Promega MSI Analysis System. FIG. 1A shows the distribution of five different MS loci (NR-21, NR-24, BAT-25, BAT-26 and MONO-27) using an MSS cell line (CCL-228). The distribution of MS loci is consistent between the test sample (MSS) and the control sample. FIG. 1B shows the distribution of three MS loci (NR-21, BAT-25 and MONO-27) using an MSI cell line (CCL-253). The distribution of MS loci is shifted to the right in the MSI cell line compared to the control sample, indicating smaller MS repeat lengths in the MSI cell line. [Diagram 2] FIG. 2 provides an example workflow of one embodiment of the present disclosure. [Diagram 3] FIG. 3 shows a flow chart including exemplary steps of a microsatellite instability computational pipeline, including training and prediction steps. [Figure 4] FIG. 4 shows an exemplary bioinformatics workflow of the microsatellite instability tissue caller component of FIG. [Diagram 5] 5A-5C are graphs showing RLD variation across microsatellite loci. FIG. 5A shows the RLD of a single MS locus in test samples and normal samples with consistent RLD. FIG. 5B shows an MS locus with an RLD from a test sample that is substantially different from the RLD obtained from a background sample. FIG. 5C shows an MS locus with RLD variation across normal samples. [Figure 6A] Figures 6A and 6B show box plots of 63 microsatellite loci from cfDNA, MSS samples, and MSI samples, as well as the distribution of the average number of repeat units at the 63 microsatellite loci. [Figure 6B]Figures 6A and 6B show box plots of 63 microsatellite loci from cfDNA, MSS samples, and MSI samples, as well as the distribution of the average number of repeat units at the 63 microsatellite loci. [Figure 7A] Figures 7A and 7B show box plots of 63 microsatellite loci from cfDNA, MSS samples, and MSI samples, as well as the distribution of skewness of the average number of repeat units at the 63 microsatellite loci. [Figure 7B] Figures 7A and 7B show box plots of 63 microsatellite loci from cfDNA, MSS samples, and MSI samples, as well as the distribution of skewness of the average number of repeat units at the 63 microsatellite loci. [Figure 8] FIG. 8 illustrates an exemplary system according to one embodiment of the present disclosure. [Figure 9] FIG. 9 illustrates another exemplary system according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] I. Introduction The present disclosure provides methods and systems for detecting microsatellite instability in a sample. The sample is optionally contacted with one or more oligonucleotide probes that are complementary across a contiguous sequence (e.g., at least 12 nucleotides) of a microsatellite locus selected from microsatellite loci: 1-170. Optionally, the sample contacted with the oligonucleotide capture probes is enriched for microsatellite loci by hybridizing the sample to the one or more oligonucleotide probes. Unbound and / or unhybridized sample is removed and the sample that hybridizes from the oligonucleotide capture probes is eluted, thereby providing enriched genomic DNA having microsatellite loci.

[0024] The present disclosure provides a method for detecting MSI in genomic DNA, comprising detecting sequencing reads from genomic DNA having a plurality of microsatellite loci, determining a repeat length distribution baseline value for each microsatellite locus with an independent threshold, quantifying the number of microsatellite loci having an RLD baseline value above the threshold, and comparing the number of microsatellite loci above the threshold to an MSI ratio threshold (if the number of microsatellite loci exceeds the MSI ratio threshold, the sample is classified as MSI).

[0025] The methods and systems described herein can improve the detection of microsatellite loci in a sample. The methods and systems described herein can improve the sensitivity and specificity of MSI detection in tissue or plasma samples. II. Definition

[0026] The terms used herein are only for the purpose of describing certain embodiments and are not intended to be limiting. The scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In this specification and the following claims, reference is made to some terms that must be defined to have the following meanings unless a contrary intention is apparent. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not be interpreted as meaning a substantial difference to the definition of the term as commonly understood in the art.

[0027] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations which are varied (+) or (-) by increments of 0.1 or 1.0, as appropriate. All numerical designations should be understood to be preceded by the term "about," although this is not always explicitly stated.

[0028] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of compounds.

[0029] The term "comprising" is intended to mean that the compounds, compositions, and methods include the recited elements, but do not exclude others. When used to define compounds, compositions, and methods, "consisting essentially of" is intended to mean excluding other elements that materially affect the basic and novel characteristics of the claimed invention. "Consisting of" is intended to mean excluding any element, step, or ingredient not specified in the claim. Embodiments defined by each of these terms are within the scope of this disclosure.

[0030] As used herein, "genomic DNA" or "gDNA" refers to the entirety of DNA in or from a cell. Most organisms contain identical copies of genomic DNA in each cell. Fragments of gDNA can be released from cells under normal cellular conditions, e.g., cell-free DNA, as discussed herein, or from tumor cells, such as circulating tumor DNA, as discussed herein. Additionally, gDNA can be extracted from cells, tissues, blood, plants, bacteria, etc., using commercially available kits (e.g., Genomic DNA Extraction Kits (Avenio ctDNA Analysis Kits) sold by Roche Sequencing Solutions, Pleasanton, CA, and PureLink™ Genomic DNA purification Kit and PureLink™ Genomic DNA Mini Kit) sold by Thermo Fisher). Genomic DNA can be isolated from intact whole cells, liquid biopsies, tissues, etc.

[0031] As used herein, "cell-free DNA" or "cfDNA" refers to fragments of genomic DNA that are derived from cells circulating in the bloodstream of a subject and are not located within the cells. Usually, cells undergo apoptosis, necrosis, and other events, which result in the release of genomic DNA fragments into the bloodstream. cfDNA has been utilized for various medical purposes, such as the detection of trisomy 21 and other genetic fetal abnormalities in maternal blood, as well as the detection of donor-derived cfDNA (dd-cfDNA) to evaluate organ transplant rejection (Bloom et al., JASN, (2017) 28: 2221-2232).

[0032] As used herein, "circulating tumor DNA" or "ctDNA" refers to tumor-derived DNA fragments that circulate in the bloodstream of a subject and are not located in circulating tumor cells. Circulating tumor cells generally occur when primary tumors release cells that enter the bloodstream or lymphatic system. These circulating tumor cells may release fragments of their genomic DNA into the bloodstream when tumor cells undergo cell death, apoptosis, or other events. Studies have shown that tumor DNA is released into the blood and is present in particularly high concentrations in plasma and serum of various types of cancer (Stroun et al., 1989 Oncology 46:318-322). Furthermore, tumor DNA released into the blood has been detected by analysis of microsatellite DNA (Hibi et al., 1998 Cancer Research 58:1405-1407; Chen et al., 1999 Clinical Cancer Research 5:2297-2303; Kopreski et al., 1999 Clinical Cancer Research 5:1961-1965; Fujiwara et al., 1999 Cancer Research 59:1567-1571). Detection of microsatellite instability in tumor DNA from plasma and serum derived from head and neck squamous cell carcinoma (Nawroz et al., 1996 Nature Med 2:1035-1037) and small cell lung carcinoma (Chen et al., 1996 Nature Med 2:1033-1035) has been shown.

[0033] As used herein, "sample" refers to tissue or bodily fluid obtained from a human or non-human mammalian subject. In some embodiments, samples include blood, blood fractions, or blood products (e.g., serum, plasma, platelets, red blood cells, peripheral blood mononuclear cells, etc.); sputum or saliva; stool, urine, other biological fluids (e.g., lymphatic fluid, saliva, prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, etc.), tissues (e.g., kidney, lung, liver, heart, brain, neural tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue), or cultured cells (e.g., primary cultures, explants, transformed cells, or stem cells). Samples can also include sections of tissues, such as biopsy and autopsy samples, and frozen sections taken, for example, for histological purposes. Samples can also include liquid biopsy samples. The sample is typically obtained from a "subject", i.e. a eukaryotic organism, most preferably a mammal such as a primate, e.g. a chimpanzee or a human, a cow, a dog, a cat, a horse, a sheep, a goat, a rodent, e.g. a guinea pig, a rat, or a mouse, a rabbit, or a bird, a reptile, or a fish.

[0034] "Microsatellite instability" or "(MSI)" as used herein refers to a form of genetic instability in which alleles of genomic DNA obtained from a sample (e.g., tissue, cell, or bodily fluid) undergo nucleotide length changes at microsatellite loci. For example, MSI can be observed upon amplification of two different samples (i.e., matched samples) of genomic DNA from a single subject, such as DNA from healthy tissue and DNA from tumor tissue, where the healthy (matched normal) sample produces amplified alleles of one or two different lengths, and the tumor sample produces amplified alleles that differ in length from the amplified alleles of the healthy sample, where at least one of the alleles is at the same locus. Generally, MSI is present as an insertion or deletion of at least one repeat unit at a microsatellite locus.

[0035] As used herein, a "microsatellite locus / locuses" refers to a region of genomic DNA that contains short repeat elements, between 1 and 6 nucleotides in length. Each repeat element is repeated one or more times within a microsatellite locus and is referred to as a "repeat unit." A microsatellite locus preferably contains at least 4 repeat units, more preferably 7 repeat units, more preferably at least 10 repeat units, and most preferably at least 20 repeat units. The term "locus" is given its ordinary meaning in the art and refers to a plurality of loci. As used herein, a "locus" refers to a unique chromosomal location that defines the location of an individual gene or DNA sequence. As used herein, a "microsatellite marker" refers to a fragment of genomic DNA that contains a microsatellite locus, and optionally includes left and right nucleic acid sequences flanking the microsatellite locus.

[0036] As used herein, "allele" refers to one of several alternative forms of a gene or DNA sequence at a particular chromosomal location (locus). At each autosomal locus, an individual has two alleles, one inherited from the paternal subject and one inherited from the maternal subject.

[0037] As used herein, "amplify" and "amplifying" and their derivatives refer to a process by which multiple copies are made from a nucleic acid locus, a target nucleic acid sequence, or a DNA fragment. Amplification can be performed by essentially any nucleic acid amplification technique, such as, but not limited to, the polymerase chain reaction (PCR) (Saiki et al. (1985) Science 230:1350-1354), reverse transcription PCR (RT-PCR) (Joyce (2002), Methods Mol Biol. 193:83-92, and Emrich et al. (2002), Methods Mol Biol. 191:99-108), ligase chain reaction (LCR) (Lee (1996) Biologicals 24(3):197-9), polymerase ligase chain reaction (Barany et al. (1991) PCR Methods Appl. 1(1):5-16), Gap-LCR (Abravaya et al. (1995) Nucleic Acids Res. 23(4):675-82), strand displacement amplification (SDA) (Walker (1993) PCR Methods Appl. 3(1):1-6), ligated linear amplification (LLA) (Killeen et al. al.(2003)Clin Chem.49(7):1050-7), rolling circle amplification (RCA) (Nilsson et al.(2002)Nucleic Acids Res.30(14):e66), transcription-mediated amplification (TMA) (Emery et al.(2000)J Clin Microbiol 38:2688-2695), nucleic acid sequence-based amplification (NASBA) (Mani et al.(1999)J Acquir Immune Defic Syndr 22:208-209 and Berndt et al.(2000)J Virol Methods 89:177-181), transcription-based amplification (Kwoh, DY, and Kwoh, TJ, American Biotechnology Laboratory, October, 1990) and self-sustained sequence replication (3SR) (Mueller et al.(1997)Histochem Cell Biol PCR can be accomplished using techniques such as PCR PCR (Dressman et al. (2003) PNAS 108:431-7) and digital PCR (Dressman et al. (2003) PNAS 15:8817-22), which use cycles of denaturation, annealing with a primer, and extension with an enzyme (usually a polymerase) to increase the number of copies of a target DNA or nucleic acid sequence to approximately 10. 6It is a technique that amplifies nucleic acids by more than 2-fold. Polymerase chain reaction processes for amplifying nucleic acids are described in U.S. Patent Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference for the description of the process. As used herein, "co-amplification" refers to a process in which multiple copies are made from two or more different loci in the same reaction mixture (e.g., the same vessel, emulsion or container) in a single amplification reaction (e.g., multiplex amplification).

[0038] As used herein, "amplicon" refers to a molecule made by copying or transcribing another molecule. Exemplary processes that can generate an amplicon include transcription, cloning, or polymerase chain reaction ("PCR") or other nucleic acid amplification techniques (e.g., strand displacement PCR amplification (SDA), duplex PCR amplification, rolling circle amplification, etc.). Typically, an amplicon is a copy of or complementary to a selected nucleic acid sequence (e.g., a template DNA, a target nucleic acid, or a genomic DNA fragment).

[0039] As used herein, the term "hybridize" refers to binding between two or more components in solution or on a solid support to form a complex. Nucleic acids "hybridize" when they bind to each other. Nucleic acids hybridize through a variety of well-characterized physicochemical forces, including hydrogen bonding, solvent exclusion, and base stacking. Hybridization can occur between fully complementary nucleic acid strands or between partially complementary nucleic acid strands that contain regions of mismatch. The degree of mismatch tolerated can generally be controlled by appropriately adjusting the hybridization conditions. Those skilled in the art of nucleic acid chemistry and molecular biology can empirically determine duplex stability by considering many variables, including, for example, the length and base pair concentration of the nucleic acid, ionic strength, and the incidence of mismatched base pairs. Extensive guides to nucleic acid hybridization are found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" (Elsevier, NY), and Ausubel (Ed.) Current Protocols in Molecular Biology, Volumes I, II, and III, (1997), each of which is incorporated by reference.Hames and Higgins (1995) Gene Probes 1 IRL Press (Oxford University Press, Oxford, England) (Hames and Higgins 1), and Hames and Higgins (1995) Gene Probes 2 IRL Press (Oxford University Press, Oxford, England) (Hames and Higgins 2) provide details regarding the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides, both of which are incorporated herein by reference. In some embodiments, hybridization can occur between primers and genomic DNA fragments, or between oligonucleotide probes and genomic DNA fragments.

[0040] As used herein, "monomorphic" refers to a locus in genomic DNA where only one allelic pattern is found to be present in the normal genomic DNA of all members of a population.

[0041] As used herein, "quasi-monomorphic" refers to a locus of genomic DNA where only one allele pattern is found to be present in the normal genomic DNA of nearly all members of a population. When a monomorphic or quasi-monomorphic locus, such as MONO-11 or MONO-27, is amplified, the resulting amplified allele size can be compared to the most frequently found allele size at that locus in the general population. In one embodiment, the method disclosed herein can detect MSI tumors of interest by amplifying two samples of genomic DNA from the same individual, where one sample is from a normal non-cancerous sample and the other sample is from a cancerous (or suspected cancerous) sample.

[0042] As used herein, "oligonucleotide" or "probe" refers to a labeled or unlabeled oligonucleotide (e.g., an oligonucleotide of at least 12, 14, 16, 18, 20, or more nucleotides) that can hybridize to genomic DNA (e.g., a human genomic DNA fragment) or a target nucleic acid under appropriate conditions. Typically, the probe is sufficiently complementary to a specific nucleic acid sequence contained within the genomic DNA to form a stable hybridization duplex with the genomic DNA under selected hybridization conditions. Hybridization assays performed using the probe under sufficiently stringent hybridization conditions allow for selective detection of specific nucleic acid sequences, such as mutants or variants of the nucleic acid sequence. The term "hybridizing region" refers to the region of the probe that is strictly or substantially complementary to, and thus hybridizes to, the genomic DNA or the target nucleic acid. The hybridizing region can refer to the entire oligonucleotide, although the probe can include additional nucleotide sequences that function, for example, as linker attachment sites that provide sites for attaching the probe sequence to a solid support or the like. In certain embodiments, probes such as 5'-nuclease probes, FRET probes, molecular beacons, etc., are included in nucleic acid molecules that include one or more labels (e.g., reporter dyes, quencher moieties, etc.) and can be utilized to detect dissociation of the probe from the target nucleic acid in a sample. In some embodiments, the hybridizing region of the probe is fully complementary to the target nucleic acid. However, in general, full complementarity is not required (i.e., the nucleic acids may be partially complementary to each other). Stable duplexes may include limited mismatched or unmatched bases. Stringency conditions may need to be modified to allow stable hybridization duplexes with one or more base pair mismatches or unmatched bases.Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001), is incorporated by reference and provides guidance for appropriate modifications. The stability of a duplex depends on many variables, including the length of the target nucleic acid, the base composition (e.g., GC content) of the target nucleic acid and the sequence of the oligonucleotide probe, temperature, and ionic conditions. Those skilled in the art will generally recognize that the exact complement of a given oligonucleotide probe is similarly useful as a probe. Those skilled in the art will also recognize that in certain embodiments, an oligonucleotide probe can also be used as a primer.

[0043] As used herein, a "primer" refers to a single-stranded oligonucleotide or DNA fragment that hybridizes to a strand of a target DNA (e.g., genomic DNA) such that the 3' end of the primer can act as a site of polymerization in the presence of a DNA polymerase enzyme. A "primer pair" refers to a pair of primers that hybridize to opposite strands of a target DNA molecule (e.g., a genomic DNA fragment) adjacent to the nucleotide region to be amplified. Typically, a "primer binding site" refers to the region of the target DNA to which the primer or primer pair hybridizes. A "locus-specific" or "target-specific" primer refers to a primer that specifically hybridizes to a portion of a described locus or its complementary strand for at least one allele of the locus and does not hybridize efficiently to other DNA sequences under the conditions used in the amplification method. Conversely, a "universal primer" refers to a primer designed to maximize random amplification of a region of DNA present in a sample without targeting a target-specific or locus-specific region of DNA. Generally, a universal primer contains a random nucleic acid sequence of about 6 to about 12 nucleotides in length and is intended to hybridize efficiently to multiple locations within genomic DNA or a selected nucleic acid molecule having a random nucleic acid sequence (e.g., an adapter oligonucleotide) under selected hybridization conditions.

[0044] As used herein, the term "identical" or "percent identity" in the context of two or more nucleic acid sequences refers to two or more sequences that are identical or have a certain percentage of identical nucleotides (i.e., % identical) when compared or aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms or by visual inspection. Exemplary algorithms that are suitable for sequence alignment and determining percent sequence identity and sequence similarity are the BLAST program, Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403-410; Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272; Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:113-141; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402; and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Res. 7:649-656, each of which is incorporated herein by reference in its entirety.

[0045] Percent identity between two nucleic acid sequences is generally calculated using standard default parameters of various methods or computer programs. As used herein, a high degree of sequence identity between two nucleic acid molecules is typically about 90% identity to 100% identity, for example, about 90% or more identity, preferably about 95% or more identity, more preferably about 98% or more identity. As used herein, a medium degree of sequence identity between two nucleic acid molecules is typically about 80% identity to about 85% identity, for example, about 80% or more identity, preferably about 85% identity. As used herein, a low degree of sequence identity between two nucleic acid molecules is typically about 50% identity to 75% identity, for example, about 50% identity, preferably about 60% identity, more preferably about 75% identity.

[0046] As used herein, "array" refers to a plurality of oligonucleotides arranged on a surface, such as, but not limited to, glass or beads, or synthesized in-situ. A DNA array typically contains a plurality of oligonucleotide probes spotted on a surface in a random or ordered manner. The oligonucleotide probes hybridize to complementary nucleic acid sequences present in a sample under high stringency conditions. Hybridization can be detected and / or quantified by various means, including fluorophores or chemiluminescence.

[0047] As used herein, a "heterologous agent" refers to an agent not found in combination with a nucleic acid molecule in nature. In one embodiment, a heterologous agent may be derived from a different organism (e.g., an antibody from a goat or mouse attached to a nucleic acid molecule of human origin). In another embodiment, a heterologous agent may be, but is not limited to, FAM or 32 In this example, the label may include FAM or 32 P is artificially linked, conjugated, bound, attached, or otherwise associated with the nucleic acid molecule. In yet another embodiment, the heterologous agent can include biotin (e.g., biotinylation of a nucleic acid). Biotin binds with high affinity and specificity to streptavidin and avidin, and these interactions can be used to separate biotinylated nucleic acid molecules from other components.

[0048] As used herein, the "repeat length distribution" or "RLD" of a locus refers to the distribution of repeat units of sequencing reads that contain the locus (e.g., determined based on alignment of reads or electropherograms). The RLD of a locus can be defined as a histogram (i.e., each length value in a range of lengths), where each length value corresponds to the number of sequencing reads that have repeat units of that particular length. The RLD is determined by analysis of sequencing reads obtained from a sample that contains a microsatellite locus. Sequence reads that contain a microsatellite locus can be aligned to a reference genome (e.g., hg38 (Human Genome version 38), a sample that lacks repeat units at the microsatellite locus, or a sample that has a known number of repeat units at the microsatellite locus). The number of repeat units present in the sequencing read can be measured by aligning the sequencing reads to the microsatellite locus of interest. If single molecule techniques are used (or at least the complete sequence is spread across the entire microsatellite locus), the number of repeat units can be readily determined from the sequencing reads after they have been aligned to a particular locus. It will be appreciated that alignment, amplification of genomic DNA fragments, or other operations / steps may introduce one or more errors into the sequencing reads, which may appear as repeat units or affect the alignment of the sequencing reads to the reference genome. For example, the RLD of locus 1 (see Table 1) is typically found to be about 13 repeat units to about 22 repeat units in MSS (e.g., non-cancerous) cells.

[0049] As used herein, "reference value" refers to a standard of measurement. In one embodiment, the reference value of the RLD is determined, for example, based on a comparison with a reference RLD. The reference value of the RLD can include one or more statistical analyses to adequately represent the RLD. In some embodiments, the reference value of the RLD is a t-statistic, such as a Student's t-test or a Wilcoxon rank test. Thus, the RLD can be represented by one or more statistical values, and these statistical values ​​of the sample RLD of the first locus can be compared to the corresponding statistical values ​​of the reference RLD. In some embodiments, the reference value is one or more summarized statistics of the RLD. Examples of such statistics include the mean, variance, skewness, kurtosis, and the like. In other embodiments, the reference value is a measure of similarity between the RLD of the tested sample and the control RLD. Examples include t-statistic, chi-squared statistic, Kolmogorov-Smirnov statistic, Kullback-Leibler information, correlation distance, cosine distance, Euclidean distance, Manhattan distance, and the like. In some embodiments, t-statistics is determined based on the RLD of test sample and the control RLD.Generally, a reference value of RLD is obtained for each microsatellite locus under evaluation.Such distance reference value can be based on the difference or ratio between the raw value of sample RLD and reference RLD, for example, the difference between probabilities that can be determined based on the normalized number of reads with a given repeat length.Here, normalization can use the total number of reads in a given measurement.Another example of normalization can be based on the total area of ​​histograms, for example, determined as electropherograms.

[0050] As used herein, "nanopore" refers to a pore, channel, or passageway having a characteristic width or diameter of about 0.1 nm to about 1,000 nm formed or otherwise provided in a membrane or other barrier material. Nanopores can be made from naturally occurring pore-forming proteins, such as α-hemolysin from Staphylococcus aureus, or mutants or variants of non-native (i.e., engineered) or naturally occurring, wild-type pore-forming proteins, such as α-HL-C46. The membrane can be an organic membrane, such as a lipid bilayer, or a synthetic membrane made from a non-naturally occurring polymeric material. The nanopore can be positioned adjacent to or in close proximity to a sensor, sensing circuitry, or an electrode coupled to a sensing circuit, such as a complementary metal-oxide semiconductor (CMOS) circuit or a field effect transistor (FET) circuit. Thus, nanopores can be used to sequence nucleic acid sequences of interest (see U.S. Patent Publication Nos. 20100331194, 20140019064, 20150176071, 20160281159, and 20170016048).

[0051] As used herein, "solid support" refers to a solid material that can be derivatized with or otherwise attached to a chemical moiety, such as an oligonucleotide probe. Exemplary solid supports include plates, beads, microbeads, tubes, fibers, whiskers, combs, hybridization chips (including microarray substrates such as those used in GeneChip™ probe arrays (Affymetrix, Inc., Santa Clara, Calif., USA)), membranes, single crystals, ceramic layers, self-assembled monolayers, and the like.

[0052] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. The term includes all cancers, whether characterized as malignant, benign, soft tissue, or solid. The term "tumor" includes all known cancers and neoplastic conditions, as well as cancers of all stages and grades, including pre- and post-metastatic cancers. Examples of various types of cancer include, but are not limited to, gastric cancer (e.g., cancer of the stomach), colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, digestive and alimentary tract cancers, such as colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, esophageal cancer, breast cancer, lung cancer, gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, prostate cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, cancer of the central nervous system, skin cancer (e.g., melanoma), lymphoma, glioma, choriocarcinoma, head and neck cancer, osteosarcoma, and blood cancer. As used herein, "tumor" includes one or more cancerous cells. In some embodiments, the cancer is colorectal cancer.

[0053] The term "agent" refers to any molecule, either natural or synthetic, such as peptides, proteins, oligopeptides (e.g., about 5 to about 25 amino acids in length, e.g., about 5, 10, 15, 20, or 25 amino acids in length), small organic molecules (e.g., organic molecules having a molecular weight of less than about 2500 daltons, e.g., less than 2000, less than 1000, or less than 500 daltons), cyclic peptides, peptidomimetics, antibodies, polysaccharides, lipids, fatty acids, inhibitory RNAs (e.g., siRNA, shRNA, or sgRNA), polynucleotides, oligonucleotides, aptamers, and drug compounds. Typically, agents are intended to have a therapeutic effect on the subject to which the agent is administered. In some cases, agents may include immune checkpoint inhibitors, such as pembrolizumab.

[0054] As used herein, the terms "treatment," "treating," and "treat" refer to any indication of success in treating or ameliorating an injury, disease, or condition, including any objective or subjective parameter, such as relief, alleviation, attenuation of symptoms or making the injury, disease, or condition more tolerable to the subject, slowing the rate of decline or decline, reducing the severity of decline at the end point of decline, and / or an improvement in the physical or mental health of the subject.

[0055] The term "pharmaceutical composition" refers to a composition suitable for administration to a subject. Typically, a pharmaceutical composition includes a pharmaceutical agent. Generally, a pharmaceutical composition is sterile and preferably free of contaminants that may induce an undesirable reaction in a subject. A pharmaceutical composition can be designed for administration to a subject in need thereof by a variety of routes of administration, including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalation, and the like.

[0056] A "therapeutic amount" or "therapeutically effective amount" of an agent (eg, an agent for treating cancer) is the amount of the agent that prevents, alleviates, relieves, or reduces the severity of symptoms of cancer in a subject.

[0057] The terms "administer," "administered," or "administering" refer to a method of delivering an agent to a desired site of biological action. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, colonic, rectal, or intraperitoneal delivery. Administration techniques that are optionally used with the agents and methods described herein include, for example, those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, PA. III. Detection Method

[0058] In one aspect, a method for detecting MSI in a sample and / or a subject is provided. In some embodiments, the described method relates to detecting MSI in a subject having or suspected of having cancer. In another aspect, the method relates to detecting a set of microsatellite loci associated with MSI tumors in a sample. The methods disclosed herein provide for the identification and / or quantification, e.g., by NGS, of genomic DNA fragments in a sample from a subject, especially when the abundance of genomic DNA fragments is low (e.g., ctDNA). The described methods ensure accurate detection and quantification of microsatellite loci in clinical samples (e.g., patient samples). This can be adversely affected if genomic DNA fragments are inaccurately normalized or quantified. Furthermore, the methods provided herein can be used to increase the yield of genomic DNA fragments with microsatellite loci, optionally through the use of oligonucleotide probes that can be attached to a solid support (e.g., selective enrichment).

[0059] As described herein, it has been found that detecting multiple microsatellite loci in a sample can predict MSI in the subject from which the sample is obtained, when compared with a predetermined threshold value.Generally, this method includes detecting sequencing reads from human genomic DNA for multiple microsatellite loci.An exemplary workflow for detecting MSI in a sample is shown in Figure 2.It will be readily apparent that some steps in the workflow of Figure 2 can be omitted and / or repeated, for example, when the sample is previously purified and prepared for sequencing.

[0060] In some embodiments, the sample is a blood sample, cell-free DNA, or a tissue sample. In one embodiment, the sample is a blood draw or liquid biopsy. In some embodiments, the sample is a tumor tissue sample. In some embodiments, the subject is a human, and the human is suspected of having cancer or has cancer. In one embodiment, the cancer is colorectal cancer. In some embodiments, the subject is treated with one or more therapeutic agents used to treat cancer. In one embodiment, the therapeutic agent is pembrolizumab.

[0061] In one aspect, the present disclosure generally relates to a method for detecting MSI, the method includes: (1) detecting sequencing reads from human genomic DNA for a plurality of microsatellite loci; (2) determining a repeat length distribution (RLD) reference value for each microsatellite locus; (3) comparing the reference value to a threshold value for each microsatellite locus, each having an independent threshold; (4) quantifying the number of microsatellite loci with RLD reference values ​​above the threshold; and (5) comparing the number of microsatellite loci with RLD reference values ​​above the threshold to a microsatellite instability (MSI) ratio threshold (if the number of microsatellite loci exceeds the MSI ratio threshold, the human has microsatellite instability).

[0062] In some embodiments, the detecting comprises generating nucleotide sequencing reads from the genomic DNA and identifying sequencing reads that comprise microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences and counting the number of repeat lengths for each sequencing read determined to comprise a microsatellite locus, thereby generating an RLD for each microsatellite locus.

[0063] In one embodiment, prior to generating nucleotide sequencing reads, the genomic DNA is enriched for microsatellite loci by hybridizing the genomic DNA to oligonucleotide probes that hybridize to microsatellite loci-containing DNA. In some embodiments, the enriched genomic DNA is washed to remove unbound or non-specific nucleic acids.

[0064] In one embodiment, the RLD criterion is a t-statistic. In some embodiments, the RLD criterion is a function of the average read length of the repeat unit and the R It may include a t-statistic based on the variance of the LD, hi some embodiments, the t-statistic is determined based on the RLD of the test sample and the control RLD.

[0065] In one embodiment, the plurality of microsatellite loci comprises a set of microsatellite loci, the set of microsatellite loci selected from the group of microsatellite loci corresponding to loci: 1-170 (see Table 1). In some embodiments, the set of microsatellite loci is selected from at least 58 microsatellite loci corresponding to the group of microsatellite loci corresponding to microsatellite loci: 1-64. In some embodiments, the set of microsatellite loci comprises the microsatellite loci of Table 2 (i.e., loci 1-4, 6-9, 11-21, 23, 25-29, 31-48 and 50-64 of Table 1). In another embodiment, the set of microsatellite loci is selected from 10 or more microsatellite loci selected from the group of microsatellite loci corresponding to loci: 1-64. In some embodiments, the set of microsatellite loci is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or more microsatellite loci selected from the group of microsatellite loci corresponding to loci: 1 to 170. In another embodiment, the set of microsatellite loci is at least 10, 20, 30, 40, or 50 microsatellite loci selected from the group of microsatellite loci corresponding to loci: 1 to 64. In one embodiment, the plurality of microsatellite loci is a group of microsatellite loci corresponding to Table 2.

[0066] In some embodiments, the plurality of microsatellite loci are selected from genomic coordinates present in one or more chromosomes of the human genome, hi another embodiment, the plurality of microsatellite loci are selected from one or more of the following genomic coordinates of microsatellite loci listed in human genome 38 (hg38): [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 2-1] [Table 2-2]

[0067] In some embodiments, the plurality of microsatellite loci comprises mono-, di-, tri-, tetra-, penta-, or hexanucleotide repeat units. In one embodiment, the plurality of microsatellite loci comprises mono-, di-, and / or trinucleotide repeat units (e.g., (A 17 ), (T 14 ), (AC 21 ), (T.G. 20 In some embodiments, where the sequencing reads are surrogates for a cell, the repeat unit length determined from the sequencing reads will be from about 2 to about 100 repeat units, from about 5 to about 80 repeat units, from about 10 to about 60 repeat units, and from about 15 to about 40 repeat units.

[0068] In some embodiments, the method includes (1) providing a sample containing human genomic DNA fragments; (2) contacting the sample with a pool of oligonucleotide probes; (3) capturing DNA fragments that contain microsatellite loci in hybridization complexes with the pooled oligonucleotide probes, thereby generating captured DNA; (4) separating the hybridization complexes from unbound nucleic acids; (5) eluting the captured DNA from the hybridization complexes; (6) sequencing the captured DNA eluted from the hybridization complexes; (7) identifying sequencing reads that contain microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences; and (8) counting the number of repeat lengths for each sequencing read determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus.

[0069] In some embodiments, the adaptor oligonucleotide is ligated, attached, or bound to the human genome fragment before the pool of oligonucleotide probes is contacted with the sample. In one embodiment, the adaptor oligonucleotide comprises one or more barcodes, a universal primer binding site, or comprises one or more barcodes and a universal primer binding site. In some embodiments, the adaptor oligonucleotide is ligated to both the 3' and 5' ends of the genomic DNA fragment to form a blunt-ended double-stranded genomic DNA fragment. The adaptor oligonucleotide with the universal primer binding site can be used in a subsequent amplification step to amplify the adaptor sequence ligated to the genomic DNA fragment.

[0070] In some embodiments, prior to sequencing, the captured DNA is amplified with a universal primer that hybridizes to a universal primer binding site present in the adapter oligonucleotide.

[0071] In some embodiments, the sequencing is nanopore-based sequencing.

[0072] In another embodiment, a method includes (1) providing a sample containing human genomic DNA fragments; (2) contacting a pool of oligonucleotide probes with the sample; amplifying DNA fragments containing microsatellite loci using oligonucleotide primers specific to the microsatellite loci to generate amplicons; (3) sequencing the amplicons; (4) identifying sequencing reads containing microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences; and (5) counting the number of repeat lengths for each sequencing read determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus. DNA extraction

[0073] Generally, any method of DNA extraction can be used to isolate genomic DNA or genomic DNA fragments for use in the present invention.In a preferred embodiment, genomic DNA is extracted from tissue, cells, liquid biopsy samples, blood or plasma samples using solution-based or solid-phase-based DNA extraction techniques.Genomic DNA extraction can include detergent-based cell lysis, denaturation of nuclear protein, and optionally removal of contaminants.For example, solution-based DNA extraction methods can include, but are not limited to, salting out or organic solvent / chaotrope methods, while solid-phase DNA extraction methods can include, but are not limited to, silica resin methods, anion exchange methods, or magnetic beads (see, for example, Chacon-Cortes and Griffiths J.Biorepository Sci App Med., (2014) 2:1-9).

[0074] Typically, DNA is extracted from a sample through a process or manipulation. Typically, a sample is treated with a lysis agent to lyse any intact cells present in the sample. DNA released from the lysed cells can be bound to a solid support, column, or membrane where it can undergo one or more washing steps to remove non-DNA components, such as contaminants and / or proteins, from the sample. Finally, the bound DNA can be released from the solid support, column, or membrane and stored in an appropriate buffer until it is ready for further processing.

[0075] In some embodiments, the genomic DNA can be sheared or broken down into smaller genomic DNA fragments, for example, by sonication, nebulization, enzymatic, or acoustic shearing methods.

[0076] In some embodiments, where the sample is a blood sample (e.g., a peripheral or whole blood draw), the sample may undergo a step of lysis of red and white blood cells, followed by binding of the released genomic DNA to a solid support, washing of the solid support with an appropriate wash buffer, and elution of the bound genomic DNA from the solid support into a suitable solution.

[0077] In some embodiments, when a sample contains circulating tumor DNA (ctDNA) (e.g., a plasma sample or a liquid biopsy sample), the ctDNA can be purified and / or concentrated to amplify the small amount of ctDNA present in the original sample. Various commercially available kits exist for isolating, purifying, and / or concentrating ctDNA from other cells or sample components, such as the AVENIO cfDNA Isolation Kit and the AVENIO ctDNA Enrichment Kit (Roche Sequencing Solutions, Pleasanton, Calif.). Nucleic acid library preparation

[0078] Once genomic DNA (e.g., cfDNA and ctDNA) is obtained, the genomic DNA , generally are subjected to one or more additional processing steps to produce a library of nucleic acid molecules that are optionally enriched prior to sequencing.

[0079] A sequencing library of nucleic acid molecules can be generated from a sample using the methods, compositions and systems provided herein or any suitable method known in the art. A variety of commercially available kits exist for sample preparation for next generation sequencing (e.g., AVENIO ctDNA Targeted Panel, Catalog Number: 08061068001 (Roche Sequencing Solutions, Pleasanton, CA); Ion Ampliseq Library Kit 2.0, ThermoFisher Scientific, Catalog Number: 4475345). In one embodiment, the sequencing library comprises a plurality of target nucleic acids (e.g., genomic DNA fragments) that are compatible with any of the sequencing systems disclosed herein or known in the art. In some embodiments, the sequencing library generated from the sample is prepared for use with an Illumina sequencing platform (e.g., HiSeq or MiSeq). Optionally, the genomic DNA fragments prepared for use in the sequencing library may include one or more oligonucleotide adaptors added to one or both ends of the genomic DNA fragments to aid in downstream analysis or classification. Optionally, the genomic DNA fragments of the sequencing library may include a barcode, for example, to distinguish one set of genomic DNA fragments from a first sample from genomic DNA fragments prepared from a second sample (e.g., samples from two different sources, or a cancerous tissue and an adjacent healthy sample (i.e., a matched normal sample) from the same source).

[0080] Steps for preparing a sequencing library may include one or more of obtaining (e.g., isolating or extracting) genomic DNA from a sample, fragmenting the genomic DNA, preparing the genomic DNA for oligonucleotide adapter ligation (e.g., end repair and polyadenylation of the 3' ends of the DNA fragments), ligating oligonucleotide adapters to one or both ends of the genomic DNA fragments, purifying the ligated genomic DNA fragments, amplifying the ligated genomic DNA fragments using one or more primers, thereby forming amplicons of the genomic DNA fragments, hybridizing the amplicons to generate captured DNA, separating the captured DNA from other unbound nucleic acids, amplifying the eluted DNA, and storing the eluted DNA for sequencing.

[0081] In other embodiments, the steps for preparing a sequencing library may include one or more of enriching the ligated oligonucleotide adaptor genomic DNA fragments, amplifying the enriched ligated oligonucleotide adaptor genomic DNA fragments using one or more primers, thereby forming amplicons of the genomic DNA fragments, and sequencing the amplicons on a sequencing platform.

[0082] In some embodiments, genomic DNA undergoes one or more of DNA polishing, end repair, and A-tailing. For example, when genomic DNA is prepared by sonication, the fragmented genomic DNA can be converted into blunt-ended genomic DNA fragments with 5' phosphate and 3' hydroxyl groups (e.g., using T4 PNK, Klenow and T4 DNA polymerase). Various commercial kits are available for processing genomic DNA, such as, but not limited to, NEB Next Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, Catalog No.: E7645S). In some embodiments, genomic DNA is processed to obtain double-stranded blunt-ended genomic DNA fragments. In a preferred embodiment, the blunt-ended genomic DNA fragments can be used for blunt-ended oligonucleotide adaptor ligation.

[0083] In some embodiments, the genomic DNA is subjected to DNA polishing, end repair, and A-tailing to obtain double-stranded genomic DNA fragments. In a preferred embodiment, the processing of the genomic DNA results in blunt-ended double-stranded nucleic acid molecules prior to ligation of one or more adaptor oligonucleotides to the 5' and / or 3' ends of the processed genomic DNA fragments. Adapter Oligonucleotides

[0084] In certain embodiments, a genomic DNA fragment (e.g., a nucleic acid molecule of interest, optionally having a double-stranded nucleic acid structure) is attached to an adaptor oligonucleotide. In some embodiments, the adaptor oligonucleotide can be designed to include an overhang or a blunt end. In a preferred embodiment, the adaptor oligonucleotide is designed to create a blunt-ended double-stranded nucleic acid molecule of interest when ligated to a genomic DNA fragment. In one embodiment, the adaptor oligonucleotide comprises a universal sequence. As used herein, a "universal sequence" refers to a sequence that can be attached to a genomic DNA fragment, for example, by ligation or other methods disclosed herein, such that the universal sequence is attached to multiple genomic DNA fragments. Thus, a universal sequence is "common" to the diverse genomic DNA fragments to which it is attached. As disclosed herein, universal sequences are particularly useful for simultaneously analyzing multiple samples. Examples of universal sequences are universal primers and universal priming sites. A universal priming site comprises a "common priming site" to which an appropriate primer (e.g., a universal primer) can hybridize and serve as a priming site for the synthesis of a nucleic acid sequence complementary to the genomic DNA fragment attached to the universal primer. Exemplary workflows and kits for performing oligonucleotide adaptor ligation include the AVENIO ctDNA Library Prep Kit (Roche Sequencing Solutions, Pleasanton, Calif.). In some embodiments, genomic DNA containing one or more oligonucleotide adaptors is purified or isolated from unligated genomic DNA fragments. amplification

[0085] In some embodiments, genomic DNA fragments, optionally including ligated adaptor oligonucleotides, are amplified before sequencing.Amplification of genomic DNA fragments is particularly useful when the starting concentration or amount of the nucleic acid of interest is low, for example, in the case of ctDNA or liquid biopsy samples.In some embodiments, amplification can be combined with the "enrichment" or "capture process" discussed below to selectively bind the nucleic acid molecule of interest.Thus, the purpose of enrichment is to concentrate the nucleic acid of interest from the sample.

[0086] A primer having a sequence complementary to the nucleic acid molecule of interest (e.g., a genomic DNA fragment) or, optionally, a primer binding site (e.g., a universal primer binding site of an adaptor oligonucleotide) is incubated with the genomic DNA fragment as a reaction mixture under conditions that allow repeated annealing, extension, and denaturation steps. Denaturation usually occurs at a temperature above 95°C, and the annealing temperature is determined based on various aspects of the genomic DNA fragment, such as, but not limited to, length, GC content, and ionic strength of the amplification reaction.

[0087] In some embodiments, the methods described herein further comprise the step of: The method includes amplifying the genomic DNA fragments (before obtaining sequencing reads). In some embodiments, the amplification includes contacting the genomic DNA fragments with one or more primers (e.g., universal primers or target-specific primers) and amplifying the genomic DNA fragments to generate amplicons. In some embodiments, the method includes detecting a nucleic acid amplification product (e.g., amplicon) generated by an amplification reaction (e.g., RT-PCR). In some embodiments, the amplification includes PCR. In some embodiments, the genomic DNA is clonally amplified in solution or in microdroplets such as an emulsion. In some embodiments, the genomic DNA is amplified by primers having nucleic acid sequences that are complementary or substantially complementary (e.g., greater than 85%) to nucleic acid sequences present in the genomic DNA fragments. In some embodiments, the genomic DNA fragments are amplified by primers having sequences complementary to primer binding sites in the genomic DNA fragments (e.g., universal primer sites in adapter oligonucleotides). In some aspects, the amplification step is an optional step that allows for the implementation of the MSI detection methods described herein in the absence of amplification of genomic DNA fragments. For example, a sample from a subject can be processed to obtain end-repaired genomic DNA fragments with 3'A tails to generate 5' phosphorylated blunt-end double-stranded nucleic acid molecules of interest. In response, adaptor oligonucleotides are ligated to one or both ends of the nucleic acid molecule, thereby forming double-stranded nucleic acid molecules with universal primer binding sites. The nucleic acid molecules with universal primer binding sites can then be concentrated from the sample (e.g., using capture probes) and denatured to form single-stranded nucleic acid molecules ready for sequencing as described below. In this example, no amplification of genomic DNA fragments is required.

[0088] In some embodiments, the second amplification of genomic DNA fragments occurs before sequencing. In one embodiment, the first amplification of genomic DNA fragments occurs before selective enrichment (e.g., using a capture probe that is complementary to the universal primer binding site of the adaptor oligonucleotide, as discussed below), and the second amplification occurs with the product of selective enrichment. In yet another embodiment, the amplification of genomic DNA fragments occurs before sequencing. In another embodiment, the amplification of genomic DNA fragments occurs prior to both selective enrichment and sequencing. nucleic acid capture

[0089] Some embodiments of the present disclosure relate to methods for selectively enriching nucleic acid molecules of interest (e.g., genomic DNA fragments, amplicons, and PCR products) from a reaction mixture. In some embodiments, the selective enrichment of nucleic acid molecules of interest utilizes a pool of oligonucleotide probes (e.g., capture probes). In some embodiments, the pool of oligonucleotide probes is available free in solution. In another embodiment, the pool of oligonucleotide probes may be attached to one or more solid supports. In one embodiment, the method relates to enriching microsatellite loci in a human genomic DNA sample.

[0090] In some embodiments, a method for enriching microsatellite loci in a human genomic DNA sample comprises (1) hybridizing human genomic DNA to an array of oligonucleotide probes, (2) removing non-hybridized genomic DNA, and (3) eluting the hybridized genomic DNA from the array of oligonucleotide probes, thereby enriching the microsatellite loci. In one embodiment, the oligonucleotide probes are linked to a solid support or a heterologous agent.

[0091] In another embodiment, the array comprises at least one microsatellite locus selected from the group consisting of microsatellite loci corresponding to loci: 1-170 (see Table 1). In one embodiment, the set of oligonucleotide probes comprises at least 10, 20, 30, 40, or 50 oligonucleotide probes each complementary to a different microsatellite locus corresponding to loci 1-64. In one embodiment, the set of oligonucleotide probes comprises at least 10, 20, 30, 40, or 50 oligonucleotide probes each complementary to a different microsatellite locus corresponding to the loci in Table 2.

[0092] In some embodiments, the oligonucleotide probes are free in solution. In other embodiments, the oligonucleotide probes are linked to a heterologous agent. In one embodiment, the heterologous agent is biotin. In other embodiments, the oligonucleotide probes are linked to the same solid support. In yet other embodiments, the oligonucleotide probes are linked to separate solid supports.

[0093] In one embodiment, the oligonucleotide probe can include modified nucleotides. In some embodiments, the oligonucleotide probe can include a hybridization region that is complementary to a portion of the nucleic acid molecule of interest (e.g., a genomic DNA fragment, an amplicon, or a related PCR product). In certain embodiments, the hybridization region can include at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, and at least about 100 nucleotides. In another embodiment, the hybridization region is about 6 to about 50, about 8 to about 40, about 10 to about 30 nucleotides in length. In yet another embodiment, the hybridization region is at least 5, 8, 10 or 12 nucleotides in length and is at least 70% complementary to the nucleic acid of interest. In one embodiment, the genomic DNA is enriched for microsatellite loci by hybridizing the genomic DNA to oligonucleotide probes that hybridize to microsatellite loci-containing DNA.

[0094] In some embodiments, the hybridization region of the oligonucleotide probe may comprise at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% complementarity to the nucleic acid molecule of interest. In some embodiments, the hybridization region comprises a nucleic acid sequence that is 100% complementary to a portion of the nucleic acid molecule of interest (e.g., a genomic DNA fragment).

[0095] Heterogeneous agents can be used in the methods described herein to detect nucleic acids. A wide variety of heterogeneous agents can be used, with the agent being selected according to the required sensitivity, the required specificity, ease of binding to the oligonucleotide probe, stability requirements, and available equipment and disposal provisions. Suitable heterologous agents include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocyanate (TRITC), Cy3, Cy3.5, Cy5, Cy5.5, JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, SYBR-Green, EtBr, and ROX, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), colorimetric labels, chemiluminescent labels, bioluminescent labels, antibodies, antigens, haptens, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, digoxigenin, metals, and the like.

[0096] Specific binding of the oligonucleotide probe to genomic DNA can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, etc. attached to the oligonucleotide probe. 32Oligonucleotide probes labeled with fluorescein (P) may be used. Chemiluminescence assays using chemiluminescent oligonucleotides specific for genomic DNA may be used. Oligonucleotide probes labeled with fluorescent dyes are also suitable. Examples of fluorescent dyes include, but are not limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and Lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease, etc. Horseradish peroxidase detection systems can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which produces a soluble product in the presence of hydrogen peroxide detectable at 450 nm. Alkaline phosphatase detection systems can be used, for example, with the chromogenic substrate p-nitrophenyl phosphate, which produces a soluble product easily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm, and a urease detection system can be used with substrates such as urea bromocresol purple (Sigma Immunochemicals; St. Louis, MO).

[0097] Signals from direct or indirect labels can be detected, for example, by spectrophotometers that detect color from chromogenic substrates; 125The assay can be performed using a radiation counter that detects radiation, such as a gamma counter for detecting I, or a fluorometer that detects fluorescence in the presence of light of a specific wavelength. For detection of enzyme-linked oligonucleotide probes, a spectrophotometer such as the EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) can be used according to the manufacturer's instructions to perform quantitative analysis. If necessary, the assay can be automated or run robotically, and signals from multiple samples can be detected simultaneously. In some embodiments, the amount of signal can be quantified using an automated high-content imaging system. High-content imaging systems are commercially available (e.g., ImageXpress, Molecular Devices Inc., Sunnyvale, CA).

[0098] As mentioned above, the oligonucleotide probe can include affinity tag. Affinity tag is useful for separating the nucleic acid molecules hybridized to hybridization region. As used herein, the term "affinity tag" and grammatical equivalents can refer to the components of a multi-component complex, where the components of the multi-component complex specifically interact or bind to each other. For example, affinity tag can include biotin that can bind to streptavidin. Other examples of multicomponent affinity tag complexes include ligands and their receptors, such as avidin-biotin, streptavidin-biotin, and derivatives of biotin, streptavidin, or avidin, such as, but not limited to, 2-iminobiotin, desthiobiotin, NeutrAvidin (Molecular Probes, Eugene, Oreg.), CaptAvidin (Molecular Probes), and the like; binding proteins / peptides, such as maltose-maltose binding protein (MBP), calcium-calcium binding protein / peptide (CBP); antigen-antibodies, such as epitope tags, including c-MYC, HA VSV-G, HSV, V5, and FLAG Tag™, and their corresponding anti-epitope antibodies; haptens, such as dinitrophenyl and digoxigenin, and their corresponding anti-epitope antibodies. These include corresponding antibodies; aptamers and their corresponding targets; fluorophores and anti-fluorophore antibodies; and others.

[0099] As mentioned above, the oligonucleotide probe can include a reporter moiety. As used herein, the term "reporter moiety" and grammatical equivalents can refer to any distinguishable tag, label, or group. Those skilled in the art will understand that different kinds of reporter moieties can be used in the methods described herein, either individually or in combination with one or more different reporter moieties. In certain embodiments, the reporter moiety can emit a signal. Examples of signals are fluorescent, chemiluminescent, bioluminescent, phosphorescent, radioactive, calorimetric, or electrochemiluminescent signals. Exemplary reporter moieties include fluorophores, radioisotopes, chromogens, enzymes, antigens with epitope tags, semiconductor nanocrystals such as quantum dots (see U.S. Pat. No. 6,544,732), heavy metals, dyes, phosphorescent groups, chemiluminescent groups, electrochemical detection moieties, binding proteins, fluorophores, rare earth chelates, transition metal chelates, near infrared dyes, electrochemiluminescent labels, and reporter moieties compatible with mass spectrometry, such as mass tags, charge tags, and isotopes. Additional reporter moieties that can be used in the methods described herein include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C. 32 Reporter moieties include spectral labels such as .P, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, etc.), spectral calorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, magnetic labels, electric labels, thermal labels, and mass tags. Reporter moieties also include enzymes (e.g., horseradish peroxidase) and magnetic particles. These and other exemplary reporter moieties are described in the 6th edition of Molecular Probes Handbook by Richard Haugland, which is incorporated herein by reference.

[0100] In some embodiments, the oligonucleotide probes can be associated with a substrate. Examples of substrates include microspheres, flat surfaces, columns, and the like. By "microspheres" or "beads" or "particles" or grammatical equivalents herein is meant small discrete particles. The composition of the substrate will vary depending on the application. Suitable compositions include those used in the synthesis of peptides, nucleic acids, and organic moieties, such as, but not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, triazoles, carbon graphite, titanium dioxide, cross-linked dextrans such as latex or Sepharose, cellulose, nylon, cross-linked micelles, and Teflon can all be used. The beads do not have to be spherical, and irregular particles may be used. Furthermore, the microspheres do not have to be consistent in size. In a preferred embodiment, one or more oligonucleotide probes are associated (e.g., bound, ligated, or conjugated) with beads that have an average particle size that varies by less than 10%. In some embodiments, the substrate can include a metal composition, such as iron, and can also include magnetic properties. One exemplary embodiment includes oligonucleotide probes comprising streptavidin-coated magnetic beads (Ito et al., (1992) PNAS 89:495-498). Additionally, the beads may be porous, thereby increasing the surface area of ​​the beads available for association with the oligonucleotide probes. Typically, the size of the beads ranges from nanometers, i.e., 1 nm to 999 nm, millimeters, i.e., 1 mm to 9 mm, and microns, i.e., 0.1 μm to about 990 μm.

[0101] In certain embodiments, the oligonucleotide probe can include a cleavable moiety, such as a cleavable linker. The cleavable moiety can include a functional group that can be cleaved in a manner that allows it to be cleaved photolytically, chemically, thermally, or enzymatically. For example, see U.S. Patent No. 5,721,099, U.S. Patent Publication No. 20040166529 and U.S. Patent Publication No. 20100236644. 00022761, as well as Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 2nd ed. Wiley, 1991.

[0102] In some embodiments, one or more oligonucleotide probes (e.g., a pool of oligonucleotide probes) can be contacted with a nucleic acid of interest (e.g., a genomic DNA fragment, an amplicon, or an associated PCR product) such that a hybridization complex is formed between the oligonucleotide probe and the nucleic acid of interest, thereby generating a captured DNA that includes a microsatellite locus. In some embodiments, the hybridization complex can be formed by denaturing the nucleic acid of interest prior to contacting with the pool of oligonucleotide probes. In a preferred embodiment, the hybridized nucleic acid of interest and the pool of oligonucleotide probes (e.g., hybridization complexes) are separated from unhybridized, unbound, and non-specifically bound nucleic acids.

[0103] In some embodiments, the nucleic acid enrichment may include binding the oligonucleotide probes to a binding moiety. The binding moiety may bind to an affinity tag and may include a ligand of such an affinity tag. The binding moiety may be attached to a substrate. In some embodiments, the nucleic acid enrichment may include removing non-hybridized nucleic acids from the hybridized nucleic acids and the oligonucleotide probes. The removing method may include, for example, washing. Methods for washing nucleic acids are well known in the art. Such methods may be applied to methods that include hybridizing nucleic acids to oligonucleotide probes. A variety of hybridization and washing conditions may be used, for example, high, medium, and low stringency conditions. For example, see Maniatis et al. al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in See, Molecular Biology, ed. Ausubel, et al. Stringent conditions will vary in different circumstances, including sequence dependent. For an extensive guide to nucleic acid hybridization, see Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993), the disclosure of which is incorporated herein by reference in its entirety. Generally, stringent conditions are those that meet the thermal melting point (T m ) is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the oligonucleotide probe hybridizes to the nucleic acid of interest. Stringent conditions include a salt concentration of less than about 1.0 M sodium ion, e.g., about 0.01 M to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and a temperature below the T of short probes (e.g., 10 to 50 nucleotides). m and the T of long probes (e.g., greater than 50 nucleotides). m At least about 6° C. lower than the

[0104] In some embodiments, the captured DNA (e.g., hybridized nucleic acid molecule and corresponding hybridized oligonucleotide probe) can be released from the hybridization complex after removal of non-specific and unbound nucleic acids. As will be understood, the method of separating the nucleic acid molecule from the hybridized oligonucleotide probe will vary depending on the type of bond between the nucleic acid molecule and the oligonucleotide probe. In some embodiments, the nucleic acid molecule can be separated from the oligonucleotide probe by denaturing the nucleic acid molecule, for example, by increasing the temperature of the reaction mixture. In some embodiments, the nucleic acid molecule can be separated from at least a portion of the oligonucleotide probe by cleaving the cleavable linker. In some embodiments, the nucleic acid molecule can be separated from at least a portion of the oligonucleotide probe by digesting at least a portion of the oligonucleotide probe, for example, an RNA oligonucleotide probe can be digested with RNAse. In some embodiments, the nucleic acid molecule can be separated from the oligonucleotide probe by adjusting the ionic strength of the reaction mixture.

[0105] In some embodiments, enrichment of the nucleic acid molecule of interest associated with one or more oligonucleotide probes can include one or more enrichment rounds. In one embodiment, the enrichment method can include (1) denaturing the nucleic acid molecule of interest and hybridizing a labeled oligonucleotide probe to the nucleic acid molecule of interest (e.g., a biotin-labeled probe hybridized to a genomic DNA fragment), (2) binding the labeled oligonucleotide probe and the hybridized nucleic acid molecule of interest to a solid support, such as magnetic streptavidin beads, (3) washing the solid support to remove unbound nucleic acids, and (4) eluting the enriched nucleic acid molecule of interest from the solid support. In a second round of enrichment, the enriched nucleic acid molecule of interest can undergo steps (1)-(4).

[0106] In some embodiments, the eluted DNA is stored in an appropriate buffer for further processing, such as sequencing. At this point, the enriched nucleic acid molecules may be referred to as a "sequencing library." In some embodiments, the sequencing library is quantified and / or pooled with other sequencing libraries prior to sequencing. Sequencing

[0107] As indicated above, the prepared nucleic acid molecules of interest (e.g., a sequencing library) are sequenced using a sequencing assay as part of a procedure for determining the sequencing reads of multiple microsatellite loci. Any of a number of sequencing technologies or sequencing assays can be utilized. As used herein, the term "next generation sequencing (NGS)" refers to a sequencing method that allows for massively parallel sequencing of clonally amplified molecules and single nucleic acid molecules.

[0108] Non-limiting examples of sequence assays suitable for use in the methods disclosed herein include nanopore sequencing (U.S. Patent Publication Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0119259, and 2015 / 0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., Nature 1999, 11:111-112 (1999)), and ELISA (Hu et al., Nature 1999, 11:111-112 (1999)). Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998), NGS methods including, but not limited to, sequencing by synthesis (e.g., HiSeq™, MiSeq™, or Genome Analyzer, each available from Illumina), sequencing by ligation (e.g., SOLiD™, Life Technologies, etc.), ion semiconductor sequencing (e.g., Ion Torrent™, Life Technologies, etc.), SMRT® sequencing (Pacific Biosciences), and pyrosequencing (e.g., 454™ sequencing, Roche Applied Science). See, e.g., Liu et al., J. Biomed Biotechnol, 2012, 2012:251364, incorporated herein by reference.

[0109] Commercially available sequencing technologies include the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.), the sequencing-by-synthesis platforms from Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.). Other sequencing technologies include, but are not limited to, Ion Torrent technology (ThermoFisher Scientific), and nanopore sequencing (Roche Sequencing Solutions, Genia Technology of Santa Clara, Cal.) and Oxford Nanopore Technologies (Oxford, United Kingdom).

[0110] In another non-limiting embodiment, the method described herein includes obtaining sequencing reads of genomic DNA using nanopore sequencing (e.g., as described in Soni GV and Meller A. Clin Chem 53:1996-2001 (2007)). Nanopore sequencing DNA analysis technology is available from a number of companies, such as Oxford Nanopore Technologies (Oxford, United Kingdom) and Sequenom (San Diego, CA). Nanopore sequencing is a single molecule sequencing technology in which a single molecule of DNA is directly sequenced as it passes through a nanopore. A nanopore is a small hole, typically on the order of one nanometer in diameter. When the nanopore is immersed in a conductive fluid and a potential (voltage) is applied across it, a small current is generated by the conduction of ions through the nanopore. The amount of current that flows is influenced by the size and shape of the nanopore. As a DNA molecule passes through the nanopore, each nucleotide of the DNA molecule blocks the nanopore to a different extent, changing the magnitude of the current through the nanopore to a different extent. This change in current as the DNA molecule passes through the nanopore thus correlates to the nucleotide that was incorporated into the synthesized DNA sequence. In one embodiment, nanopore sequencing can be performed using the Genia technology from Roche Sequencing Solutions (Santa Clara, CA). The Genia technology uses a protein nanopore in a lipid bilayer for each well, e.g., hundreds of thousands or millions of wells, on a substrate chip. The well contains a polymerase and a DNA template, and nucleotides are added to the DNA template by polymerization with the polymerase. Unique tags are attached to the four nucleotides, corresponding to adenine (A), thymine (T), cytosine (C), and guanine (G). Once a nucleotide is added (catalyzed) to the DNA template, the tag is threaded into the nanopore, resulting in a unique signal (i.e., current) that corresponds to the particular nucleotide that was incorporated.Specifically, an alternating current (AC) signal is applied across the nanopore, causing the tag to enter and exit the nanopore multiple times and data to be acquired multiple times during the process (see, e.g., U.S. Patent No. 2015 / 0119259, U.S. Patent No. 2016 / 0178577 and U.S. Patent No. 2017 / 0254797).

[0111] Typically, nanopore sequencing (nanopore sequencing by synthesis) involves synthesizing a DNA strand complementary to a template nucleic acid using a DNA polymerase or other chain-extending enzyme, and determining the identity of each nucleotide as it is added to the growing strand, thereby determining the template sequence. Each added nucleotide is detected by monitoring the flow of current over time through a nanopore located adjacent to the polymerase active site as the strand is synthesized. Obtaining an accurate signal requires proper positioning of the polymerase active site near the nanopore, and the use of a tag for each added nucleotide that can enter the nanopore and impart a discernible change in the current through the nanopore. To provide accurate nanopore sequencing, the tag must enter the nanopore, reside there for a sufficient amount of time (i.e., "dwell time"), and impart a sufficiently detectable and discernible disruption of the current through the nanopore (i.e., "current disruption") while present in the nanopore such that the particular nucleotide associated with the tag can be clearly distinguished from other tagged nucleotides.

[0112] In another non-limiting embodiment, the method described herein includes obtaining sequence reads of genomic DNA using a chemically sensitive field effect transistor (chemFET) array (e.g., as described in US Patent Application Publication No. 2009 / 0026082). In one example of this technology, a nucleic acid molecule of interest can be placed in a reaction chamber, where the nucleic acid molecule is hybridized to a sequencing primer in the presence of a polymerase. The incorporation of one or more triphosphates at the 3' end of the sequencing primer can be identified by the chemFET as a change in current.

[0113] In another embodiment, Ion Torrent™ sequencing, which combines semiconductor technology with label-free sequencing chemistry, can be used to directly translate chemically end-coded information (A, C, G, T) into digital information (0, 1) on a semiconductor chip. Ion Torrent uses a high-density array of microfabricated wells to perform massively parallel incorporation of nucleotides into the 3' end of a sequencing primer, simultaneously releasing a hydrogen ion for each nucleotide that is incorporated. Each well holds a single nucleic acid molecule of interest, and beneath the well is an ion-sensitive layer (ISFET) and an ion sensor. When a nucleotide is incorporated into the 3' end of the sequencing primer, a hydrogen ion is released, causing a slight change in the pH of the solution, which can be detected by the ion sensor. The Ion Torrent sequencer then flows each nucleotide into the chip in large quantities, followed by a wash step to remove any unincorporated nucleotides. If no nucleotides are incorporated during application to the chip, there is no change in voltage and no record of the incorporated nucleotide.

[0114] Any detection method suitable for the sequencing assay used can be used. In some embodiments, the sequencing assay can use a label in the detection method. As used herein, the term "label" refers to a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent dyes, luminescent agents, radioisotopes (e.g., 32 P, 3 H), electron-dense reagents, enzymes, biotin, digoxigenin, or haptens and proteins, or other entities that can be made detectable, for example, by incorporating a radioactive label into an oligonucleotide. Exemplary detection methods include radioactive detection (e.g., 32P), optical absorption detection, e.g., UV-visible absorption detection, and luminescence detection, e.g., fluorescence or chemiluminescence. For example, genomic DNA fragments can be detected using a sequencing platform by simultaneously or sequentially scanning all or part of each labeled genomic DNA fragment, amplicon, or associated PCR product, depending on the sequencing platform and method used. Radioactive signals (e.g., 32 In the case of GFP (P), a phosphorimager device can be used (Johnston et al., 1990; Drmanac et al., 1992; 1993). In another embodiment, the genomic DNA fragments can be unlabeled and their production can be detected by the release of hydrogen ions during the incorporation of each nucleotide during DNA synthesis (i.e., Ion Torrent sequencing and, for example, U.S. Pat. Nos. 9,139,874, 9,309,557 and 9,657,281). In another embodiment, the sequencing assay can include nanopore sequencing, such as, but not limited to, the sequencing methods disclosed in U.S. Pat. Nos. 8,852,864, 8,968,540, 9,121,059, 9,279,153 and 9,542,527.

[0115] In some embodiments, the signal of any detection method employed can be measured and / or analyzed manually or by suitable computational methods to provide sequencing data that is preferably translated into sequencing reads. In some embodiments, computational methods can be used to translate the sequencing reads via a computer system.

[0116] In some embodiments of the methods described herein, the sequencing reads obtained from a sequencing assay comprise a length of about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, about 60 nucleotides, about 65 nucleotides, about 70 nucleotides, about 75 nucleotides, about 80 nucleotides, about 85 nucleotides, about 90 nucleotides, about 95 nucleotides, about 100 nucleotides, about 120 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, or about 500 nucleotides. In some embodiments, the sequencing reads obtained from the sequencing assay include about 20bp, about 25bp, about 30bp, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about 90bp, about 95bp, about 100bp, about 110bp, about 120bp, about 130, about 140bp, about 150bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp, or about 500bp. In some embodiments, the sequencing reads include at least 10 or more nucleotides identified in a single sequencing reaction. Therefore, it is not necessary for each sequencing reaction to be complete, and it is not necessary for each sequencing read to be unambiguous.

[0117] Any suitable method, calculation, or threshold can be used to determine whether an alignment of a sequencing read aligns with a microsatellite locus (e.g., Li et al., Bioinformatics, (2009) 25(14):1754-1760). Preferably, at least 8, 10, 20, 30, 40, 50, or more consecutive nucleotides from each sequencing read align with the corresponding microsatellite locus. In one embodiment, each sequencing read includes 10 or more consecutive nucleotides corresponding to the microsatellite locus of interest. In some embodiments, the sequencing reads may overlap (tile) across a genome, gene, or domain of interest (e.g., a region of a chromosome, an entire chromosome, an exon, an intron, etc.). In some embodiments, the sequencing reads include at least 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or more of the genome. In another embodiment, the sequencing reads align to one, more than one, or all of the set of microsatellite loci under evaluation.

[0118] In one embodiment, the sequencing reads can be mapped to a reference genome such as the GRCh37 / hg19 sequence available on the world wide web at http: / / hgdownload.cse.ucsc.edu / golden / path / hg19 / bigzips / , or the GRCh38 / hg38 sequence available on the world wide web at http: / / hgdownload.cse.ucsc.edu / golden / path / hg38 / bigzips / . Other sources of public sequence information include GenBank, dbEST, dbSTS, EMBL (the European Molecular Biology Laboratory), and DDBJ (DNA Data Bank of Japan). Includes:

[0119] Many computer algorithms are available for aligning nucleotide sequences such as sequencing reads described herein, including, but not limited to, BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), FASTA (Person & Lipman, 1988), BOWTIE (Langmead et al., Genome Biology 10:R25.1-R25.10

[2009] ), or ELAND (Illumina, Inc., San Diego, Calif., USA).

[0120] A number of sequencing reads are obtained per sequencing assay, as evidenced by the amount of genomic DNA fragments typically present in a sample. In some embodiments, at least about 3×10 6 Sequencing reads of at least approximately 5 x 10 6 Sequencing reads of at least approximately 8 x 10 6 Sequencing reads of at least approximately 10 x 10 6 Sequencing reads of at least approximately 15 x 10 6 Sequencing reads of at least approximately 20x10 6 Sequencing reads of at least approximately 30x10 6 Sequencing reads of at least approximately 40x10 6 of sequencing reads, or at least approximately 50x10 6 In some embodiments, the sequencing reads comprise about 20 to about 500 nucleotides per sequencing read. In one embodiment, each sequencing read or a portion (e.g., about 10% or more) of the total sequencing reads are mapped to a reference genome. In another embodiment, the sequencing reads that are mapped to the reference genome are retained and utilized for further processing (e.g., to determine a baseline value of RLD), while the sequencing reads that do not map or align to the reference genome and / or microsatellite loci are discarded. analysis

[0121] In some embodiments, the data obtained from the sequencing reaction or assay is in the form of nucleotide sequences representing the sequencing reads obtained from the sample. In some embodiments, the sequencing assay generates 1-100 billion, 50-10 billion, and 100-1 billion sequencing reads. In some embodiments, the sequencing reads (e.g., raw data) can be refined to remove poor or low quality sequencing reads. In some embodiments, the sequencing assay provides more than 100 sequencing reads and less than 1 million sequencing reads per microsatellite locus. In another embodiment, the sequencing reads can be de-duplicated to remove duplicate reads from the sequencing data. In another embodiment, the sequencing reads can be refined to remove adapter oligonucleotides, universal primer binding sites, and / or barcode sequences from the sequencing reads. In some embodiments, the sequencing reads can be stored in a suitable bioinformatics format, such as, but not limited to, FASTA and FASTQ formats. An exemplary workflow for processing sequencing reads obtained by the methods of detecting microsatellite instability disclosed herein is provided in FIG. 4.

[0122] After sequencing reads are generated, sequencing reads that contain microsatellite loci are identified (e.g., by aligning the sequencing reads to a reference genome or a set of known microsatellite loci). Typically, the number of repeat units detected in the sequencing reads at the microsatellite loci can be determined (e.g., counted), thereby generating a repeat length distribution (RLD) across the sequencing reads for each microsatellite locus under evaluation.

[0123] The sequencing reads are aligned to the microsatellite loci, so that the number of repeat units of the sequencing reads can be determined. For example, a microsatellite locus with a dinucleotide repeat of "GT" can be confirmed as GT6 in the first sequencing read, as GT9 in the second sequencing read, as GT6 in the third sequencing read, and as GT7 in the fourth sequencing read. Any method can be used to determine the repeat unit length of the microsatellite locus in the sequencing read. In a preferred method, the determination of the repeat unit length is performed by computer analysis (e.g., software analysis or algorithm).

[0124] Typically, the number of repeat unit lengths in microsatellite loci in MSS or normal (non-cancerous cells) is stable and consistent (see, e.g., Figures 1A and 1B). However, the number of repeat unit lengths of individual microsatellite loci may vary between different cells. Some cells contain microsatellite loci with identical repeat unit lengths for cells of the same type (e.g., healthy non-cancerous cells), while other cells (e.g., CRC cells) may contain microsatellite loci with shorter or longer repeat unit lengths. In some embodiments, when sequencing reads are surrogates for cells, the repeat unit length determined from the sequencing reads will be about 2 to about 100 repeat units, about 5 to about 80 repeat units, about 10 to about 60 repeat units, and about 15 to about 40 repeat units.

[0125] Once the RLDs of the microsatellite loci are generated, the reference value of the RLDs can be determined. In some embodiments, the reference value of the RLDs is a t-statistic. In one embodiment, the reference value of the RLDs is a t-statistic, such as, but not limited to, a Student's t-test or a Wilcoxon rank test. In another embodiment, the reference value of the RLDs is determined based on the average read length of a given microsatellite locus. In yet another embodiment, the reference value of the RLDs is determined based on the skewness of the RLDs among the sequencing reads of a given microsatellite locus. In some embodiments, the reference value of the RLDs is a t-statistic determined based on the average read length and the variance of the RLDs. In another embodiment, the reference value of the RLDs of each microsatellite locus under evaluation is based on a two-sample t-test between the test sample (e.g., ctDNA from a sample suspected of having CRC) and a normal background sample (e.g., a reference sample or a matched healthy sample). In some embodiments, the t-statistic is determined based on the RLDs of the test sample and the control RLDs. In some embodiments, the baseline value of the RLD is determined in the absence of a matched sample by reference to a known sample or a reference sample, for example, where the baseline value of the RLD for each microsatellite locus under evaluation is known. In some embodiments, the baseline value of the RLD is determined by comparing the RLD of the microsatellite under evaluation to a pre-calculated background RLD for a given microsatellite locus.

[0126] Thus, one or more statistical values ​​can be determined for the sample RLD and the pre-calculated normal background RLD, and the statistical values ​​can be compared, for example, as a t-statistic. In other embodiments, the raw values ​​of the two RLDs can be compared, for example, to determine a distance.

[0127] In other embodiments, the criterion value is a measure of similarity between the RLD of the tested sample and the control RLD.Distance criterion values ​​include, but are not limited to, Kullback-Leibler divergence, correlation distance, cosine distance, Euclidean distance, Manhattan distance, and Kolmogorov-Smirnov statistic.Distance criterion value can be used to evaluate the similarity between two RLDs.For example, the difference between the values ​​(counts) at each length of two RLDs can be taken as, for example, the difference of numerical vectors, where each vector is an array of counts of the number of reads with a given repeat length.

[0128] In some embodiments, inconsistent RLDs across a single microsatellite locus can be filtered using a distance metric to measure the similarity between two RLDs. The distance metric can be determined using pairwise distances, e.g., the difference or ratio of corresponding values ​​of each RLD of a pair of RLDs. For example, a first RLD can have a first probability that the read is of a first length (e.g., as determined from a normalized count of reads having the first length), and a second RLD can have a second probability that the read is of the first length.

[0129] In some embodiments, a microsatellite locus is considered to have an inconsistent RLD if the majority of pairwise distances from the confirmed RLD are "longer" than any distance criterion tested. As used herein, an RLD is "long" if it is greater than the median plus two standard deviations distance from all sample pairs of all microsatellite loci. In some embodiments, the RLD is measured using the median rather than the mean to reduce the influence of outliers.

[0130] In some embodiments, when filtering inconsistent samples for a given microsatellite locus, Euclidean distance may be preferred to measure the similarity between two RLDs. In one embodiment, a sample is considered inconsistent compared to other samples if its Euclidean distance to a normal background sample is greater than the median plus two standard deviation distance from all samples for all microsatellite loci.

[0131] In one embodiment, a sample with MSI comprises RLDs of one or more microsatellite loci that are different from the RLDs of the same microsatellite loci identified in the MSS sample. In one embodiment, the MSI sample may comprise RLDs with longer and / or greater mean read length skewness compared to the RLDs of the same microsatellite loci in the MSS sample. For example, Figures 1A and 1B demonstrate that the microsatellite locus NR-21 is identified to have greater read length and skewness in the MSI cell line compared to the RLD of NR-21 in the MSS cell line.

[0132] In some embodiments, the reference value is prepared by comparing the RLD of a given microsatellite locus to a pre-calculated normal background RLD of the corresponding microsatellite locus. In some embodiments, the pre-calculated normal background is based on a set of normal samples. The reference value is then compared to a threshold value for the corresponding microsatellite locus. In some embodiments, the threshold value is selected to maximize the difference between the true positive rate and the false positive rate of the training set. In some embodiments, each microsatellite locus has its own independent threshold value to which the calculated RLD reference value is compared. In some embodiments, the threshold value comprises a repeat length distribution typically seen in MSS samples. In some embodiments, the threshold value comprises a repeat length distribution of normal matched samples. In some embodiments, the threshold value comprises an RLD reference value obtained from an MSS cell line. In some embodiments, the threshold value is a value at which the microsatellite locus is identified as MSI. In some embodiments, if the RLD reference value exceeds the threshold value for the corresponding microsatellite locus, the microsatellite locus under evaluation is identified as MSI.

[0133] In some embodiments, one or more microsatellite genes obtained from the sample The RLD reference value of the locus is compared to the threshold value of the corresponding microsatellite locus. In one embodiment, microsatellite loci in the sample having RLD reference values ​​exceeding the threshold value of the corresponding microsatellite locus are identified (e.g., MONO-27, NR-21 and BAT-25). In another embodiment, the RLD reference value of the microsatellite locus from the sample is compared to the threshold value of the corresponding microsatellite locus, and the number of microsatellite loci exceeding the corresponding threshold is quantified (e.g., 58 microsatellite loci from the set of 64 microsatellite loci evaluated (e.g., microsatellite loci corresponding to loci: 1-64) have RLD reference values ​​exceeding the corresponding microsatellite locus threshold).

[0134] In some embodiments, the number of microsatellite loci with RLD reference values ​​above the corresponding threshold (e.g., 58 microsatellite loci out of a total of 64 microsatellite loci) is compared to a microsatellite instability ratio threshold (MSI-PT). In one embodiment, if the number of microsatellite loci with RLD reference values ​​above the corresponding threshold (e.g., 58 microsatellite loci) exceeds the MSI-PT, the subject from whom the sample was obtained is classified as microsatellite unstable (MSI). In another embodiment, if the number of microsatellite loci with RLD reference values ​​above the corresponding threshold (e.g., 58 microsatellite loci) is equal to or less than the MSI-PT, the subject from whom the sample was obtained is classified as microsatellite stable (MSS).

[0135] The microsatellite instability ratio threshold (MSI-PT) represents a fraction or percentage of the total number of microsatellite loci evaluated. The MSI-PT threshold can be a predefined value or range. In some embodiments, the MSI-PT is at least greater than 35%. The purpose of the MSI-PT is to accurately separate or differentiate samples into MSS or MSI status. In one embodiment, the MSI-PT is at least about 40%, 50%, 60%, 70%, 75%, or more. In the above example, 58 microsatellite loci from a pool of 64 microsatellite loci are determined to have RLD reference values ​​exceeding the threshold of the corresponding microsatellite loci. Therefore, the 58 microsatellite loci are determined to be MSI loci, and the remaining 6 microsatellite loci are determined to be MSS. Furthermore, 58 microsatellite loci / 64 microsatellite loci is equivalent to 90% of the microsatellite loci being classified as MSI. Since 90% is greater than 35% of the MSI-PTs, the sample is classified as MSI. In some embodiments, if the sample has less than 35% of the MSI-PTs, the sample is classified as MSS. Reference Value

[0136] In one embodiment, the sequencing reads that contain microsatellite loci are compared with a threshold value to determine whether the microsatellite loci are MSI.The amount of MSI loci compared with the total number of evaluated microsatellite loci is then compared with MSI-PT.MSI-PT can be determined using various methods.

[0137] In one embodiment, MSI-PT corresponds to a reference value for a microsatellite locus (e.g., loci: 1-170) and is determined by evaluating the levels of the microsatellite loci from a population of subjects (e.g., healthy subjects) known to be cancer-free. As a non-limiting example, in one embodiment, a population of subjects (e.g., 10, 20, 50, 100, 200, 500 or more subjects) are known to be cancer-free and a sample from each subject is analyzed for the level of the microsatellite locus for each sample.

[0138] In another embodiment, the MSI-PT corresponds to a reference value for a microsatellite locus (e.g., microsatellite locus: 1-170) and is determined by assessing the level of the microsatellite locus in a sample from a population of subjects with cancer (e.g., colorectal cancer).

[0139] In another non-limiting example, a population of subjects (e.g., 10, 20, 50, 100, 200, 500 or more subjects) have colorectal cancer and a sample from each subject is analyzed for microsatellite loci (e.g., microsatellite loci: 1-170). In some embodiments, the population of subjects is matched to the subject under study according to one or more patient characteristics, such as age, sex, race, or other criteria. In some embodiments, the reference value is established using a sample from the population of subjects of the same type as the sample used to detect MSI in the subject under study (e.g., a sample including plasma or colorectal tissue sample).

[0140] Reference value can be determined using any suitable method (e.g., collect sample from subject and determine microsatellite locus level). It will be understood that in making such determination, standard statistical methods can be adopted by experts. For example, see Principles of Biostatistics by Marcello Pagano et al. (Brook Cole; 2000) and Fundamentals of Biostatistic by Bernard Rosner (Duxbury Press, 5th Ed, 1999).

[0141] In another embodiment, the threshold value of a particular microsatellite locus (e.g., locus:1) of a sample from a subject under investigation can be compared to a control sample (e.g., a matched healthy sample) to determine whether the microsatellite locus is MSI. In some embodiments, the control sample is a sample from a subject that does not exhibit any clinical symptoms of cancer. In some embodiments, the control sample is a sample from a subject that has been clinically diagnosed with cancer (e.g., colorectal cancer). In some embodiments, the subject from which the control sample is obtained is the same age, or about the same age, and / or the same gender as the subject under investigation. Subjects and Samples

[0142] In some embodiments, the sample is from an individual human subject (i.e., a human subject being evaluated for cancer). In some embodiments, the subject is an adult. In some embodiments, the subject is a child. In some embodiments, the subject is a human who has been diagnosed with cancer. In some embodiments, the subject is a human who is suspected of having cancer. In some cases, the subject is a dog, cat, horse, sheep, mouse, rat, rabbit, monkey, or human.

[0143] In some embodiments, the sample from the subject comprises whole blood (e.g., blood draw), serum, plasma, saliva, urine, cerebrospinal fluid, or a tissue sample (e.g., colon biopsy). In some embodiments, the sample comprises cells obtained from a tumor. In some embodiments, the sample is a colon tissue sample. In some embodiments, the sample is a cell-free DNA sample. In yet another embodiment, the sample is a circulating tumor DNA (ctDNA) sample. In preferred embodiments, the sample comprises genomic DNA from the subject. In some embodiments, the genomic DNA is derived from an individual subject, preferably a mammal. In some aspects, the sample may comprise any tissue, cell, or biological fluid from the subject that contains genomic DNA. In some embodiments, the sample is a blood sample. In another embodiment, the sample is a tissue sample obtained from a tumor.

[0144] In some embodiments, the sample is a formalin-fixed paraffin-embedded tissue sample (FFPE). Tissue samples from biopsies are usually stored in FFPE for long-term storage. Formalin forms crosslinks in tissue samples that can be difficult to break, potentially resulting in reduced DNA yield. Another problem associated with formalin-fixed paraffin-embedded samples is often the amplification of longer nucleic acid fragments. When DNA from FFPE samples is used in multiplex amplification reactions, fragment sizes often increase and peak heights are often significantly reduced. The microsatellite instability method of the present invention is preferably designed to amplify and analyze genomic DNA from FFPE tissue samples. In one embodiment, the method disclosed herein for detecting MSI includes at least one genomic DNA fragment from a tumor of interest. In another embodiment, the method disclosed herein does not require a matched sample from healthy tissue to detect MSI. IV. Computer Systems

[0145] In one aspect, the present disclosure relates to a system for detecting microsatellite instability. In one embodiment, the system includes a processor and a non-transitory computer-readable medium coupled to the processor, the non-transitory computer-readable medium includes a code executable by the processor to execute a method, wherein the method includes: (1) receiving sequencing reads of a plurality of microsatellite loci from human genomic DNA; (2) determining a repeat length distribution (RLD) for each microsatellite locus; (3) generating a reference value of RLD for each microsatellite locus; (4) comparing the reference value of each microsatellite locus with an independent threshold value to a threshold value of the microsatellite locus; (5) quantifying the number of microsatellite loci with RLD reference values ​​above the threshold value that are detected; and (6) (i) comparing the number of microsatellite loci with RLD reference values ​​above the threshold value to (ii) a ratio threshold value of a set of loci, and if the number of microsatellite loci exceeds the ratio threshold value of the set of loci, the system classifies the sample from the human as MSI. In some embodiments, if the number of microsatellite loci above the threshold is less than the ratio threshold of the set of loci, the system classifies the sample from the human as MSS. In some embodiments, the system can simultaneously determine the MSI status of one or more samples. In a preferred embodiment, the system can simultaneously determine the MSI status of two or more samples from two or more subjects.

[0146] In one embodiment, the system further comprises at least one device configured to assay a plurality of MSI loci in a sample to determine the RLD of the plurality of MSI loci. In some embodiments, the reference value of the RLD of each microsatellite locus can include a t-statistic, such as a Student's t-test or a Wilcoxon rank test. In some embodiments, the t-statistic of a locus is determined based on the mean read length and variance of the RLD of the locus. In some embodiments, the t-statistic is determined based on the RLD of the test sample and the control RLD.

[0147] In some embodiments, the computer readable medium, which may include a collection of various storage devices, includes a database including a list of therapeutics available according to MSI status, instructions for inputting a number of microsatellite loci with RLD reference values ​​above a threshold, and a function for comparing the number of microsatellite loci with RLD reference values ​​above the threshold to a ratio threshold for the set of loci. In some embodiments, the computer readable medium further includes computer readable code having instructions for generating a report including a list of therapeutics, where the comparison of the set of loci to the ratio threshold indicates a likely benefit of at least one therapeutic in the database. In some embodiments, the likely benefit of the at least one therapeutic is determined based on one or more known or documented features in human subjects diagnosed with MSI. Including curative, positive, or ameliorative responses. In some embodiments, the at least one therapeutic agent is an antibody, a small molecule, a chemotherapeutic agent, or a combination thereof. In one embodiment, the at least one therapeutic agent is pembrolizumab.

[0148] In some embodiments, the system further includes instructions for identifying sequencing reads that contain microsatellite loci by aligning the sequencing reads to the set of microsatellite locus DNA sequences, and for counting the number of repeat lengths of each sequencing read determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus.

[0149] Conventional data networking, application development, and other functional aspects of a computer system (and components of individual operating components of a computer system) are not described in detail herein, but are considered part of this disclosure. Furthermore, the connecting lines shown in the various figures contained herein are intended to represent example functional relationships and / or physical connections between the various elements. It should be noted that many alternative or additional functional relationships or physical connections may exist in a computer system.

[0150] The various computer system components discussed herein may include one or more of: a host server or other computing system including a processor for processing digital data; a memory coupled to the processor for storing digital data; an input digitizer coupled to the processor for inputting digital data; an application program stored in the memory and accessible by the processor for directing the processing of the digital data by the processor; a display device coupled to the processor and the memory for displaying information derived from the digital data processed by the processor; and a plurality of databases.

[0151] The various databases used herein may include patient data such as family history, demographic and environmental data, biological sample data, past treatment and protocol data, patient clinical data, molecular profiling data of biological samples, data regarding therapeutic and / or investigational drugs, gene libraries, disease libraries, drug libraries, patient tracking data, file management data, financial management data, billing data, and / or similar data useful in the operation of the system.

[0152] As one skilled in the art will appreciate, a computer system may include an operating system (e.g., Windows NT, 95 / 98 / 2000, OS2, UNIX, Linux, Solaris, MacOS, etc.) as well as various conventional support software and drivers typically associated with a computer. The computer system may include any suitable personal computer, network computer, workstation, minicomputer, mainframe, etc. The computer system may be located in a home, medical or business environment with access to a network. In an exemplary embodiment, access is via a network or the Internet via a commercially available web browser software package.

[0153] As used herein, the term "network" is intended to include any electronic communication means incorporating both its hardware and software components. Communication between a user and other parties can be accomplished through any suitable communication channel, such as, for example, a telephone network, an extranet, an intranet, the Internet, a point of interaction device, a personal digital assistant, a mobile phone, a kiosk, online communication, satellite communication, offline communication, wireless communication, transponder communication, a local area network (LAN), a wide area network (WAN), a network or connection device, a keyboard, a mouse, and / or any suitable communication or data entry modality. Specific information regarding protocols, standards, and application software utilized in connection with the Internet is generally known to those skilled in the art and therefore need not be detailed herein. See, for example, Dilip Naik, Internet Standards and Protocols (1998), Java 2 complete by multiple authors (Sybex 1999), and Deborah Ray and Eric Ray, Mastering html 4.0 (1997).

[0154] The various computer system components may be suitably networked via data links, either independently, individually, or collectively. Furthermore, the system contemplates the use, sale, or distribution of any goods, services, or information (e.g., sequencing reads or sequencing data) over any network having similar functionality as described herein.

[0155] As used herein, "transmitting" may include sending electronic data from one system component to another over a network connection. Additionally, as used herein, "data" may include generic information such as commands, queries, files, data for storage, etc., in digital or any other form.

[0156] Any of the computer systems referred to herein may utilize any suitable number of subsystems. Examples of such subsystems are shown in FIG. 8. In some embodiments, computer system 10 includes a single computer device, where the subsystems may be components of the computer device. In other embodiments, the computer system may include multiple computer devices, each with its own internal components, each of which is a subsystem.

[0157] The subsystems shown in FIG. 8 are interconnected via a system bus 75. Additional subsystems are shown, such as a printer 74, a keyboard 78, a storage device 79, and a monitor 76 connected to a display adapter 82. Peripherals and input / output (I / O) devices are connected to an I / O controller 71 and can be connected to the computer system by several means known in the art, such as a serial port 77. For example, the serial port 77 or an external interface 81 (e.g., Ethernet, Wi-Fi, etc.) can be used to connect the computer system 10 to a wide area network, such as the Internet, a mouse input device, or a scanner. Through the interconnection via the system bus 75, the central processing unit 73 can communicate with each subsystem and control the execution of instructions from the system memory 72 or storage device 79 (e.g., fixed disk), as well as the exchange of information between the subsystems. The system memory 72 and / or the storage device 79 can embody a computer-readable medium. Any of the values ​​described herein can be output from one component to another and can be output to a user.

[0158] A computer system may include multiple identical components or subsystems, connected together, for example, by external interfaces 81 or internal interfaces. In some embodiments, computer systems, subsystems, or devices may communicate over a network. In such a case, one computer may be considered a client and another computer may be considered a server, each of which may be part of the same computer system. The client and server may each include multiple systems, subsystems, or components.

[0159] It should be understood that any of the embodiments of the present invention can be implemented using hardware (e.g., application specific integrated circuits or field programmable gate arrays) and / or using computer software, typically with programmable processors in a modular or integrated manner, in the form of control logic. As used herein, a processor includes a multi-core processor on the same integrated chip, or multiple processing units on a single circuit board or networked. Based on the disclosure and teachings provided herein, one of ordinary skill in the art will know and understand other means and / or methods for implementing embodiments of the present invention using hardware and combinations of hardware and software.

[0160] Any of the software components or functions described in this application can be implemented as software code executed by a processor using any suitable computer language, such as Java, C++ or Perl, for example using conventional or object-oriented techniques. The software code can be stored as a sequence of instructions or commands on a computer-readable medium for storage and / or transmission, suitable media including random access memory (RAM), read-only memory (ROM), magnetic media such as hard drives or floppy disks, or optical media such as compact disks (CDs) or DVDs (digital versatile disks), flash memory, and the like. The computer-readable medium can be any combination of such storage or transmission devices. The computer-executable instructions can be written in a suitable computer language or a combination of several languages. Basic computational biology methods are described, for example, in Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Genes and Proteins (Wiley & Sons, Inc., 2.sup.nd ed., 2001). See, e.g., U.S. Patent No. 6,420,108.

[0161] Such programs may also be encoded and transmitted using carrier signals adapted for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet. Thus, computer-readable media according to embodiments of the present invention may be created using data signals encoded with such programs. Computer-readable media encoded with program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Such computer-readable media may reside on or within a single computer program product (e.g., a hard drive, CD, or an entire computer system), or may reside on or within different computer program products within a system or network. The computer system may include a monitor, printer, or other suitable display for providing a user with any of the results described herein.

[0162] Any of the methods described herein can be performed in whole or in part using a computer system including one or more processors that can be configured to perform the steps. Thus, the embodiments can relate to a computer system configured to perform the steps of any of the methods described herein, potentially comprising various components performing each step or each group of steps. Although presented as numbered steps, the steps of the methods herein can be performed simultaneously or in different orders. Furthermore, some of these steps can be used with some of other steps from other methods. Also, all or some of the steps can be optional. Furthermore, any steps of any method can be performed using modules, circuits, or other means for performing these steps.

[0163] As will be appreciated by those skilled in the art, a computer system may be embodied as a customization of an existing system, an add-on product, upgraded software, a standalone system, a distributed system, a method, a data processing system, a device for data processing, and / or a computer program product. Thus, a computer system may take the form of an entirely software embodiment, an entirely hardware embodiment, or an embodiment combining both software and hardware aspects.

[0164] FIG. 9 shows a block diagram of an exemplary embodiment of a system 40 for detecting MSI in a sample from a subject utilizing reference values ​​or MSI-PTs of RLD for each microsatellite locus and set of loci. The system 40 includes a user interface 12, a host server 14 including a processor 16 for processing data, a memory 18 connected to the processor, an application program 20 stored in the memory 18 and accessible by the processor 16 to direct processing of the data, the processor 16, a number of internal databases 22 and an external database 24, and an interface with a wired or wireless communication network 26 (such as the Internet). The system 10 may also include an input digitizer 28 connected to the processor 16 for inputting digital data from data received from the user interface 12. The user interface 12 includes an input device 30 and a display 32 for inputting data into the system 10 and for displaying information derived from data processed by the processor 16. The user interface 12 may also include a printer 34 for printing information derived from data processed by the processor 16, such as a report that may include MSI test results and suggested therapeutics based on the test results. The internal databases 22 may include, but are not limited to, patient sample / specimen information and tracking, clinical data, patient data, file management, research protocols, and patient test results. The external databases 24 may include, but are not limited to, drug libraries, disease libraries, and public and private databases such as NCBI, PubMed, UniGene, OMIM, GO, TIGR, GenBank, KEGG, and Biocarta.

[0165] Furthermore, the present disclosure relates to embodiments including methods for providing genetic information, such as MSI, via a network, such as the Internet, as shown in US Publication No. 20020183936. For example, one or more methods disclosed herein can be performed in one location, such as a city, state, country, or continent, and the results can be transmitted to another city, state, country, or continent. Treatment selection can be made in whole or in part at the second location. The methods of the present invention include transmission of MSI information between different locations. V. Treatment method

[0166] In another aspect, methods of treating a subject diagnosed with MSI (e.g., colorectal cancer) are provided. In some embodiments, the methods described relate to cancer. In some embodiments, the methods include treating a subject by slowing or reversing the progression of cancer.

[0167] In some embodiments, the method includes determining whether the subject has MSI. In some embodiments, the method includes determining the RLD reference value of a plurality of microsatellite loci in a sample from the subject, comparing the RLD reference value with a threshold value of the plurality of microsatellite loci, quantifying the number of microsatellite loci with RLD reference value above the threshold, comparing the number of microsatellite loci with RLD reference value above the threshold with an MSI ratio threshold (if the number of microsatellite loci exceeds the MSI ratio threshold, the subject has MSI), and administering one or more agents to the subject. In some embodiments, the RLD reference value is prepared by determining the sample RLD of each microsatellite locus and comparing it with a pre-calculated background RLD for each microsatellite locus to generate the RLD reference value of each microsatellite locus under evaluation.

[0168] Agents described herein can be administered, for example, intravenously, intrathecally, intraspinally, intraperitoneally, intramuscularly, intranasally, subcutaneously, orally, topically, and / or by inhalation.

[0169] The agent is administered in a manner compatible with the dosage form and in such amount as is therapeutically effective. The term "therapeutically effective amount" refers to an amount of the agent (e.g., a compound or pharmaceutical composition described herein) administered that will treat to some extent a disease, disorder, or condition, e.g., reduce one or more symptoms of the disease or symptoms that the subject is at risk of developing. In some embodiments, dosages of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg per day can be used. However, dosages can vary depending on the condition of the patient, the severity of the condition being treated, and the therapeutic agent being used. Dosages will also be determined by the existence, nature, and extent of adverse side effects associated with the administration of a particular compound in a particular patient. Determination of the dosage appropriate for a particular situation is within the skill of the practitioner. Often, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, dosages are increased by small increments until the optimum effect is obtained under the circumstances. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.

[0170] In some embodiments, the methods disclosed herein include treating the subject with an agent if the subject is diagnosed with MSI. In one embodiment, the agent is pembrolizumab.

[0171] In some embodiments, the subject has or is suspected of having cancer. In some embodiments, the subject with MSI has or is suspected of having colorectal cancer (CRC). Thus, in some embodiments, the method disclosed herein comprises treating the subject with a known CRC drug, surgery, chemotherapy, and / or radiation therapy. In one embodiment, the treatment of the subject with CRC is selected from the group consisting of surgery, radiation therapy, drugs, and combinations thereof.

[0172] In some embodiments, the method of treating colon and / or rectal cancer includes one or more agents approved by the Food and Drug Administration (FDA) for colon and / or rectal cancer. In some embodiments, the one or more agents are selected from the following list: In one embodiment, treating CRC includes administering a therapeutic amount of MDG007 (MacroGenics) to a human in need thereof.

[0173] In some embodiments, drugs approved for the treatment of colon cancer include: , but not limited to these. Avastin (bevacizumab) Bevacizumab Camptosar (Irinotecan hydrochloride) Capecitabine Cetuximab Cyramza (ramucirumab) Eloxatin (oxaliplatin) Erbitux (cetuximab) 5-FU (fluorouracil injection) Fluorouracil injection Irinotecan Hydrochloride Leucovorin calcium Lonsurf (trifluridine and tipiracil hydrochloride) Nivolumab Opdivo (nivolumab) Oxaliplatin Panitumumab Ramucirumab Regorafenib Stivarga (regorafenib) Trifluridine and Tipiracil Hydrochloride Vectibix (panitumumab) Wellcovorin (Leucovorin Calcium) Xeloda (Capecitabine) Zaltrap (Ziv-Aflibercept) Ziv-Aflibercept

[0174] In another embodiment, drug combinations approved for the treatment of colon cancer include, but are not limited to, the following: Capox; Folfiri; Folfiri-bevacizumab; Folfiri-cetuximab; Folfox; Fu-Lv; Xeliri; Xelox;

[0175] In some embodiments, drugs approved for the treatment of rectal cancer include, but are not limited to, the following: Avastin (bevacizumab); Bevacizumab; Camptosar (irinotecan hydrochloride); Capecitabine; Cetuximab; Cyramza (ramucirumab); Eloxatin (oxaliplatin); Erbitux (cetuximab); 5-FU (fluorouracil injection); Irinotecan hydrochloride; Leucovorin calcium; Lonsurf (trifluridine and tipiracil hydrochloride); Nivolumab Opdivo (nivolumab); Oxaliplatin; Panitumumab; Ramucirumab; Regorafenib; Stivarga (regorafenib); Trifluridine and tipiracil hydrochloride; Vectibix (panitumumab); Wellcovorin (leucovorin calcium); Xeloda (capecitabine); Zaltrap (Ziv-aflibercept); and Ziv-Aflibercept

[0176] In some embodiments, drug combinations approved for the treatment of rectal cancer include, but are not limited to, the following: Capox; Folfiri; Folfiri-bevacizumab; Folfiri-cetuximab; Folfox; Fu-Lv; Xeliri; and Xelox

[0177] In some embodiments, the subject is treated with one or more agents that are specific or reactive to the type of cancer that the subject has or is suspected of having. VI. Kit

[0178] The present disclosure also provides a kit for carrying out the method of the present invention, comprising the primers, adaptor oligonucleotides, and oligonucleotide probes described herein. In some embodiments, the oligonucleotide probes are provided in solution, while in other embodiments, they are provided attached to a solid support. Preferably, the primers and adaptor oligonucleotides are provided in solution at known concentrations. The kit also includes instructions for detecting the amplification of genomic DNA fragments, ligation of adaptor oligonucleotides to genomic DNA fragments, hybridization of oligonucleotide probes to genomic DNA fragments, and separation of oligonucleotide probes from genomic DNA fragments and amplicons.

[0179] In certain embodiments, the kit also includes instructions for one or more of obtaining genomic DNA fragments, amplifying genomic DNA, binding oligonucleotide probes to genomic DNA, and changing at least one condition (e.g., temperature condition as part of melting curve analysis). In addition, the kit optionally includes one or more components selected from, for example, nucleotides, nucleotide analogs, enzymes, salts, buffers, cofactors, etc. In some embodiments, the kit also includes nucleic acid molecules that act as positive and / or negative controls for the amplification process, the sequencing process, and / or the reagents thereof. Typically, the kit also includes at least one container for packaging primers, adapter oligonucleotides, and oligonucleotide probes, instructions, and / or one or more other components. EXAMPLES

[0180] Example 1 - Determining Repeat Length Distribution A microsatellite locus consists of a left-handed sequence, a number of repeat units, and a right-handed sequence. An example of a microsatellite locus is shown below. (Left nucleotide sequence)-(GT) 24 -(Right nucleotide sequence)

[0181] We assume that samples generally contain a variety of repeat unit lengths. The repeat length distribution can be defined as R S , L , where S is the sample, L is the locus, and R is the repeat length distribution.

[0182] Therefore, R S , L can be expressed as follows: R S , L = <n1、n2、…n k >, where n1 is the number of reads that support the presence of i repeat units. For a particular locus L, R S , L is quasi-monomorphic for healthy individuals in the population. If instability exists, RS , L The mean of is significantly shifted from the normal background sample. This shift is detectable because it has been determined that it is larger in magnitude than the shift caused by PCR stutter.

[0183] 1A and 1B are electropherograms showing the fragment distribution of microsatellite loci in MSS (FIG. 1A) or MSI (FIG. 1B) cell lines. The thick lines in the electropherograms represent the internal control and the light lines represent the test samples. As can be seen in FIG. 1A (MSS cell line), the MSS test samples and the internal control appear to migrate with similar fragment size distributions.

[0184] In contrast, MSI cell line samples at all three microsatellite loci were observed to migrate at smaller fragment sizes (i.e., shifting the migration pattern of MSI test samples to the left) compared to the internal controls, thus allowing MSI samples to be distinguished from MSS samples based on repeat length distribution. Distribution of the average number of repeat units in microsatellite loci of Examples 2-64

[0185] In this example, a set of 64 microsatellite loci was evaluated from colorectal and endometrial cancers. Cell-free DNA samples from seven healthy normal subjects, four MSS cell lines, and four MSI cell lines were evaluated. For each of the 15 samples, R S , L was calculated.

[0186] Box plots were generated for the average number of repeat units per locus across the 15 samples, separated by sample type (e.g., cfDNA, unstable or stable cell lines). The mean of the repeat length distribution is given for each locus and cell type.

[0187] Figure 6A and Figure 6B show a series of 63 plots, each corresponding to a unique microsatellite locus. In the cfDNA samples, the number of repeat units per locus is stable and remains at 58 / 63 loci. Furthermore, the distribution of the average number of repeat units of these 58 loci is completely overlapping or closely approximating the distribution of the average number of repeat units of samples from MSS cell lines. Thus, this data demonstrates that it is possible to calculate a background signal that represents the MSS sample, which can be used as a reference for the test sample. Furthermore, it was confirmed that the distribution of the average number of repeat units of MSI cell lines is not overlapping and is significantly different from the cfDNA / MSS samples. Example 3 - Skewness as a component of repeat length distribution

[0188] As in Example 2, the same 64 loci were evaluated for skewness of the average number of repeat units. Cell-free DNA samples from 7 healthy normal subjects, 4 MSS cell lines and 4 MSI cell lines were evaluated. Skewness was calculated for each of the 15 samples.

[0189] Box plots were generated for the skewness of the average number of repeat units per locus across the 15 samples, separated by sample type (e.g., cfDNA, unstable or stable cell lines).

[0190] Figures 7A and 7B show a series of 64 plots, each corresponding to a unique microsatellite locus. In the cfDNA samples, the skewness of the average number of repeat units per locus is stable and remains at approximately 37 microsatellite loci. Thus, this data demonstrates that it is possible to calculate a background signal, representative of the MSS samples that can be used as a reference for test samples.Furthermore, we confirmed that the distributions of skewness of the mean number of repeat units in MSI cell lines are non-overlapping and significantly different from the cfDNA / MSS samples. Example 4 - Computational process for MSI detection

[0191] The detection of microsatellite instability in a sample is based on the MSI ratio threshold obtained for a set of microsatellite loci. Here, the sample is sequenced or sequencing reads for a sample containing one or more microsatellite loci are obtained (e.g., from a sequencing data file or database). The RLD of each DNA fragment containing a microsatellite locus is determined. Then, a RLD reference value is determined for each microsatellite locus. The RLD reference value is compared to a threshold value (e.g., a normal background sample of the same microsatellite locus, or a pre-calculated normal background RLD), and the number of microsatellite loci that exceed the threshold value for the corresponding microsatellite locus is determined.

[0192] The amount of microsatellite loci with RLD criteria above the threshold is then compared to an MSI ratio threshold (e.g., 35%). If the amount of microsatellite loci with RLD criteria above the threshold is greater than the MSI-PT, the sample is classified as MSI. The above steps can be performed using a computer system to determine the MSI or MSS status in a sample. An exemplary workflow of such a computer process is shown in FIG. 3 and is referred to herein as the "MSI caller pipeline." The MSI caller pipeline functions in four main stages.

[0193] First, msisensor (Niu et al. 2013) is used to summarize the RLDs obtained from the sequencing read alignment (pathway a).

[0194] Second, a set of microsatellite loci that exhibit preserved RLD within a set of normal cell-free DNA samples from healthy donors is selected (pathway b).

[0195] Third, a binarized classifier is trained for each microsatellite locus to discriminate MSI status from a set of training samples with known microsatellite stability status (pathway c).

[0196] Fourth, when a test sample is applied, a computer process determines whether the sample is an MSI or an MSS (path d). Microsatellite loci panel

[0197] The exemplary MSI detection panel included 64 microsatellite loci, referred to herein as microsatellite loci 1-64 (see Table 1). These microsatellite loci are known to provide information on microsatellite stability status for colorectal, endometrial, and prostate cancer. The initial microsatellite panel was subsequently expanded to a size of 170 loci (see Table 1, Microsatellite Loci: 1-170). RLD summary from msisensor

[0198] The above computational pipeline uses "msisensor" to calculate RLDs in BAM formatted read alignment files. This process typically involves executable code compiled for msisensor, a microsatellite panel file that defines the locations of included microsatellite markers, and a BAM file of sequencing read alignments. Msisensor outputs an RLD file that summarizes the RLDs of included microsatellite loci based on the sequencing read alignments. Background buildup from normal samples

[0199] The MSI caller pipeline can include pre-calculated "normal" RLD values. This works particularly well when microsatellite loci contain consistent RLDs across microsatellite stable (MSS) samples (e.g., Figure 5A). We determined that repeat length variation is primarily a side effect of PCR stutter during sequencing and can be extracted from a set of normal samples. Therefore, we calculated the average RLD from a set of normal samples (CFDNA in Figures 6A and 6B and Figures 7A and 7B) to represent the pre-calculated normal background RLD for a given microsatellite locus.

[0200] However, variable repeat lengths at the heterozygous and homozygous levels (e.g., Figure 5C) should be excluded from downstream analyses, as they may be caused by actual germline mutations rather than PCR stutter. Additionally, microsatellite loci that differ significantly from the norm (e.g., Figure 5B) should be excluded from the background, as they may imply germline mutations from minority groups.

[0201] Inconsistent RLDs across microsatellite loci can be filtered using a distance criterion to measure the similarity between two RLDs. A microsatellite locus is considered to have an inconsistent RLD if the majority of pairwise distances from the RLD are "longer" than any of the distance criterion values ​​tested. As used herein, an RLD is long if it is greater than the median plus two standard deviation distance from all sample pairs at all microsatellite loci. In some cases, the median is used rather than the average to reduce the influence of outliers.

[0202] Various distance metrics can be used to assess the similarity between two RLDs, such as Kullback-Leibler divergence, correlation distance, cosine distance, Euclidean distance, Manhattan distance, and Kolmogorov-Smirnov statistic.

[0203] When filtering out inconsistent samples for a given microsatellite locus, Euclidean distance may be preferred to measure the similarity between two RLDs. A sample is considered to be similar to other samples if its Euclidean distance to a normal background sample is greater than the median plus two standard deviations distance from all samples at all locations. is seen as comparatively inconsistent. MSI Caller

[0204] The prediction of MSI by the MSI caller pipeline is determined by the percentage or proportion of MSI loci under evaluation that are greater than a defined threshold (i.e., MSI-PT). The classifier training step learns these thresholds from a set of training samples, and the MSI caller uses these thresholds to classify samples as MSI or MSS. The t-statistic is calculated as follows: [ka]

[0205] where μf and S f 2 are the mean and variance of the sample RLD, while μb and S b 2 are the mean and variance of the background RLD, and n f is the total number of reads in a sample that contain a particular microsatellite locus. In one embodiment, the maximum Youden's index is selected to maximize the difference between the true positive rate and the false positive rate.

[0206] Normal RLD background samples are prepared from cfDNA of healthy non-cancerous samples. Samples can be filtered using the distance criteria described above to reduce RLD variation between samples (data not shown). conclusion

[0207] We created a computational pipeline that allows for the detection of MSI status in tissue samples. We expanded an existing microsatellite instability detection panel from 5 to 170 and used NGS to simultaneously monitor MSI status from all markers at scale. We eliminated the requirement for a matched normal sample by extracting a common background of repeat length distribution from healthy samples. The absence of a matched normal tissue sample represents an important advance in MSI detection, as it eliminates another form of invasive surgery and associated risks that occur when collecting a matched normal tissue sample. We also tested sequencing-based assays and bioinformatics workflows on plasma samples obtained from blood draws and found that plasma samples can be used to classify samples as MSI or MSS. These findings represent another important advance in that blood draws can be used to detect MSI and / or monitor MSI status before, during, and after a treatment regimen. When applied to large clinical cohorts, a 35% MSI score threshold can be used as guidance for determining MSS / MSI representation of samples.

[0208] The sequencing-based assay and bioinformatics workflow presented here provides robust analytical performance for MSI detection in FFPE tissue samples and plasma, even in samples with low content of circulating tumor DNA (ctDNA).

Claims

1. 1. A method for detecting microsatellite instability, comprising: detecting sequencing reads from a sample of human genomic DNA for a plurality of microsatellite loci; determining a baseline value for the repeat length distribution (RLD) of each microsatellite locus, said baseline value being a t-statistic determined by comparing the RLD of a given microsatellite locus to a pre-calculated normal background RLD of the corresponding microsatellite locus, and said normal background RLD is prepared from cfDNA from a healthy non-cancerous sample; comparing said reference value with thresholds for said microsatellite loci, each having an independent threshold, and said thresholds are selected as the maximum Youden's index that maximizes the difference between the true positive rate and the false positive rate. quantitating the number of microsatellite loci having RLD reference values ​​above said threshold; and comparing the ratio of numbers of the microsatellite loci having an RLD reference value above the threshold to a microsatellite instability (MSI) ratio threshold, wherein if the ratio of numbers exceeds the MSI ratio threshold, the human has microsatellite instability. The method, wherein the sample is cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA).

2. 2. The method of claim 1, wherein the plurality of microsatellite loci are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 microsatellite loci selected from the group consisting of microsatellite loci: 1-170 of Table 1.

3. 2. The method of claim 1, wherein the plurality of microsatellite loci are at least 58 microsatellite loci selected from the group consisting of microsatellite loci: 1-64 of Table 1.

4. 2. The method of claim 1, wherein the plurality of microsatellite loci correspond to the microsatellite loci in Table 2.

5. The method of claim 1, wherein the reference value of RLD is determined in the absence of a healthy sample, the healthy sample being obtained from the same subject as the sample of claim 1.

6. 2. The method of claim 1, wherein the reference value for a microsatellite locus is a t-statistic determined using one or more statistical values ​​of RLD for the microsatellite locus in a human and one or more statistical values ​​of a reference RLD.

7. The method of claim 6, wherein the t-statistic is determined based on the mean read length and variance in RLD.

8. 2. The method of claim 1, wherein the detecting comprises generating nucleotide sequencing reads from genomic DNA and identifying sequencing reads that contain microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences and counting the number of repeat lengths of each sequencing read determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus.

9. 9. The method of claim 8, wherein prior to generating nucleotide sequencing reads, the human genomic DNA is enriched for microsatellite loci by hybridizing the human genomic DNA to oligonucleotide probes that hybridize to microsatellite loci-containing DNA.

10. 2. The method of claim 1 , Providing a sample containing human genomic DNA fragments; contacting the sample with a pool of oligonucleotide probes; capturing DNA fragments comprising microsatellite loci in hybridization complexes with said pool oligonucleotide probes, thereby generating captured DNA; separating the hybridization complexes from unbound nucleic acid; eluting the captured DNA from the hybridization complex; and sequencing the captured DNA eluted from the hybridization complex; and identifying sequencing reads that include microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences; and counting the number of repeat lengths for each sequencing read determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus.

11. 2. The method of claim 1 , Providing a sample containing human genomic DNA fragments; amplifying a DNA fragment comprising a microsatellite locus using oligonucleotide primers specific to the microsatellite locus to generate an amplicon; sequencing the amplicons; and identifying sequencing reads that include microsatellite loci by aligning the sequencing reads to a set of microsatellite locus DNA sequences; and counting the number of repeat lengths for each sequencing read determined to contain a microsatellite locus, thereby generating an RLD for each microsatellite locus.

12. 1. A system for detecting microsatellite instability, comprising: A processor; and a non-transitory computer-readable medium coupled to the processor, the non-transitory computer-readable medium comprising: receiving sequencing reads for a plurality of microsatellite loci from a sample of human genomic DNA; determining the repeat length distribution (RLD) of each microsatellite locus; generating a reference value of the RLD for each microsatellite locus, the reference value being a t-statistic determined by comparing the RLD of a given microsatellite locus to a pre-calculated normal background RLD of the corresponding microsatellite locus, and the normal background RLD is prepared from cfDNA from a healthy, non-cancerous sample; comparing the reference value for each microsatellite locus with a threshold value for the microsatellite locus, each microsatellite having an independent threshold value; and selecting the threshold value as the maximum Youden's index that maximizes the difference between the true positive rate and the false positive rate. quantitating the number of detected loci having RLD reference values ​​above the threshold; and (ii) comparing a ratio of numbers of microsatellite loci having an RLD reference value above the threshold to a ratio threshold for a set of loci, wherein if the ratio of numbers exceeds the ratio threshold for the set of loci, the human has microsatellite instability. The system, wherein the sample is cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA).

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