Patent Publication Number: US-2023160002-A1

Title: Process for Microsatellite Instability Detection

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. Ser. No. 16/204,642, filed Nov. 29, 2018, which claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Ser. No. 62/593,664 filed Dec. 1, 2017, and of U.S. Ser. No. 62/741,448 filed Oct. 4, 2018. The entire content of each of the aforementioned applications is herein incorporated by reference for all purposes. 
    
    
     TECHNICAL FIELD 
     The present invention relates generally to the detection, monitoring, and treatment of cancer and more specifically to determining the MSI status of a patient by liquid biopsy. 
     BACKGROUND 
     Cancer causes more than a half a million deaths each year in the United States alone. The success of current treatments depends on the type of cancer and the stage at which it is detected. Many treatments include costly and painful surgeries and chemotherapies, and are often unsuccessful. Early and accurate detection of mutations is essential for effective cancer therapy. 
     Many cancers involve the accumulation of mutations that results from failure of the DNA mismatch-repair (MMR). One important marker of MMR deficiency is microsatellite instability (MSI), a polymorphism of tandem nucleotide repeat lengths ubiquitously distributed throughout the genome. The presence of MMR-deficiency or MSI may serve as a marker for immunotherapy response with checkpoint inhibition. Knowledge of MSI status is thus important and valuable for the treatment of cancer. While it may be possible to determine MSI status by sequencing DNA from a tumor sample, such as a formalin-fixed paraffin-embedded (FFPE) tumor tissue specimen, there are patients for whom tumor material is not readily obtained. 
     Absent a fixed tissue specimen, a potential source for tumor information is through the analysis of circulating tumor DNA (ctDNA). ctDNA is released from tumor tissue into the blood and can be analyzed by liquid biopsy. Liquid biopsies potentially allow for the detection and characterization of cancer. However, liquid biopsies present their own inherent challenges associated with low circulating tumor DNA (ctDNA) levels as well as problems with faithfully amplifying and sequencing regions of DNA characterized by tracts of mononucleotide repeats. 
     SUMMARY OF THE INVENTION 
     The present invention is based on the seminal discovery that a circulating tumor DNA based approach is useful for the detection of high tumor mutation burden and microsatellite instability in cancer patients with advanced disease and can be used to predict responders to immune checkpoint blockade. 
     The invention provides methods for determining the MSI status of a patient by liquid biopsy. Methods include a sample preparation using hybrid capture and non-unique barcodes. The sample preparation both compensates for errors such as sequencing artifacts and polymerase slippage and provides for the successful capture of target DNA even when present only at a very low fraction of total DNA. Methods include sequencing tracts of mononucleotide repeats within captured sample and modelling the distribution of lengths of those tracts. A peak-finding operation evaluates peaks in the modelled distribution and reveals MSI in the patient when the peaks deviate from a reference distribution (e.g., such as by indicating that the tracts of mononucleotide repeats in the patient&#39;s DNA are markedly shorter than in healthy DNA). 
     Methods of the disclosure are amenable to implementation in conjunction with other genomic screenings such as screening panels of markers, genes, or whole genomes to report mutations or mutational burden. Methods may be implemented by including MSI markers within any suitable liquid-biopsy based sequencing assay and may evaluate MSI status by interrogating MSI markers such as BAT-25, BAT-26, MONO-27, NR-21, and NR-24, BAT-40, TGFβ RII, IGFIIR, hMSH3, BAX and dinucleotide D2S123, D9S283, D9S1851 and D18S58 loci, by way of example, or by modeling distributions of lengths of any other suitable set(s) of repeats in the genome. 
     In certain aspects, the invention provides a method of detecting microsatellite instability (MSI). The method includes obtaining cell-free DNA (cfDNA) from a sample of plasma from a patient and sequencing portions of the cfDNA to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA. A report is provided describing an MSI status in the patient when a distribution of lengths of the plurality of tracts has peaks that deviate significantly from peaks in a reference distribution. Obtaining the cfDNA may include capturing target portions of DNA with probes, fragmenting the target portions to yield fragments, and attaching barcodes to the fragments. In preferred embodiments, the barcodes are non-unique barcodes that include duplicates such that different ones of the fragments are attached to identical barcodes. 
     The method may include amplifying the fragments to produce amplicons that include barcode information and copies of the fragments, wherein the sequencing step comprises sequencing the amplicons. In one aspect, the sequencing is next-generation, short-read sequencing. The obtained sequences may include a plurality of sequence reads and the method may include aligning the sequence reads to a reference, and identifying groups of sequence reads that originated from a unique segment of the cfDNA by means of the barcode information and position or content of the sequence reads. 
     The use of the non-unique barcodes to identify groups of sequence reads that originated from a unique segment of the cfDNA allows for the lengths of the plurality of tracts to be determined correctly by correcting for errors introduced by sequencing artifacts or polymerase slippage during the amplifying step. 
     Preferably, the target portions are markers for MSI such as one or more of BAT25, BAT26, MON027, NR21, NR24, Penta C, and Penta D. For example, the markers may include all of BAT25, BAT26, MON027, NR21, and NR24. In certain embodiments, each of the microsatellite markers is selected from the group consisting of BAT-25, BAT-26, MONO-27, NR-21, NR-24, Penta C, and Penta D, BAT-40, TGFβ RII, IGFIIR, hMSH3, BAX and dinucleotide D2S123, D9S283, D9S1851 and D18S58 loci, by way of example. 
     In some embodiments, the method includes recommending a treatment for the patient based on the MSI status. Where the MSI status indicates that the patient is microsatellite instable, the treatment may include an immune checkpoint inhibitor. In certain embodiments, the method includes administering the treatment (e.g., the immune checkpoint inhibitor) to the patient. The immune checkpoint inhibitor may be, for example, an antibody such as an anti-PD-1 antibody; an anti-IDO antibody; anti-CTLA-4 antibody; an anti-PD-L1 antibody; or an anti-LAG-3 antibody. 
     Related aspects provide a method of detecting microsatellite instability (MSI) that includes obtaining a sample comprising fragments of cell-free DNA from a patient; attaching barcodes to the fragments, wherein at least some of the barcodes are not unique; sequencing the barcodes to obtain sequences of a plurality of markers in the DNA; determining a distribution of lengths of the plurality of markers; and providing a report describing MSI in the patient when peaks in the distribution deviate significantly from expected peaks in a modeled healthy distribution. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    diagrams a method for determining MSI status. 
         FIG.  2    shows a system for performing methods of the invention. 
         FIG.  3    shows a model of length distribution of mononucleotide repeats. 
         FIG.  4    shows a report provided by systems and methods of the invention. 
         FIGS.  5 A- 5 D  Plasma-Based Detection of Microsatellite Instability. (A) Prior to error correction and Digital Peak Finding (DPF)(light pink), the mononucleotide count distribution demonstrated high background noise due to sequencing related aberrations and polymerase slippage in library preparation PCR and sequencing. These are subsequently resolved after error correction and DPF (dark pink) to create distinct distributions for MSI and MSS alleles. (B) Across the BAT25, BAT26, MON027, NR21, and NR24 mononucleotide loci in 163 healthy donor plasma specimens, the error corrected mononucleotide count distribution was assessed with a DPF algorithm to identify mononucleotide alleles and determine MSI status. Prior to error correction and DPF (light pink), the majority of healthy donor samples exhibit alleles below the MSI cutoff (hashed line). Kaplan-Meier curves for progression free survival (C) and overall survival (D) among patients with progressive metastatic carcinoma were determined using MSI status from pre-treatment plasma specimens. In MSI patients (n=9*), median progression free survival was 16.17 months, while median overall survival was not reached. In MSS patients (n=7*), median progression free survival and median overall survival were 2.81 and 7.6 months, respectively. *Three patients with a tissue enrollment status of MSI-H were classified as MSS using pre-treatment baseline cfDNA obtained from plasma. 
         FIGS.  6 A- 6 E  Plasma-Based Detection of High Tumor Mutation Burden. (A) Using whole exome sequencing data derived from The Cancer Genome Atlas (TCGA), a significant positive correlation between the tumor mutation burden (TMB) evaluated in the 98 kb targeted regions compared to the whole exome analyses was observed (r=0.91, p&lt;0.0001; Pearson correlation). (B) Comparison of the accuracy for determination of the TMB derived from the targeted panel in plasma compared to whole-exome analyses of matched archival tissue samples in 13 patients yielded a significant positive correlation (r=0.693, p=0.007; Pearson correlation). (C) The overall TMB status at baseline was assigned as TMB-High or TMB-Low using a cutoff of 50.8 mutations/Mbp sequenced. In total, six patients were categorized as TMB-High and ten patients as TMB-Low, with a median load of 132 mutations/Mbp sequenced and 15.2 mutations/Mbp sequenced, respectively. Additionally, 163 healthy donor cases were evaluated, all of which were determined to be TMB-Low, with a median load of 0 mutations/Mbp sequenced across the panel. Kaplan-Meier curves for progression free survival (D) and overall survival (E) among this same cohort of patients were determined using TMB status from pre-treatment plasma specimens with a cutoff of 50.8 mutations/Mbp sequenced. In TMB-High patients (n=6), median progression free survival and median overall survival were not reached. In TMB-Low patients (n=10), median progression free survival and median overall survival were 2.84 and 7.62 months, respectively. 
         FIGS.  7 A- 7 F  Serial Plasma-Based Overall Survival Analysis for Patients Treated with Immune Checkpoint Blockade. (A) Evaluation of overall survival with the protein biomarker level at last dose (CEA or CA19-9). A significant inverse correlation was observed between the overall survival in months when compared to the residual protein biomarker (r=−0.99, p=&lt;0.001; Pearson correlation). (B) Kaplan-Meier curves for overall survival among patients with tissue enrollment status of MSI and detectable protein biomarker levels (n=8). For patients with &gt;80% reduction in protein biomarker levels (n=4), median overall survival was not reached. For patients with ≤80% reduction in protein biomarker levels (n=4), median overall survival was 5.26 months. (C) Evaluation of overall survival compared to residual MSI allele levels at last dose. A significant inverse correlation was observed between the overall survival when compared to the residual MSI allele levels (r=−0.70, p=0.034; Pearson correlation). (D) Kaplan-Meier curves for overall survival among patients with tissue enrollment status of MSI and detectable MSI status at baseline (n=9). For patients with two consecutive timepoints displaying no residual MSI alleles (n=4) median overall survival was not reached. For patients with multiple timepoints containing residual MSI alleles (n=5) median overall survival was 7.64 months. (E) Evaluation of overall survival compared to residual TMB levels at last dose. A significant inverse correlation was observed between the overall survival in months when compared to the residual TMB levels (r=−0.95, p=&lt;0.001; Pearson correlation). (F) Kaplan-Meier curves for overall survival among patients with tissue enrollment status of MSI and detectable TMB levels at baseline (n=11). For patients with &gt;90% reduction in TMB levels (n=4), median overall survival was not reached. For patients with ≤90% reduction in TMB levels (n=7), median overall survival was 7.64 months. “/” indicates a censored datapoint; “*” indicates cases where baseline protein biomarker, MSI or TMB was not detected and were not included in the subsequent analyses; In cases where residual protein biomarker, MSI or TMB levels increased when compared to baseline, values of greater than 100% are indicated. 
         FIGS.  8 A- 8 D  Monitoring of Patients During Immune Checkpoint Blockade. For three patients with a complete response to immune checkpoint blockade (CS97 (A), CS98 (B), and CS00 (C) and one patient with progressive disease (C505 (D)), circulating protein biomarkers (CEA, ng/mL and CA19-9, units/mL), residual alleles exhibiting MSI, and TMB levels were evaluated over time during treatment. In each case exhibiting a complete response, residual MSI and TMB alleles were reduced to 0% mutant allele fraction (MAF) between 0.6 and 4.8 months after first dose. 
         FIGS.  9 A- 9 D  Archival Tissue-Based Detection of Microsatellite Instability and High Tumor Mutation Burden. Kaplan-Meier curves for progression free survival (A) and overall survival (B) among patients with progressive metastatic carcinoma were determined using MSI status from archival tissue. In MSI patients (n=12), median progression free survival and median overall survival were 4.23 and 20.69 months, respectively. In MSS patients (n=4), median progression free survival and median overall survival were 2.81 and 6.31 months, respectively. Kaplan-Meier curves for progression free survival (C) and overall survival (D) among patients with progressive metastatic carcinoma were determined. In TMB-High patients (n=10), median progression free survival was 10.81 months, while median overall survival was not reached. In TMB-Low patients (n=3), median progression free survival and median overall survival were 2.81 and 5.02 months, respectively. 
         FIGS.  10 A- 10 F  Plasma-Based Progression Free Survival Analysis for Patients Treated with Immune Checkpoint Blockade. (A) Evaluation of progression free survival with the protein biomarker level at last dose (CEA or CA19-9). An inverse correlation was observed between the progression free survival in months when compared to the residual protein biomarker (r=−0.92, p=0.001; Pearson correlation). (B) Kaplan-Meier curves for progression free survival among patients with tissue enrollment status of MSI and detectable protein biomarker levels (n=8). For patients with &gt;80% reduction in protein biomarker levels (n=4), median progression free survival was not reached. For patients with ≤80% reduction in protein biomarker levels (n=4), median progression free survival was 2.63 months. (C) Evaluation of progression free survival compared to residual MSI allele levels at last dose. A significant inverse correlation was observed between the progression free survival in months when compared to the residual MSI allele levels (r=−0.84, p=0.004; Pearson correlation). (D) Kaplan-Meier curves for progression free survival among patients with tissue enrollment status of MSI and detectable MSI status at baseline (n=9). For patients with two consecutive timepoints displaying no residual MSI alleles (n=4) median progression free survival was not reached. For patients with multiple timepoints containing residual MSI alleles (n=5) median progression free survival was 3.01 months. (E) Evaluation of progression free survival compared to residual TMB levels at last dose. A significant inverse correlation was observed between the progression free survival in months when compared to the residual TMB levels (r=−0.98, p=&lt;0.001; Pearson correlation). (F) Kaplan-Meier curves for progression free survival among patients with tissue enrollment status of MSI and detectable TMB levels at baseline (n=11). For patients with &gt;90% reduction in TMB levels (n=4), median progression free survival was not reached. For patients with ≤90% reduction in TMB levels (n=7), median progression free survival was 2.88 months. “/” indicates a censored datapoint; “*” indicates cases where baseline protein biomarker, MSI or TMB was not detected and were not included in the subsequent analyses; In cases where residual protein biomarker, MSI or TMB levels increased when compared to baseline, values of greater than 100% are indicated. 
         FIG.  11    Radiographic Imaging of Case CS98 Displaying a Complete Response to Immune Checkpoint Blockade. After 20 weeks of treatment with immune checkpoint blockade, radiographic imaging was performed and revealed potential lesions in the liver, but later disappeared, so likely instead represented inflammatory liver nodules. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to the discovery that microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade. The present invention shows a plasma-based approach for detection of MSI and TMB-High in patients with advanced cancer. To detect sequence alterations across a 98 kilobase panel, including those in microsatellite regions, the inventors developed an error correction approach with specificities &gt;99% (n=163) and sensitivities of 75% (n=12) and 60% (n=10), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, the data demonstrate that MSI and TMB-High in pre-treatment plasma predicted progression-free survival (hazard ratios 0.2 and 0.12, p=0.01 and 0.004, respectively). The data shows the results when plasma during therapy was analyzed in order to develop a prognostic signature for patients who achieved durable response to PD-1 blockade. These analyses demonstrate the feasibility of non-invasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade. 
     The disclosure provides for the detection of MSI by liquid biopsy. While plasma is the illustrative example provided herein, it is understood that a liquid biopsy can be performed with a biological sample including blood, plasma, saliva, urine, feces, tears, mucosal secretions and other biological fluids. 
     In particular, methods of the disclosure provide and include the analytical validation of an integrated NGS-based liquid biopsy approach for the detection of microsatellite instability associated with cancers such as pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, or prostate cancer, as well as hematopoietic cancers, among others. Failure of the DNA mismatch repair (MMR) pathway during DNA replication in cancer leads to the increased accumulation of somatic mutations. One important marker of MMR deficiency is microsatellite instability (MSI), which presents as polymorphism of tandem nucleotide repeat lengths ubiquitously distributed throughout the genome. Methods of the disclosure are offered to assay for and detect those markers via liquid biopsy. Additionally, since the presence of MMR-deficiency or MSI may serve as a marker for immunotherapy response with checkpoint inhibition, methods may be used to determine a course of treatment such as immunotherapy or the administration of a checkpoint inhibitor. 
     Microsatellite instability (MSI) and mismatch repair (MMR) deficiency have recently been demonstrated to predict immune checkpoint blockade response. The checkpoint inhibitor pembrolizumab is now indicated for the treatment of adult and pediatric patients with any unresectable or metastatic solid tumors identified as having either of these biomarkers. This indication covers patients with solid tumors that have progressed following prior treatment and have no satisfactory alternative treatment options. 
     Cancer is characterized by the accumulation of somatic mutations that have the potential to result in the expression of neoantigens, which may elicit T-cell-dependent immune responses against tumors. MMR is a mechanism by which post-replicative mismatches in daughter DNA strands are repaired and replaced with the correct DNA sequence. MMR deficiency results in both MSI and high tumor mutation burden (TMB-High), which increases the likelihood that acquired somatic mutations may be transcribed and translated into proteins that are recognized as immunogenic neoantigens. Historically, testing for MSI has been restricted to screening for Hereditary Non-Polyposis Colorectal Cancer (HNPCC), which is often characterized by early age onset colorectal cancer and endometrial cancer, as well as other extracolonic tumors. HNPCC, commonly referred to as Lynch Syndrome, is caused by mutations in the DNA mismatch repair genes (MLH1, MSH2, MSH6 and PMS2), as well as the more recently described, EPCAM (16). In addition to familial conditions, MSI can occur sporadically in cancer, and both hereditary and sporadic MSI patients respond to immune checkpoint blockade (1,2). A recent study, conducted across 39 tumor types and 11,139 patients to determine the landscape of MSI prevalence, concluded that 3.8% of these cancers across 27 tumor types displayed MSI, including 31.4% of uterine/endometrial carcinoma, 19.7% of colon adenocarcinoma, and 19.1% of stomach adenocarcinoma. 
     MSI can be detected through alterations in the length of microsatellite sequences typically due to deletions of repeating units of DNA to create novel allele lengths in tumor-derived DNA when compared to a matched-normal or a reference population. Current methods for MSI testing, using tissue biopsies and resection specimens, include PCR-based amplification followed by capillary electrophoresis, and more recently, next-generation sequencing (NGS) based approaches, which are used to quantify microsatellite allele lengths. The challenge associated with application of the former approach are polymerase induced errors (stutter bands), particularly in samples with low tumor purity, such as cell-free DNA (cfDNA), which can mask true biological alleles exhibiting MSI. In the case of NGS based approaches, sensitivity is typically limited by the accuracy for determination of homopolymer lengths. A novel method was recently described for determination of MSI using pre-PCR elimination of wild-type DNA homopolymers in liquid biopsies. However, given the low prevalence of MSI across cancer, it would be preferable to develop an NGS profiling approach which can include other clinically actionable alterations in cancer, including TMB, sequence mutations, copy number alterations, and translocations. 
     In addition to the technical challenges associated with MSI detection, it is often not possible to readily obtain biopsy or resection tissue for genetic testing due to insufficient material (biopsy size and tumor cellularity), exhaustion of the limited material available after prior therapeutic stratification, logistical considerations for tumor and normal sample acquisition after initial diagnosis, or safety concerns related to additional tissue biopsy interventions (26). In contrast, plasma-based approaches offer the unique opportunity to obtain a rapid and real-time view of the primary tumor and metastatic lesions along with associated response to therapy. Circulating tumor DNA can be used to monitor and assess residual disease in response to clinical intervention, such as surgery or chemotherapy (27-33), which can directly impact patient care. To determine the clinical impact of identifying tumors that harbor MSI or TMB-High using cfDNA, we developed and applied a 98 kb 58-gene targeted panel to cancer patients with advanced disease treated with PD-1 blockade.  FIG.  1    diagrams a method  101  of detecting microsatellite instability (MSI). The method  101  includes obtaining  107  cell-free DNA (cfDNA) from a sample of plasma from a patient. Preferably, non-unique barcode are attached  111 . Portions of the cfDNA are sequenced  115  to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA. The method  101  includes modeling  121  a distribution of lengths of tracts of nucleotide repeats. A report is provided  125  describing an MSI status in the patient when a distribution of lengths of the plurality of tracts has peaks that deviate significantly from peaks in a reference distribution. Obtaining the cfDNA may include capturing target portions of DNA with probes, fragmenting the target portions to yield fragments, and attaching barcodes to the fragments. 
     Briefly, cell-free DNA may be extracted from cell line or blood or plasma specimens and prepared into a genomic library suitable for next-generation sequencing with oligonucleotide barcodes through end-repair, A-tailing and adapter ligation. An in-solution hybrid capture, utilizing for example, 120 base-pair (bp) RNA oligonucleotides may be performed. 
     In one embodiment, at least about 10-100 ng, such as 50 ng of DNA in 100 microliters of TE is fragmented in a sonicator to a size of about 150-450 bp. To remove fragments smaller than 150 bp, DNA may be purified using Agencourt AMPure XP beads (Beckman Coulter, Ind.) in a ratio of 1.0 to 0.9 of PCR product to beads twice and, e.g., washed using 70% ethanol per the manufacturer&#39;s instructions. Purified, fragmented DNA is mixed with H2O, End Repair Reaction Buffer, End Repair Enzyme Mix (cat #E6050, NEB, Ipswich, Mass.). The mixture is incubated then purified using Agencourt AMPure XP beads (Beckman Coulter, Ind.) in a ratio of 1.0 to 1.25 of PCR product to beads and washed using 70% ethanol per the manufacturer&#39;s instructions. To A-tail, end-repaired DNA is mixed with Tailing Reaction Buffer and Klenow (exo-) (cat #E6053, NEB, Ipswich, Mass.). The mixture is incubated at 37 degree C. for 30 min and purified using Agencourt AMPure XP beads (Beckman Coulter, Ind.) in a ratio of 1.0 to 1.0 of PCR product to beads and washed using 70% ethanol per the manufacturer&#39;s instructions. For adaptor ligation, A-tailed DNA is mixed with H2O, PE-adaptor (Illumina), Ligation buffer and Quick T4 DNA ligase (cat #E6056, NEB, Ipswich, Mass.). The ligation mixture was incubated, then amplified. 
     Exonic or targeted regions were captured in solution using the Agilent SureSelect v.4 kit according to the manufacturer&#39;s instructions (Agilent, Santa Clara, Calif.). The captured library was then purified with a Qiagen MinElute column purification kit. To purify PCR products, a NucleoSpin Extract II purification kit (Macherey-Nagel, PA) may be used before sequencing. 
     Targeted sequencing is performed. Two technical challenges to implementing these approaches in the form of a liquid biopsy include the limited amount of DNA obtained and the low mutant allele frequency associated with the MSI markers. It may be that as few as several thousand genomic equivalents are obtained per milliliter of plasma, and the mutant allele frequency can range from &lt;0.01% to &gt;50% total cfDNA. see Bettegowda, 2014, Detection of circulating tumor DNA in early- and late-stage human malignancies, Sci Trans Med 6(224): 224ra24, incorporated by reference. The disclosed techniques overcome such problems and improve test sensitivity, optimized methods for conversion of cell-free DNA into a genomic library, and digital sequencing approaches to improve the specificity of next-generation sequencing approaches. 
     Methods may include extracting and isolating cell-free DNA from a blood or plasma sample and assigning an exogenous barcode to each fragment to generate a DNA library. The exogenous barcodes are from a limited pool of non-unique barcodes, for example 8 different barcodes. The barcoded fragments are differentiated based on the combination of their exogenous barcode and the information about the reads that results from sequencing such as the sequence of the reads (effectively, an endogenous barcode) or position information (e.g., stop and/or start position) of the read mapped to a reference. The DNA library is redundantly sequenced  115  and the sequences with matching barcodes are reconciled. The reconciled sequences may be aligned to a human genome reference. 
     The invention recognizes that completely unique barcode sequences are unnecessary. Instead, a combination of predefined set of non-unique sequences together with the endogenous barcodes can provide the same level of sensitivity and specificity that unique barcodes could for biologically relevant DNA amounts and can, in-fact, correct for sequencing artifacts or polymerase slippage. A limited pool of barcodes is more robust than a conventional unique set and easier to create and use. Methods include obtaining a sample comprising nucleic acid fragments, providing a plurality of sets of non-unique barcodes, and tagging  111  the nucleic acid fragments with the barcodes to generate a genomic library, wherein each nucleic acid fragment is tagged with the same barcode as another different nucleic acid fragment in the genomic library. 
     In embodiments, the plurality of sets is limited to twenty or fewer unique barcodes. In other embodiments, the plurality of sets is limited to ten or fewer unique barcodes. 
     According to the present invention, a small pool of non-unique exogenous barcodes can be used to provide a robust assay that achieves levels of sensitivity that are comparable to traditional, more complex barcoding schemes, while vastly reducing cost and complication. 
     After processing steps such as those described above, nucleic acids can be sequenced. Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes. 
     A sequencing technique that can be used includes, for example, use of sequencing-by-synthesis systems sold under the trademarks GS JUNIOR, GS FLX+and 454 SEQUENCING by 454 Life Sciences, a Roche company (Branford, Conn.), and described by Margulies, M. et al., Genome sequencing in micro-fabricated high-density picotiter reactors, Nature, 437: 376-380 (2005); U.S. Pat. Nos. 5,583,024; 5,674,713; and 5,700,673, the contents of which are incorporated by reference herein in their entirety. 
     Other examples of DNA sequencing techniques include SOLiD technology by Applied Biosystems from Life Technologies Corporation (Carlsbad, Calif.) and ion semiconductor sequencing using, for example, a system sold under the trademark ION TORRENT by Ion Torrent by Life Technologies (South San Francisco, Calif.). Ion semiconductor sequencing is described, for example, in Rothberg, et al., An integrated semiconductor device enabling non-optical genome sequencing, Nature 475: 348-352 (2011); U.S. Pub. 2010/0304982; U.S. Pub. 2010/0301398; U.S. Pub. 2010/0300895; U.S. Pub. 2010/0300559; and U.S. Pub. 2009/0026082, the contents of each of which are incorporated by reference in their entirety. 
     Another example of a sequencing technology that can be used is Illumina sequencing. Illumina sequencing is based on the amplification of DNA on a solid surface using fold-back PCR and anchored primers. Adapters are added to the 5′ and 3′ ends of DNA that is either naturally or experimentally fragmented. DNA fragments that are attached to the surface of flow cell channels are extended and bridge amplified. The fragments become double stranded, and the double stranded molecules are denatured. Multiple cycles of the solid-phase amplification followed by denaturation can create several million clusters of approximately 1,000 copies of single-stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA polymerase and four fluorophore-labeled, reversibly terminating nucleotides are used to perform sequential sequencing. After nucleotide incorporation, a laser is used to excite the fluorophores, and an image is captured and the identity of the first base is recorded. The 3′ terminators and fluorophores from each incorporated base are removed and the incorporation, detection and identification steps are repeated. Sequencing according to this technology is described in U.S. Pat. Nos. 7,960,120; 7,835,871; 7,232,656; 7,598,035; 6,911,345; 6,833,246; 6,828,100; 6,306,597; 6,210,891; U.S. Pub. 2011/0009278; U.S. Pub. 2007/0114362; U.S. Pub. 2006/0292611; and U.S. Pub. 2006/0024681, each of which are incorporated by reference in their entirety. 
     Preferably sequencing is done redundantly for deep coverage, preferably at least 30× coverage or 100×. DNA libraries may be sequenced using paired-end 111umina HiSeq 2500 sequencing chemistry to an average target total coverage of either &gt;20,000-fold or &gt;5,000-fold coverage for each targeted base. Sequence data may be mapped to the reference human genome. Preferably, the sequencing is next-generation, short-read sequencing. The obtained sequences may include a plurality of sequence reads and the method may include aligning the sequence reads to a reference, and identifying groups of sequence reads that originated from a unique segment of the cfDNA by means of the barcode information and position or content of the sequence reads. Primary processing of sequence data may be performed using Illumina CASAVA software (v1.8), including masking of adapter sequences. Sequence reads may bealigned against the human reference genome (version hg18) using ELAND with additional realignment of select regions using the Needleman-Wunsch method. 
     In some embodiments, the barcodes are non-unique barcodes that include duplicates such that different ones of the fragments are attached to identical barcodes. The high clinical efficacy of MSI status now requires a fast, objective, highly sensitive screening method, particularly in late-stage patients where tumor material may not be readily obtained. However, to extend this approach to a liquid biopsy panel requires technological advances to both overcome the inherent challenges associated with low circulating tumor DNA (ctDNA) levels which is compounded by polymerase slippage in mononucleotide repeat regions during PCR amplification as well as other sequencing artifacts. 
     To overcome these limitations, we applied error correction approach using molecular barcoding together with high sequencing depth and a novel peak finding algorithm to more accurately identify the specific mononucleotide sequences in cell-free DNA (cfDNA) analyses of a 64 gene panel, by way of illustration. The MSI markers can be sequenced in conjunction with such 64 gene panel, or in isolation (e.g., just sequence the markers) or in conjunction with any other gene panel (e.g., &gt;300 genes) or with whole genome or whole exome sequencing. 
     The method may include amplifying the fragments to produce amplicons that include barcode information and copies of the fragments, wherein the sequencing step comprises sequencing the amplicons. 
     The use of the non-unique barcodes to identify groups of sequence reads that originated from a unique segment of the cfDNA allows for the lengths of the plurality of tracts to be determined correctly by correcting for errors introduced by sequencing artifacts or polymerase slippage during the amplifying step. By eliminating a significant majority of sequencing errors and polymerase slippage artifacts, we were able to reduce background error rates by &gt;90%. Combined with implementation of a distribution modeling and a peak finding algorithm, we were able to accurately sequence the mononucleotide tracts to minimize false discovery rates for cfDNA analyses. 
       FIG.  2    shows a system  901  for performing methods of the disclosure. The system  901  includes a computer  933 , and may optionally include a server computer  909 . In certain embodiments, the system  901  includes a sequencing instrument  955  (such as an Illumina HiSeq device) which may itself include an instrument computer  951  (e.g., onboard in the sequencing instrument). Any of the computers may communicate via network  915 . Each computer preferably includes at least one tangible, non-transitory memory device  975  and any input/output devices coupled to a processor. The memory may include instructions executable by the processor(s) to perform methods such as a method of detecting microsatellite instability (MSI) that includes obtaining a sample comprising fragments of cell-free DNA from a patient; attaching barcodes to the fragments, wherein at least some of the barcodes are not unique; sequencing the barcodes to obtain sequences of a plurality of markers in the DNA; determining a distribution of lengths of the plurality of markers; and providing a report describing MSI in the patient when peaks in the distribution deviate significantly from expected peaks in a modeled healthy distribution. 
       FIG.  3    illustrates distribution modeling for peak finding. In the illustrated distribution model  301 , a model  307  of a distribution of lengths of tracts of nucleotide repeats is determined. It may be compared to a reference distribution  305  and an operation may be performed to find a peak  313  for the patient data  307  and/or the reference distribution  305  (which may be from patient healthy sample DNA or from a human genome reference or any other suitable source. In some embodiments, when the peak finding operation determines that the patient peak  313  is sufficiently deviant from a location of a reference peak, the method and system report the patient as MSI (microsatellite instable) for the relevant marker. Most preferably, the peak finding and distribution modeling is performed for each MSI marker. A benefit of the described method is that the distribution modeling and peak finding may be reliably implemented and automated in a high-throughput system. 
     MSI may be assayed by hybrid capture and NGS to address such markers as mononucleotide repeat markers such as BAT25, BAT26, MON027, NR21, and NR24. See U.S. Pub. 2017/0267760, incorporated by reference. Knowledge of MSI status is important and valuable in the treatment of many cancers, and there are patients for whom tumor material is not readily obtained. Tumors deficient in mismatch repair are particularly susceptible to a particular form of immunotherapy because this phenotype results in ongoing accumulation of mutations at a high frequency. Methods may include recommending or administering treatment for cancer patients that display the microsatellite instability phenotype or other high mutational burden. The treatment involves an inhibitory antibody for an immune checkpoint. Such checkpoints include PD-1, IDO, CTLA-4, PD-L1, and LAG-3 by way of example. Other immune checkpoints can be used as well. Antibodies can be administered by any means that is convenient, including but not limited to intravenous infusion, oral administration, subcutaneous administration, sublingual administration, ocular administration, nasal administration, and the like. 
     Preferably, the method  101  includes providing  125  a report with MSI status. 
       FIG.  4    shows a report  410  that includes a status of “instable” for certain MSI markers. Preferably, the target portions are markers for MSI such as one or more of BAT25, BAT26, MON027, NR21, NR24, Penta C, and Penta D. For example, the markers may include all of BAT25, BAT26, MON027, NR21, and NR24. In certain embodiments, each of the microsatellite markers is selected from the group consisting of BAT-25, BAT-26, MONO-27, NR-21, NR-24, Penta C, and Penta D. 
     In some embodiments, the method includes recommending a treatment for the patient based on the MSI status. Where the MSI status indicates that the patient is microsatellite instable, the treatment may include an immune checkpoint inhibitor. In certain embodiments, the method includes administering the treatment (e.g., the immune checkpoint inhibitor) to the patient. The immune checkpoint inhibitor may be, for example, an antibody such as an anti-PD-1 antibody; an anti-IDO antibody; anti-CTLA-4 antibody; an anti-PD-L1 antibody; or an anti-LAG-3 antibody. Types of antibodies which can be used include any that are developed for the immune checkpoint inhibitors. These can be monoclonal or polyclonal. They may be single chain fragments or other fragments of full antibodies, including those made by enzymatic cleavage or recombinant DNA techniques. They may be of any isotype, including but not limited to IgG, IgM, IgE. The antibodies may be of any species source, including human, goat, rabbit, mouse, cow, chimpanzee. The antibodies may be humanized or chimeric. The antibodies may be conjugated or engineered to be attached to another moiety, whether a therapeutic molecule or a tracer molecule. The therapeutic molecule may be a toxin, for example. The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. The following examples are intended to illustrate but not limit the invention. 
     EXAMPLES 
     Example 1 
     Methods 
     Patients and Sample Collection 
     Formalin fixed paraffin embedded (FFPE) tumor and matched normal buffy coat specimens (n=61) from individuals with cancer were obtained after surgical resection through commercial biorepositories from BioIVT (Hicksville, N.Y., USA), Indivumed (Hamburg, Germany), and iSpecimen (Lexington, Mass., USA). Plasma samples from healthy individuals (n=163) were procured through BioIVT (Hicksville, N.Y., USA) during routine screening with negative results and no prior history of cancer. Human cells from previously characterized MSI cell lines were obtained from ATCC (Manassas, Va., USA) (n=5; LS180, LS411N, SNU-C2B, RKO, and SNU-C2A). Finally, baseline and serial plasma samples from cancer patients with progressive metastatic carcinoma (n=16; 11 colorectal, 3 ampullary, and 2 small intestine) were obtained while patients were enrolled in a phase 2 clinical trial to evaluate immune checkpoint blockade with pembrolizumab (1,2). Radiographic and serum protein biomarker data for CEA and CA19-9 were collected as a part of routine clinical care. All samples were obtained under Institutional Review Board approved protocols with informed consent for research. 
     Orthogonal Testing of FFPE Tissue for MSI Status 
     The Promega MSI analysis system (Madison, Wis., USA) was used to assess MSI status in DNA derived from FFPE tumor tissue together with matched normal buffy coat by multiplex PCR and fluorescent capillary electrophoresis. Tumors were classified as MSI if two or more of the five mononucleotide markers (BAT25, BAT26, MON027, NR21, and NR24) had significant length differences compared to the matched normal allele lengths. Additionally, 2-pentanucleotide repeat loci (PentaC and PentaD) were used to confirm case identity between normal and tumor samples. 
     Sample Preparation and Next-Generation Sequencing 
     FFPE Tumor and Normal Analyses 
     Sample processing from tissue or buffy coat, library preparation, hybrid capture, and sequencing were performed as previously described at Personal Genome Diagnostics (Baltimore, Md.) (34,36). Briefly, DNA was extracted from FFPE tissue and matched normal buffy coat cells using the Qiagen FFPE Tissue Kit and DNA Blood Mini Kit, respectively (Qiagen, Hilden, Germany). Genomic DNA was sheared using a Covaris sonicator (Woburn, Mass., USA) to a size range of 150-450 bp, and subsequently used to generate a genomic library using the New England Biolabs (Ipswich, Mass., USA) end-repair, A-tailing, and adapter ligation modules. Finally, genomic libraries were amplified and captured using the Agilent SureSelect XT in-solution hybrid capture system with a custom 120 bp RNA panel targeting the pre-defined regions of interest across 125 genes (Table 1). Captured libraries were sequenced on the Illumina HiSeq 2000 or 2500 (Illumina, San Diego, Calif., USA) with 100 bp paired end reads. 
     Plasma Analyses 
     Sample processing from plasma, library preparation, hybrid capture, and sequencing were performed as previously described at Personal Genome Diagnostics (Baltimore, Md.) (34). Briefly, blood was collected in EDTA tubes and centrifuged at 800 g for 10 minutes at 4° C. to separate plasma from white blood cells. Cell-free DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Libraries were prepared with 5-250 ng of cfDNA using the NEBNext DNA Library Prep Kit (New England Biolabs, Ipswich, Mass., USA). After end repair and a-tailing, a pool of eight unique Illumina dual index adapters with 8 bp barcodes were ligated to cfDNA to allow for accurate error correction of duplicate reads, followed by 12 cycles of amplification. Targeted hybrid capture was performed using Agilent SureSelect XT in-solution hybrid capture system with a custom 120 bp RNA panel targeting the pre-defined regions of interest across 58 genes (Table 4) according to the manufacturer protocol (Agilent Technologies, Santa Clara, Calif., USA). Captured libraries were sequenced on the Illumina HiSeq 2000 or 2500 (Illumina, San Diego, Calif., USA) with 100 bp paired end reads. 
     Example 2 
     Microsatellite Instability Analyses by Next-Generation Sequencing 
     Sequence data were aligned to the human reference genome assembly (hg19) using BWA-MEM (37). Reads mapping to microsatellites were excised using Samtools (38) and analyzed for insertion and deletion events (indels). In most cases, alignment and variant calling did not generate accurate indel calls in repeated regions due to low quality bases surrounding the microsatellites. Therefore, a secondary local realignment and indel quantitation was performed. Reads were considered for an expanded indel analysis if (i) the mononucleotide repeat was contained to more than eight bases inside of the start and end of the read, (ii) the indel length was &lt;12 bases from the reference length, (iii) there were no single base changes found within the repeat region, (iv) the read had a mapping score of 60, and (v) ≤20 bases of the read were soft clipped for alignment. After read specific mononucleotide length analysis, error correction was performed to allow for an aggregated and accurate quantitation among duplicated fragments using molecular barcoding. Reads were aggregated into barcode families by using the ordered and combined read 1 and read 2 alignment positions with the molecular barcode. Barcode families were considered for downstream analysis if they comprised of at least 2 reads and &gt;50% of reads had consistent mononucleotide lengths. The error corrected mononucleotide length distribution was subjected to a peak finding algorithm where local maxima were required to be greater than the error corrected distinct fragment counts of the adjacent lengths ±2 bp. Identified peaks were further filtered to only include those which had &gt;3 error corrected distinct fragments at ≥1% of the absolute coverage. The shortest identified mononucleotide allele length was compared to the hg19 reference length. If the allele length was ≥3 bp shorter than the reference length, the given mononucleotide loci was classified as exhibiting instability. This approach was applied across all mononucleotide loci. Samples were classified as MSI-H if ≥20% of loci were MSI. In the targeted 58 gene plasma panel, BAT25, BAT26, MON027, NR21, and NR24 mononucleotide loci were for the determination of MSI status. In the targeted 125 gene targeted tissue panel, an additional 65 microsatellite regions were used for MSI classification. 
     Example 3 
     Tumor Mutational Burden Analyses by Next-Generation Sequencing 
     Next generation sequencing data were processed and variants were identified using the VariantDx custom software as previously described (34). A final set of candidate somatic mutations were selected for tumor mutational burden analyses based on: (i) variants enriched due to sequencing or alignment error were removed (≤5 observations or &lt;0.30% mutant allele fraction), (ii) nonsynonymous and synonymous variants were included, but variants arising in non-coding regions were removed, (iii) hotspot variants annotated in COSMIC (version 72) were not included to reduce bias toward driver alterations, (iv) common germline SNPs found in dbSNP (version 138) were removed as well as variants deemed private germline variants based on the variant allele frequency, and (v) variants associated with clonal hematopoietic expansion were not included in the candidate variant set (39). 
     In Silico TCGA Analyses 
     In order to evaluate the accuracy of the 98 kb targeted panel for prediction of TMB, a comparison to whole-exome sequencing data derived from The Cancer Genome Atlas (TCGA) (35) was performed by considering synonymous and nonsynonymous alterations, excluding known hotspot mutations which may not be representative of TMB in the tumor. The cutoff for consideration as TMB-High was set to 5 candidate variants (50.8 mutations/Mbp sequenced) based on in silico analyses utilizing the TCGA data to achieve &gt;95% accuracy (&gt;36 mutations/Mbp). 
     Statistical Analyses 
     Due to small sample size, Firth&#39;s Penalized Likelihood was used to evaluate significant differences between Kaplan-Meier curves for progression free survival and overall survival with the classifiers baseline MSI status, baseline TMB status, two consecutive timepoints with &gt;80% reduction in baseline protein biomarker levels, two consecutive timepoints with 0% residual MSI alleles on treatment, and two consecutive timepoints with &gt;90% reduction in baseline TMB levels. Pearson correlations were used to evaluate significant association between TMB in the 58 gene targeted panel compared to whole-exome analyses, progression free and overall survival compared to residual protein biomarker levels, and progression free and overall survival compared to residual MSI and TMB allele levels. A student t-test was used to evaluate significant differences between the mean TMB level in TMB-High and TMB-Low patients. Response rate was calculated as the number of patients exhibiting a complete or partial response as a proportion of the total patients considered, and then evaluated using a Fisher&#39;s exact test. 
     Example 4 
     Development of an Assay to Identify MSI in Cell-Free DNA 
     To identify MSI in tumor-derived cfDNA, a method to detect length polymorphisms in mononucleotide tract alleles in circulating tumor DNA (ctDNA), which occur at low frequency in plasma, is needed. To overcome this issue, we developed a highly sensitive error-correction approach incorporating the commonly-used mononucleotide tracts BAT25, BAT26, MON027, NR21, and NR24 for the determination of MSI status in tissue and plasma specimens using NGS. DNA was converted into an NGS compatible library using molecular barcoding, after which these targeted microsatellite loci were enriched using in-solution hybrid capture chemistry together with the regions associated with other clinically relevant genomic alterations. 
     To address the technical challenges associated with detection of low level allele length polymorphisms obtained from NGS, we combined an error correction approach for accurate determination of insertions and deletions (indels) present in the cfDNA fragments, together with a digital peak finding (DPF) method for quantification of MSI and MSS alleles. Redundant sequencing of each cfDNA fragment was performed, and reads were aligned to the five microsatellite loci contained in the human reference genome (hg19). cfDNA sequences were then analyzed for indels through a secondary local alignment at these five microsatellite loci to more accurately determine the indel length. To perform the error correction, duplicated reads associated with each cfDNA molecule were consolidated, only recognizing indels present throughout barcoded DNA fragment replicates obtained through redundant sequencing. Finally, the DPF approach was applied across the error corrected distribution of indels to identify high confidence alleles which exhibit microsatellite instability ( FIGS.  5 A and  5 B ). 
     To demonstrate the capability of this approach, we first evaluated the performance of the method for detection of MSI in formalin fixed, paraffin embedded (FFPE) tumor tissue specimens obtained from 31 MSI-High (MSI-H) and 30 microsatellite stable (MSS) tumors previously characterized with the PCR-based Promega MSI analysis system. In addition to these five mononucleotide markers, we sequenced 125 selected cancer genes which harbor clinically actionable genetic alterations consisting of sequence mutations (single base substitutions and indels), copy number alterations, and gene rearrangements in cancer (Table 1). Analyses of these 61 colorectal tumors yielded 193 Gb of total sequence data, corresponding to 832-fold distinct coverage on average across the 979 kb panel (Table 2). Analysis of these five mononucleotide loci, together with 65 additional microsatellite regions contained within the 125 gene panel resulted in 100% sensitivity ( 31/31) and 100% specificity ( 30/30) for determination of MSI status using the patient-matched tumor and normal samples (Table 3). Similarly, analysis of tumor NGS data using the DPF approach without the patient-matched normal sample yielded 100% concordance ( 61/61). 
     Next, we evaluated the signal-to-noise ratio in homopolymer regions from next-generation sequencing data obtained using cfDNA extracted from plasma. Together with the five mononucleotide loci, we developed a 98 kb, 58 gene panel for sequence mutation (single base substitutions and indels) analyses of clinically actionable genetic alterations in cancer (Table 4). To demonstrate the specificity of this approach for direct detection of MSI, we first obtained plasma from healthy donors (n=163), all of which would be expected to be tumor-free and MSS. These analyses yielded over 1.2 Tb of total sequence data, corresponding to 2,600-fold distinct coverage on average across the 98 kb targeted panel, and resulted in a per-patient specificity of 99.4% ( 162/163) for determination of MSI status ( FIG.  5 B , Tables 5 and 6). 
     Because ctDNA, even in patients with advanced cancer, may be present at mutant allele fractions (MAFs) less than 5%, we characterized the ability of DPF for sensitive and reproducible detection of MSI at low MAFs. Five previously characterized MSI cell line samples obtained from ATCC (LS180, LS411N, SNU-C2B, RKO, and SNU-C2A) were sheared to a fragment profile simulating cfDNA and diluted with normal DNA to yield a total of 25 ng evaluated at 1% MAF. Additionally, three of these cell lines (LS180, LS411N, and SNU-C2B) were evaluated at 1% MAF in triplicate within, and triplicate across library preparation and sequencing runs (Table 5). Based on the MAF observed in the parental cell line, the cases detected as MSI were computationally confirmed to contain MSI allele MAFs of 0.35%-1.87%, with a median MSI allele MAF of 0.92%. In total, MSI was detected in 90% ( 18/20) of samples and demonstrated 93.3% ( 14/15) repeatability and reproducibility within and across runs (Table 6). For one case which was not detected as MSI, one MSI allele was identified at 0.33% MAF and for the other case, no MSI alleles were detected. 
     Example 5 
     Assessment of MSI in CFDNA in Patients Treated With PD-1 Blockade 
     To evaluate the analytical and clinical performance of this approach for determination of MSI in cfDNA from patients with late-stage cancers, we obtained baseline and serial plasma from patients with metastatic cancers (including 11 colorectal, 3 ampullary, and 2 small intestine), with or without MMR deficiency, while enrolled in a clinical trial to evaluate immune checkpoint blockade with the PD-1 blocking antibody, pembrolizumab (1,2) (Table 7). In total, 12 MSI-H cases and 4 MSS cases, determined through archival tissue-based analyses, were evaluated across at least two timepoints, including baseline, and after approximately 2 weeks, 10 weeks, 20 weeks, and &gt;100 weeks. 
     Patients with MSI tumors as determined by archival tissue analyses had improved progression-free survival (hazard ratio, 0.25; p=0.05, likelihood ratio test) and overall survival (hazard ratio, 0.24; p=0.041, likelihood ratio test) ( FIGS.  9 A and  9 B  and Table 8). In cfDNA, we could detect MSI in 75% ( 9/12) of the previously characterized MSI-H patients, and correctly identified 100% ( 4/4) of the MSS patients (Table 6). Of the three cases that were MSI in the tumor tissue and MSS in the cfDNA, one was a colorectal tumor (patient exhibited progressive disease) and two were small intestinal tumors (one patient exhibited a partial response and one exhibited progressive disease) with relatively low levels of ctDNA with MAF of 0.4%, 1.1%, and no detectable ctDNA in the third case (34) (Table 7). 
     We then evaluated pre-treatment MSI status in ctDNA to predict response and clinical outcome to treatment with PD-1 blockade. We assessed radiographic response, progression-free and overall survival to predict clinical outcome. When compared to progression free survival, direct detection of MSI in baseline cfDNA could be used to predict response to immune checkpoint blockade (hazard ratio, 0.2; p=0.01, likelihood ratio test) ( FIGS.  5 C and  5 D ). 
     Estimating Tumor Mutation Burden in ctDNA 
     In addition to MSI status, we also evaluated the ability of our cfDNA panel to predict TMB across a range of tumor types, using whole exome sequencing data derived from The Cancer Genome Atlas (TCGA) (35). We considered synonymous and nonsynonymous alterations identified by TCGA and excluded known hotspot mutations which may not be representative of TMB in the tumor. These analyses demonstrated a positive correlation between predicted TMB from our targeted 58 gene plasma panel compared to the TCGA whole exome analyses (r=0.91, p&lt;0.0001; Pearson correlation) ( FIG.  6 A ). We determined that a cutoff of five mutations (50.8 mutations/Mbp sequenced) in the targeted plasma panel could be used to identify tumors with exceptionally high TMB related to MMR deficiency (&gt;36 mutations/Mbp) at &gt;95% accuracy. 
     Patients with TMB-High tumors as determined by archival tissue analyses (≥10 mutations/Mbp) had improved progression-free survival (hazard ratio, 0.19; p=0.041, likelihood ratio test) and overall survival (hazard ratio, 0.18; p=0.047, likelihood ratio test) ( FIGS.  9 C and  9 D ). We also evaluated the accuracy of TMB derived from the targeted panel in 13 baseline plasma cases, compared to whole-exome analyses of tumor and matched normal tissue in the same patients(1,2), and a similar correlation was identified (r=0.69, p=0.007; Pearson correlation) ( FIG.  6 B ). These patients were classified as either TMB-High or TMB-Low using a cutoff of 50.8 mutations/Mbp sequenced, which captured six of the ten tumors categorized as TMB-High by archival tissue and provided a statistically significant difference in the TMB classification (p=0.0072, t-test) ( FIG.  6 C ). This algorithm was applied to the same 163 healthy donor plasma samples and 100% ( 163/163) were determined to be TMB-Low ( FIG.  6 C ). When considering TMB classification as a predictor of clinical outcome from the same phase 2 study cohort, TMB-High status was associated with favorable progression free survival (hazard ratio, 0.12; p=0.004 likelihood ratio test) and overall survival (hazard ratio, 0.16; p=0.014, likelihood ratio test) ( FIGS.  6 D and  6 E ). Interestingly, all four MSI-H enrolled patients exhibiting a complete response were classified as TMB-High, and all five enrolled MSI-H patients with progressive disease were classified as TMB-Low (Table 7). 
     Example 6 
     Assessment of Molecular Remission and Biomarker Dynamics in Patients Treated With PD-1 Blockade 
     In addition to baseline plasma analyses, we also hypothesized that the molecular remission, as measured by ctDNA during treatment, would be predictive of long term durable response to immune checkpoint blockade. We first evaluated the utility of monitoring serum tumor protein biomarkers CEA or CA19-9 for determination of response and found that multiple consecutive timepoints with a &gt;80% reduction in the baseline protein biomarker level resulted in improved overall and progression free survival (hazard ratio, 0.05; p=0.01 and hazard ratio, 0.05; p=0.01, likelihood ratio test, respectively) ( FIGS.  7 A and  7 B  and  FIGS.  10 A and  10 B ). When evaluating the on-treatment serial plasma samples for residual ctDNA levels, there was a significant inverse correlation between the overall and progression free survival when compared to the residual MSI allele levels at last dose (r=−0.70, p=0.034 and r=−0.84, p=0.004, respectively; Pearson correlation) ( FIGS.  7 C  and  FIG.  10 C ). We were able to correctly identify four of the six MSI patients who would achieve a long term durable clinical response requiring multiple consecutive on-treatment time points with 0% residual alleles displaying MSI, all four of which displayed a complete response (hazard ratio, 0.09; p=0.032, likelihood ratio test for overall survival) ( FIG.  7 D  and  FIG.  10 D ). A similar trend was observed when considering patients with a &gt;90% decrease in overall TMB across two timepoints when compared to baseline (hazard ratio, 0.07; p=0.013, likelihood ratio test for overall survival) ( FIGS.  7 E and  7 F  and  FIGS.  10 E and  10 F ). 
     Additionally, for three patients (CS97, CS98, and CS00) with a complete response to immune checkpoint blockade, and one patient (CS05) without a response to immune checkpoint blockade, circulating protein biomarkers (CEA, ng/mL or CA19-9, units/mL) and residual alleles exhibiting MSI and TMB were evaluated over time during treatment ( FIG.  8   ). In each of the patients exhibiting a complete response, there was a concurrent decrease in the circulating protein biomarker levels, the residual MSI alleles, and TMB levels, which correlated with reduced overall tumor volume as assessed by radiographic imaging. Protein biomarker levels decreased by more than 80% between 1.3 to 2.3 months after first dose. Residual MSI alleles and TMB levels were reduced by &gt;90% between 0.6 and 4.8 months after first dose for these three cases. However, for patient CS05 with progressive disease, the protein biomarker levels remained relatively constant, but there was an increase in residual alleles exhibiting MSI and TMB of 78% and 50%, respectively, at 4.8 months. This correlated with a 13% increase in tumor volume as assessed by radiographic imaging at 5 months. 
     Patient CS97 demonstrated a partial radiographic response at 10.6 months, however, achieved a 100% reduction in residual MSI and TMB levels at 2.8 months. CS97 then went on to a complete radiographic response at 20.2 months (Table 7). A different patient, CS98, appeared to develop new liver lesions at 20 weeks suggestive of progressive disease ( FIG.  11   ). However, following an initial spike, protein biomarkers and residual MSI and TMB levels demonstrated a biochemical tumor response at 1.3 and 4.8 months. A liver biopsy demonstrated only inflammatory changes in the location where new lesions were noted, suggesting checkpoint therapy induced inflammation. Radiographic imaging finally demonstrated resolution of any hepatic lesions and a 100% reduction in tumor volume at 16.8 months. A similar pattern was observed for patient CS00 where significant reduction in protein biomarker and residual MSI and TMB levels occurred at 1.5 and 0.6 months, respectively, however, radiographic imaging did not demonstrate a 100% reduction in tumor volume until 17 months. These data suggest that the residual MSI allele burden and TMB prognostic signature are indicative of overall tumor response to immune checkpoint blockade. 
     Discussion 
     The checkpoint inhibitor pembrolizumab is now indicated for the treatment of adult and pediatric patients with unresectable or metastatic solid tumors identified as having MSI or MMR deficiency (1,2). This represents the first pan-cancer biomarker indication, and now covers patients with solid tumors that have progressed following prior treatment and have no satisfactory alternative treatment options, as well as patients with colorectal cancer that have progressed following treatment with certain chemotherapy drugs. However, it is often not possible to readily obtain biopsy or resection tissue for genetic testing due to insufficient material, exhaustion of the limited material available after prior therapeutic stratification, logistical considerations for tumor and normal sample acquisition after initial diagnosis, or safety concerns related to additional tissue biopsy interventions (26). 
     We have described the development of a method for simultaneous detection of MSI and TMB-High directly from cfDNA and demonstrated proof of concept for the clinical utility afforded through these analyses for the prediction of response to immune checkpoint blockade. Additionally, given the concordance with circulating protein biomarker data while these patients were on treatment, these data suggest that the residual MSI allele burden and TMB prognostic signature could be applied to other tumor types where standardized protein biomarkers do not exist and may be an earlier predictor of response than radiographic imaging. 
     These methods described herein provide feasibility for a viable diagnostic approach for screening and monitoring of patients who exhibit MSI or TMB-High and may respond to immune checkpoint blockade. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 125 Gene List for FFPE Tissue Analyses 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Sequence  
                 Trans- 
                 Amplifi- 
               
               
                   
                 Gene 
                 Mutations 
                 locations 
                 cations 
               
               
                   
                 (n = 125) 
                 (n = 117) 
                 (n = 29) 
                 (n = 41) 
               
               
                   
                   
               
               
                   
                 ABL1 
                 Yes 
                 — 
                 — 
               
               
                   
                 AKT1 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 ALK 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 AR 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 ATM 
                 Yes 
                 — 
                 — 
               
               
                   
                 ATRX 
                 Yes 
                 — 
                 — 
               
               
                   
                 AXL 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 BCL2 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 BCR 
                 — 
                 Yes 
                 — 
               
               
                   
                 BRAF 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 BRCA1 
                 Yes 
                 Yes 
                 — 
               
               
                   
                 BRCA2 
                 Yes 
                 Yes 
                 — 
               
               
                   
                 CBFB 
                 — 
                 Yes 
                 — 
               
               
                   
                 CCND1 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 CCND2 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 CCND3 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 CDK4 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 CDK6 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 CDKN2A 
                 Yes 
                 — 
                 — 
               
               
                   
                 CHEK2 
                 Yes 
                 — 
                 — 
               
               
                   
                 CREBBP 
                 Yes 
                 — 
                 — 
               
               
                   
                 CSF1R 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 CTNNB1 
                 Yes 
                 — 
                 — 
               
               
                   
                 DDR2 
                 Yes 
                 — 
                 — 
               
               
                   
                 DNMT3A 
                 Yes 
                 — 
                 — 
               
               
                   
                 EGFR 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 EP300 
                 Yes 
                 — 
                 — 
               
               
                   
                 EPHA2 
                 Yes 
                 — 
                 — 
               
               
                   
                 ERBB2 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 ERBB3 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 ERBB4 
                 Yes 
                 — 
                 — 
               
               
                   
                 ERCC3 
                 Yes 
                 — 
                 — 
               
               
                   
                 ERG 
                 Yes 
                 Yes 
                 — 
               
               
                   
                 ESR1 
                 Yes 
                 — 
                 — 
               
               
                   
                 ETV1 
                 — 
                 Yes 
                 — 
               
               
                   
                 ETV4 
                 — 
                 Yes 
                 — 
               
               
                   
                 ETV5 
                 — 
                 Yes 
                 — 
               
               
                   
                 ETV6 
                 — 
                 Yes 
                 — 
               
               
                   
                 EWSR1 
                 — 
                 Yes 
                 — 
               
               
                   
                 EZH2 
                 Yes 
                 — 
                 — 
               
               
                   
                 FANCA 
                 Yes 
                 — 
                 — 
               
               
                   
                 FANCD2 
                 Yes 
                 — 
                 — 
               
               
                   
                 FANCG 
                 Yes 
                 — 
                 — 
               
               
                   
                 FBXW7 
                 Yes 
                 — 
                 — 
               
               
                   
                 FGFR1 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 FGFR2 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 FGFR3 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 FGFR4 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 FLT1 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 FLT3 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 FLT4 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 FOXL2 
                 Yes 
                 — 
                 — 
               
               
                   
                 GNA11 
                 Yes 
                 — 
                 — 
               
               
                   
                 GNAQ 
                 Yes 
                 — 
                 — 
               
               
                   
                 GNAS 
                 Yes 
                 — 
                 — 
               
               
                   
                 HDAC2 
                 Yes 
                 — 
                 — 
               
               
                   
                 HNF1A 
                 Yes 
                 — 
                 — 
               
               
                   
                 HRAS 
                 Yes 
                 — 
                 — 
               
               
                   
                 IDH1 
                 Yes 
                 — 
                 — 
               
               
                   
                 IDH2 
                 Yes 
                 — 
                 — 
               
               
                   
                 JAK1 
                 Yes 
                 — 
                 — 
               
               
                   
                 JAK2 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 JAK3 
                 Yes 
                 — 
                 — 
               
               
                   
                 KDR 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 KEAP1 
                 Yes 
                 — 
                 — 
               
               
                   
                 KIT 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 KMT2A 
                 Yes 
                 — 
                 — 
               
               
                   
                 KRAS 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 MAP2K1 
                 Yes 
                 — 
                 — 
               
               
                   
                 MAP2K2 
                 Yes 
                 — 
                 — 
               
               
                   
                 MEN1 
                 Yes 
                 — 
                 — 
               
               
                   
                 MET 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 MLH1 
                 Yes 
                 — 
                 — 
               
               
                   
                 MLH3 
                 Yes 
                 — 
                 — 
               
               
                   
                 MPL 
                 Yes 
                 — 
                 — 
               
               
                   
                 MRE11A 
                 Yes 
                 — 
                 — 
               
               
                   
                 MSH2 
                 Yes 
                 — 
                 — 
               
               
                   
                 MSH6 
                 Yes 
                 — 
                 — 
               
               
                   
                 MST1R 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 MTOR 
                 Yes 
                 — 
                 — 
               
               
                   
                 MYC 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 MYCN 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 MYD88 
                 Yes 
                 — 
                 — 
               
               
                   
                 NBN 
                 Yes 
                 — 
                 — 
               
               
                   
                 NF1 
                 Yes 
                 — 
                 — 
               
               
                   
                 NOTCH1 
                 Yes 
                 — 
                 — 
               
               
                   
                 NPM1 
                 Yes 
                 — 
                 — 
               
               
                   
                 NRAS 
                 Yes 
                 — 
                 — 
               
               
                   
                 NTRK1 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 NTRK2 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 NTRK3 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 PALB2 
                 Yes 
                 — 
                 — 
               
               
                   
                 PDGFRA 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 PDGFRB 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 PIK3CA 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 PIK3CB 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 PIK3R1 
                 Yes 
                 — 
                 — 
               
               
                   
                 PMS2 
                 Yes 
                 — 
                 — 
               
               
                   
                 POLD1 
                 Yes 
                 — 
                 — 
               
               
                   
                 POLE 
                 Yes 
                 — 
                 — 
               
               
                   
                 PTCH1 
                 Yes 
                 — 
                 — 
               
               
                   
                 PTEN 
                 Yes 
                 — 
                 — 
               
               
                   
                 PTPN11 
                 Yes 
                 — 
                 — 
               
               
                   
                 RAD51 
                 Yes 
                 — 
                 — 
               
               
                   
                 RAF1 
                 Yes 
                 Yes 
                 — 
               
               
                   
                 RARA 
                 Yes 
                 Yes 
                 — 
               
               
                   
                 RB1 
                 Yes 
                 — 
                 — 
               
               
                   
                 RET 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 RNF43 
                 Yes 
                 — 
                 — 
               
               
                   
                 ROS1 
                 Yes 
                 Yes 
                 Yes 
               
               
                   
                 RUNX1 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 SDHB 
                 Yes 
                 — 
                 — 
               
               
                   
                 SMAD4 
                 Yes 
                 — 
                 — 
               
               
                   
                 SMARCB1 
                 Yes 
                 — 
                 — 
               
               
                   
                 SMO 
                 Yes 
                 — 
                 — 
               
               
                   
                 SRC 
                 Yes 
                 — 
                 — 
               
               
                   
                 STK11 
                 Yes 
                 — 
                 — 
               
               
                   
                 TERT 
                 Yes 
                 — 
                 — 
               
               
                   
                 TET2 
                 Yes 
                 — 
                 — 
               
               
                   
                 TMPRSS2 
                 — 
                 Yes 
                 — 
               
               
                   
                 TP53 
                 Yes 
                 — 
                 — 
               
               
                   
                 TSC1 
                 Yes 
                 — 
                 — 
               
               
                   
                 TSC2 
                 Yes 
                 — 
                 — 
               
               
                   
                 VEGFA 
                 Yes 
                 — 
                 Yes 
               
               
                   
                 VHL 
                 Yes 
                 — 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Summary of Next Generation Sequencing Statistics 
                   
                 DPF 
                   
               
               
                 for FFPE Tumor and Matched Normal Samples 
                 Orthogonal 
                 Matched 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Bases 
                   
                   
                 Promega 
                 Tumor 
                 DPF 
               
               
                   
                   
                   
                   
                   
                   
                 Mapped to 
                 Average 
                 Average 
                 MSI 
                 and 
                 Tumor 
               
               
                   
                   
                   
                   
                   
                 Bases 
                 Targeted 
                 Total 
                 Distinct 
                 Analysis 
                 Normal 
                 Only 
               
               
                 Case 
                 Sample 
                 Tumor 
                 Tumor 
                 Bases 
                 Mapped to 
                 Regions of 
                 Coverage 
                 Coverage 
                 System 
                 Analysis 
                 Analysis 
               
               
                 ID 
                 Type 
                 Type 
                 Purity 
                 Sequenced 
                 Genome 
                 Interest 
                 (Fold) 
                 (Fold) 
                 Result 
                 Result 
                 Result 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 T1 
                 Tumor 
                 Colorectal 
                 40% 
                 3,166,928,200 
                 2,938,394,500 
                 1,273,077,559 
                 1,258 
                 927 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T2 
                 Tumor 
                 Colorectal 
                 40% 
                 2,956,194,800 
                 2,731,020,900 
                 1,118,791,878 
                 1,102 
                 769 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T3 
                 Tumor 
                 Colorectal 
                 80% 
                 4,620,105,200 
                 4,266,153,700 
                 1,719,208,838 
                 1,705 
                 1,151 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T4 
                 Tumor 
                 Colorectal 
                 60% 
                 3,830,551,400 
                 3,577,875,700 
                 1,587,036,697 
                 1,584 
                 1,047 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T5 
                 Tumor 
                 Colorectal 
                 60% 
                 3,694,440,800 
                 3,417,070,000 
                 1,421,110,311 
                 1,423 
                 1,021 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T6 
                 Tumor 
                 Colorectal 
                 70% 
                 2,781,902,600 
                 2,581,541,800 
                 1,314,484,935 
                 1,308 
                 509 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T7 
                 Tumor 
                 Colorectal 
                 50% 
                 2,946,039,800 
                 2,766,061,900 
                 1,287,341,543 
                 1,287 
                 870 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T8 
                 Tumor 
                 Colorectal 
                 40% 
                 3,418,941,400 
                 3,141,032,200 
                 1,134,791,347 
                 1,128 
                 699 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T9 
                 Tumor 
                 Colorectal 
                 60% 
                 2,554,068,000 
                 2,397,514,900 
                 1,059,806,697 
                 1,065 
                 789 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T10 
                 Tumor 
                 Colorectal 
                 70% 
                 2,490,357,800 
                 2,325,119,000 
                 1,041,133,466 
                 1,045 
                 577 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T11 
                 Tumor 
                 Colorectal 
                 70% 
                 2,802,989,000 
                 2,574,326,700 
                 1,021,889,116 
                 1,028 
                 611 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T12 
                 Tumor 
                 Colorectal 
                 60% 
                 2,732,188,800 
                 2,532,625,600 
                 1,102,256,555 
                 1,106 
                 809 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T13 
                 Tumor 
                 Colorectal 
                 60% 
                 3,374,700,400 
                 3,160,846,000 
                 1,444,383,958 
                 1,452 
                 856 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T14 
                 Tumor 
                 Colorectal 
                 30% 
                 4,449,316,000 
                 4,158,857,900 
                 1,912,478,277 
                 1,908 
                 1,254 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T15 
                 Tumor 
                 Colorectal 
                 40% 
                 3,221,878,600 
                 2,990,670,400 
                 1,289,368,393 
                 1,297 
                 984 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T16 
                 Tumor 
                 Colorectal 
                 30% 
                 2,706,508,600 
                 2,523,131,100 
                 1,106,624,877 
                 1,112 
                 859 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T17 
                 Tumor 
                 Colorectal 
                 25% 
                 3,251,114,200 
                 2,856,483,800 
                 961,918,119 
                 966 
                 736 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T18 
                 Tumor 
                 Colorectal 
                 30% 
                 3,231,913,800 
                 3,009,768,900 
                 1,360,021,648 
                 1,348 
                 991 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T19 
                 Tumor 
                 Colorectal 
                 25% 
                 3,363,038,600 
                 3,113,118,000 
                 1,384,620,931 
                 1,376 
                 997 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T20 
                 Tumor 
                 Colorectal 
                 25% 
                 2,438,680,600 
                 2,276,538,900 
                 1,062,441,883 
                 1,068 
                 664 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T21 
                 Tumor 
                 Colorectal 
                 50% 
                 3,835,047,200 
                 3,616,937,100 
                 1,599,268,481 
                 1,585 
                 1,070 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T22 
                 Tumor 
                 Colorectal 
                 50% 
                 3,571,104,800 
                 3,364,850,000 
                 1,560,304,548 
                 1,549 
                 1,027 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T23 
                 Tumor 
                 Colorectal 
                 50% 
                 3,358,858,000 
                 3,148,152,000 
                 1,543,361,455 
                 1,531 
                 381 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T24 
                 Tumor 
                 Colorectal 
                 30% 
                 3,800,714,600 
                 3,454,271,300 
                 1,451,100,665 
                 1,437 
                 1,021 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T25 
                 Tumor 
                 Colorectal 
                 70% 
                 2,786,623,600 
                 2,616,808,300 
                 1,300,533,976 
                 1,308 
                 839 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T26 
                 Tumor 
                 Colorectal 
                 70% 
                 2,745,441,000 
                 2,560,749,200 
                 1,144,854,802 
                 1,150 
                 831 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T27 
                 Tumor 
                 Colorectal 
                 50% 
                 2,718,178,000 
                 2,492,681,200 
                 1,007,856,362 
                 1,009 
                 772 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T28 
                 Tumor 
                 Colorectal 
                 50% 
                 3,811,856,800 
                 3,469,104,100 
                 1,178,261,647 
                 1,164 
                 881 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T29 
                 Tumor 
                 Colorectal 
                 60% 
                 2,836,284,200 
                 2,639,980,200 
                 1,070,525,027 
                 1,076 
                 803 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T30 
                 Tumor 
                 Colorectal 
                 60% 
                 3,054,498,800 
                 2,864,552,700 
                 1,219,192,787 
                 1,225 
                 918 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T31 
                 Tumor 
                 Colorectal 
                 80% 
                 2,580,688,000 
                 2,400,044,300 
                 974,909,901 
                 980 
                 778 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T32 
                 Tumor 
                 Colorectal 
                 25% 
                 2,794,799,600 
                 2,572,462,100 
                 1,100,684,869 
                 1,106 
                 852 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T33 
                 Tumor 
                 Colorectal 
                 25% 
                 4,145,168,800 
                 3,782,601,800 
                 1,600,830,943 
                 1,581 
                 1,023 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T34 
                 Tumor 
                 Colorectal 
                 30% 
                 2,805,656,200 
                 2,574,761,000 
                 1,165,019,629 
                 1,164 
                 830 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T35 
                 Tumor 
                 Colorectal 
                 25% 
                 3,314,533,600 
                 3,084,210,800 
                 1,337,527,973 
                 1,324 
                 978 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T36 
                 Tumor 
                 Colorectal 
                 40% 
                 3,083,111,800 
                 2,855,861,400 
                 1,234,493,152 
                 1,237 
                 871 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T37 
                 Tumor 
                 Colorectal 
                 50% 
                 2,944,656,600 
                 2,738,021,600 
                 1,185,096,762 
                 1,184 
                 871 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T38 
                 Tumor 
                 Colorectal 
                 50% 
                 2,753,927,200 
                 2,556,267,100 
                 1,111,967,617 
                 1,107 
                 826 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T39 
                 Tumor 
                 Colorectal 
                 50% 
                 2,909,479,000 
                 2,736,312,200 
                 1,240,864,480 
                 1,245 
                 880 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T40 
                 Tumor 
                 Colorectal 
                 50% 
                 2,861,106,600 
                 2,664,312,500 
                 1,238,921,489 
                 1,235 
                 816 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T41 
                 Tumor 
                 Colorectal 
                 50% 
                 3,067,986,400 
                 2,803,426,000 
                 1,187,719,134 
                 1,191 
                 807 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T42 
                 Tumor 
                 Colorectal 
                 50% 
                 2,575,126,400 
                 2,352,723,700 
                 985,780,477 
                 986 
                 729 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T43 
                 Tumor 
                 Colorectal 
                 50% 
                 3,553,245,200 
                 3,291,761,000 
                 1,519,022,764 
                 1,520 
                 569 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T44 
                 Tumor 
                 Colorectal 
                 50% 
                 3,879,433,600 
                 3,543,367,700 
                 1,412,136,767 
                 1,401 
                 951 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T45 
                 Tumor 
                 Colorectal 
                 50% 
                 2,906,836,200 
                 2,640,548,900 
                 1,084,265,479 
                 1,083 
                 708 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T46 
                 Tumor 
                 Colorectal 
                 50% 
                 3,691,316,800 
                 3,373,293,300 
                 1,490,830,422 
                 1,477 
                 633 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T47 
                 Tumor 
                 Colorectal 
                 50% 
                 3,682,074,800 
                 3,431,016,500 
                 1,578,799,330 
                 1,565 
                 1,099 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T48 
                 Tumor 
                 Colorectal 
                 50% 
                 3,219,857,800 
                 2,968,614,400 
                 1,345,719,299 
                 1,346 
                 953 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T49 
                 Tumor 
                 Colorectal 
                 50% 
                 3,682,200,200 
                 3,314,391,200 
                 1,446,490,248 
                 1,448 
                 827 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T50 
                 Tumor 
                 Colorectal 
                 50% 
                 2,698,383,600 
                 2,488,106,900 
                 1,176,178,010 
                 1,178 
                 824 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T51 
                 Tumor 
                 Colorectal 
                 50% 
                 3,319,692,800 
                 2,956,351,200 
                 1,252,235,401 
                 1,242 
                 798 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T52 
                 Tumor 
                 Colorectal 
                 50% 
                 3,360,317,400 
                 3,095,067,800 
                 1,411,042,834 
                 1,410 
                 1,024 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T53 
                 Tumor 
                 Colorectal 
                 25% 
                 2,961,310,600 
                 2,731,451,400 
                 1,142,603,080 
                 1,149 
                 597 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T54 
                 Tumor 
                 Colorectal 
                 25% 
                 2,777,509,400 
                 2,589,331,600 
                 1,149,189,533 
                 1,155 
                 769 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T55 
                 Tumor 
                 Colorectal 
                 30% 
                 2,639,198,800 
                 2,442,326,100 
                 911,827,087 
                 916 
                 617 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T56 
                 Tumor 
                 Colorectal 
                 20% 
                 2,755,033,400 
                 2,531,769,000 
                 1,000,935,424 
                 1,007 
                 681 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 T57 
                 Tumor 
                 Colorectal 
                 20% 
                 2,447,814,600 
                 2,287,722,900 
                 1,015,055,726 
                 1,020 
                 696 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T58 
                 Tumor 
                 Colorectal 
                 20% 
                 3,286,578,200 
                 3,057,086,600 
                 1,307,312,335 
                 1,314 
                 961 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T59 
                 Tumor 
                 Colorectal 
                 25% 
                 2,903,957,600 
                 2,684,504,100 
                 1,230,635,449 
                 1,238 
                 569 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T60 
                 Tumor 
                 Colorectal 
                 30% 
                 3,057,011,000 
                 2,834,612,800 
                 1,299,500,617 
                 1,298 
                 678 
                 MSS 
                 MSS 
                 MSS 
               
               
                   
                   
                 Cancer 
               
               
                 T61 
                 Tumor 
                 Colorectal 
                 30% 
                 3,524,443,800 
                 3,332,286,300 
                 1,616,082,336 
                 1,617 
                 890 
                 MSI-H 
                 MSI-H 
                 MSI-H 
               
               
                   
                   
                 Cancer 
               
               
                 N1 
                 Normal 
                 NA 
                 NA 
                 1,544,823,000 
                 1,450,418,000 
                 585,339,892 
                 586 
                 521 
                 NA 
                 NA 
                 NA 
               
               
                 N2 
                 Normal 
                 NA 
                 NA 
                 1,902,151,400 
                 1,773,538,400 
                 686,222,130 
                 686 
                 604 
                 NA 
                 NA 
                 NA 
               
               
                 N3 
                 Normal 
                 NA 
                 NA 
                 1,845,286,600 
                 1,717,939,600 
                 660,169,442 
                 663 
                 574 
                 NA 
                 NA 
                 NA 
               
               
                 N4 
                 Normal 
                 NA 
                 NA 
                 1,747,777,400 
                 1,604,382,200 
                 578,803,459 
                 581 
                 507 
                 NA 
                 NA 
                 NA 
               
               
                 N5 
                 Normal 
                 NA 
                 NA 
                 1,358,892,200 
                 1,270,257,700 
                 532,386,922 
                 536 
                 466 
                 NA 
                 NA 
                 NA 
               
               
                 N6 
                 Normal 
                 NA 
                 NA 
                 1,403,909,400 
                 1,328,105,100 
                 603,030,506 
                 606 
                 525 
                 NA 
                 NA 
                 NA 
               
               
                 N7 
                 Normal 
                 NA 
                 NA 
                 1,477,544,600 
                 1,386,426,800 
                 600,947,522 
                 604 
                 517 
                 NA 
                 NA 
                 NA 
               
               
                 N8 
                 Normal 
                 NA 
                 NA 
                 1,922,041,400 
                 1,784,316,400 
                 701,537,824 
                 704 
                 613 
                 NA 
                 NA 
                 NA 
               
               
                 N9 
                 Normal 
                 NA 
                 NA 
                 1,389,792,400 
                 1,302,753,500 
                 551,063,430 
                 556 
                 478 
                 NA 
                 NA 
                 NA 
               
               
                 N10 
                 Normal 
                 NA 
                 NA 
                 1,368,669,200 
                 1,282,523,900 
                 527,619,827 
                 533 
                 468 
                 NA 
                 NA 
                 NA 
               
               
                 N11 
                 Normal 
                 NA 
                 NA 
                 1,124,099,400 
                 1,056,703,600 
                 434,115,592 
                 439 
                 390 
                 NA 
                 NA 
                 NA 
               
               
                 N12 
                 Normal 
                 NA 
                 NA 
                 1,297,100,600 
                 1,221,405,100 
                 504,944,038 
                 510 
                 450 
                 NA 
                 NA 
                 NA 
               
               
                 N13 
                 Normal 
                 NA 
                 NA 
                 1,320,243,600 
                 1,222,723,200 
                 477,732,672 
                 482 
                 362 
                 NA 
                 NA 
                 NA 
               
               
                 N14 
                 Normal 
                 NA 
                 NA 
                 2,096,304,800 
                 1,924,550,100 
                 629,989,948 
                 634 
                 563 
                 NA 
                 NA 
                 NA 
               
               
                 N15 
                 Normal 
                 NA 
                 NA 
                 1,857,918,400 
                 1,749,514,000 
                 741,540,523 
                 745 
                 637 
                 NA 
                 NA 
                 NA 
               
               
                 N16 
                 Normal 
                 NA 
                 NA 
                 1,296,158,400 
                 1,225,401,300 
                 539,144,010 
                 545 
                 481 
                 NA 
                 NA 
                 NA 
               
               
                 N17 
                 Normal 
                 NA 
                 NA 
                 1,172,080,200 
                 1,072,384,000 
                 351,008,283 
                 354 
                 321 
                 NA 
                 NA 
                 NA 
               
               
                 N18 
                 Normal 
                 NA 
                 NA 
                 2,197,386,400 
                 2,043,193,200 
                 792,440,480 
                 793 
                 683 
                 NA 
                 NA 
                 NA 
               
               
                 N19 
                 Normal 
                 NA 
                 NA 
                 1,126,031,600 
                 1,057,174,000 
                 429,799,839 
                 435 
                 388 
                 NA 
                 NA 
                 NA 
               
               
                 N20 
                 Normal 
                 NA 
                 NA 
                 2,203,340,000 
                 2,079,985,600 
                 923,434,168 
                 927 
                 780 
                 NA 
                 NA 
                 NA 
               
               
                 N21 
                 Normal 
                 NA 
                 NA 
                 1,999,881,200 
                 1,849,375,700 
                 663,902,271 
                 666 
                 578 
                 NA 
                 NA 
                 NA 
               
               
                 N22 
                 Normal 
                 NA 
                 NA 
                 1,919,525,200 
                 1,795,389,600 
                 721,484,943 
                 723 
                 621 
                 NA 
                 NA 
                 NA 
               
               
                 N23 
                 Normal 
                 NA 
                 NA 
                 1,331,809,200 
                 1,260,705,600 
                 596,587,867 
                 602 
                 517 
                 NA 
                 NA 
                 NA 
               
               
                 N24 
                 Normal 
                 NA 
                 NA 
                 1,903,783,600 
                 1,792,126,800 
                 790,507,492 
                 792 
                 691 
                 NA 
                 NA 
                 NA 
               
               
                 N25 
                 Normal 
                 NA 
                 NA 
                 1,386,738,000 
                 1,304,498,200 
                 573,306,885 
                 579 
                 511 
                 NA 
                 NA 
                 NA 
               
               
                 N26 
                 Normal 
                 NA 
                 NA 
                 1,288,502,200 
                 1,211,465,700 
                 518,972,645 
                 524 
                 465 
                 NA 
                 NA 
                 NA 
               
               
                 N27 
                 Normal 
                 NA 
                 NA 
                 2,531,366,000 
                 2,385,834,400 
                 1,019,543,903 
                 1,027 
                 883 
                 NA 
                 NA 
                 NA 
               
               
                 N28 
                 Normal 
                 NA 
                 NA 
                 1,992,785,000 
                 1,873,292,800 
                 802,670,846 
                 804 
                 687 
                 NA 
                 NA 
                 NA 
               
               
                 N29 
                 Normal 
                 NA 
                 NA 
                 1,455,104,600 
                 1,366,776,900 
                 575,386,950 
                 581 
                 510 
                 NA 
                 NA 
                 NA 
               
               
                 N30 
                 Normal 
                 NA 
                 NA 
                 1,664,936,200 
                 1,561,641,100 
                 646,468,058 
                 652 
                 572 
                 NA 
                 NA 
                 NA 
               
               
                 N31 
                 Normal 
                 NA 
                 NA 
                 1,200,639,800 
                 1,128,641,300 
                 463,140,630 
                 467 
                 416 
                 NA 
                 NA 
                 NA 
               
               
                 N32 
                 Normal 
                 NA 
                 NA 
                 1,167,761,400 
                 1,092,348,500 
                 438,958,934 
                 442 
                 396 
                 NA 
                 NA 
                 NA 
               
               
                 N33 
                 Normal 
                 NA 
                 NA 
                 1,665,825,400 
                 1,549,475,700 
                 609,072,888 
                 612 
                 544 
                 NA 
                 NA 
                 NA 
               
               
                 N34 
                 Normal 
                 NA 
                 NA 
                 1,367,489,200 
                 1,290,133,900 
                 556,470,787 
                 563 
                 499 
                 NA 
                 NA 
                 NA 
               
               
                 N35 
                 Normal 
                 NA 
                 NA 
                 1,503,631,400 
                 1,412,540,500 
                 580,358,931 
                 585 
                 509 
                 NA 
                 NA 
                 NA 
               
               
                 N36 
                 Normal 
                 NA 
                 NA 
                 1,549,988,400 
                 1,458,406,100 
                 628,637,957 
                 634 
                 560 
                 NA 
                 NA 
                 NA 
               
               
                 N37 
                 Normal 
                 NA 
                 NA 
                 1,568,304,000 
                 1,468,430,300 
                 616,176,975 
                 620 
                 519 
                 NA 
                 NA 
                 NA 
               
               
                 N38 
                 Normal 
                 NA 
                 NA 
                 1,701,739,600 
                 1,592,992,100 
                 659,495,963 
                 662 
                 556 
                 NA 
                 NA 
                 NA 
               
               
                 N39 
                 Normal 
                 NA 
                 NA 
                 1,309,687,400 
                 1,234,750,100 
                 546,420,462 
                 551 
                 464 
                 NA 
                 NA 
                 NA 
               
               
                 N40 
                 Normal 
                 NA 
                 NA 
                 1,712,442,800 
                 1,608,493,700 
                 702,640,817 
                 705 
                 588 
                 NA 
                 NA 
                 NA 
               
               
                 N41 
                 Normal 
                 NA 
                 NA 
                 1,191,012,000 
                 1,122,882,200 
                 485,771,595 
                 491 
                 415 
                 NA 
                 NA 
                 NA 
               
               
                 N42 
                 Normal 
                 NA 
                 NA 
                 2,147,527,600 
                 2,012,745,300 
                 865,817,634 
                 870 
                 721 
                 NA 
                 NA 
                 NA 
               
               
                 N43 
                 Normal 
                 NA 
                 NA 
                 2,632,733,800 
                 2,480,587,000 
                 1,109,697,317 
                 1,120 
                 878 
                 NA 
                 NA 
                 NA 
               
               
                 N44 
                 Normal 
                 NA 
                 NA 
                 1,425,864,600 
                 1,323,295,400 
                 519,416,405 
                 524 
                 445 
                 NA 
                 NA 
                 NA 
               
               
                 N45 
                 Normal 
                 NA 
                 NA 
                 1,155,307,800 
                 1,076,440,400 
                 436,138,888 
                 439 
                 377 
                 NA 
                 NA 
                 NA 
               
               
                 N46 
                 Normal 
                 NA 
                 NA 
                 1,221,955,800 
                 1,150,302,700 
                 518,176,709 
                 523 
                 444 
                 NA 
                 NA 
                 NA 
               
               
                 N47 
                 Normal 
                 NA 
                 NA 
                 1,869,437,000 
                 1,735,921,400 
                 728,621,647 
                 732 
                 602 
                 NA 
                 NA 
                 NA 
               
               
                 N48 
                 Normal 
                 NA 
                 NA 
                 1,587,657,000 
                 1,487,987,900 
                 631,794,049 
                 637 
                 537 
                 NA 
                 NA 
                 NA 
               
               
                 N49 
                 Normal 
                 NA 
                 NA 
                 1,781,366,200 
                 1,673,812,500 
                 729,333,555 
                 734 
                 619 
                 NA 
                 NA 
                 NA 
               
               
                 N50 
                 Normal 
                 NA 
                 NA 
                 1,148,277,400 
                 1,076,124,300 
                 342,181,739 
                 346 
                 294 
                 NA 
                 NA 
                 NA 
               
               
                 N51 
                 Normal 
                 NA 
                 NA 
                 1,904,405,800 
                 1,764,410,700 
                 680,778,768 
                 687 
                 576 
                 NA 
                 NA 
                 NA 
               
               
                 N52 
                 Normal 
                 NA 
                 NA 
                 1,640,495,200 
                 1,534,588,100 
                 662,986,346 
                 669 
                 549 
                 NA 
                 NA 
                 NA 
               
               
                 N53 
                 Normal 
                 NA 
                 NA 
                 1,495,963,000 
                 1,388,748,600 
                 604,616,038 
                 612 
                 448 
                 NA 
                 NA 
                 NA 
               
               
                 N54 
                 Normal 
                 NA 
                 NA 
                 1,413,743,800 
                 1,318,632,100 
                 570,759,302 
                 578 
                 432 
                 NA 
                 NA 
                 NA 
               
               
                 N55 
                 Normal 
                 NA 
                 NA 
                 1,254,353,600 
                 1,146,429,600 
                 439,784,552 
                 445 
                 346 
                 NA 
                 NA 
                 NA 
               
               
                 N56 
                 Normal 
                 NA 
                 NA 
                 1,358,890,600 
                 1,256,367,000 
                 500,055,838 
                 506 
                 375 
                 NA 
                 NA 
                 NA 
               
               
                 N57 
                 Normal 
                 NA 
                 NA 
                 1,257,193,000 
                 1,177,020,700 
                 528,523,113 
                 535 
                 410 
                 NA 
                 NA 
                 NA 
               
               
                 N58 
                 Normal 
                 NA 
                 NA 
                 1,315,380,800 
                 1,233,333,300 
                 528,722,180 
                 535 
                 432 
                 NA 
                 NA 
                 NA 
               
               
                 N59 
                 Normal 
                 NA 
                 NA 
                 1,275,383,800 
                 1,172,703,600 
                 504,548,697 
                 511 
                 383 
                 NA 
                 NA 
                 NA 
               
               
                 N60 
                 Normal 
                 NA 
                 NA 
                 2,296,267,800 
                 2,109,785,900 
                 916,969,966 
                 925 
                 645 
                 NA 
                 NA 
                 NA 
               
               
                 N61 
                 Normal 
                 NA 
                 NA 
                 1,433,371,200 
                 1,350,514,800 
                 632,414,511 
                 639 
                 475 
                 NA 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Comparison of Microsatellite Status  
               
               
                 Determined through FFPE Tissue Analyses 
               
            
           
           
               
               
               
            
               
                   
                   
                 Promega MSI  
               
               
                   
                   
                 Analysis System 
               
            
           
           
               
               
               
               
            
               
                   
                 FFPE Tissue Analysis 
                 MSI-H 
                 MSS 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 125 Gene  
                 MSI 
                 31 
                 0 
               
               
                   
                 Targeted Panel 
                 MSS 
                 0 
                 30 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 58 Gene List for Plasma Analyses 
               
            
           
           
               
               
               
            
               
                   
                 Gene (n = 58) 
                 Sequence Region Covered 
               
               
                   
                   
               
               
                   
                 AKT1 
                 Hot Exon Analysis 
               
               
                   
                 ALK 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 AR 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 ATM 
                 Hot Exon Analysis 
               
               
                   
                 BRAF 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 BRCA1 
                 Hot Exon Analysis 
               
               
                   
                 BRCA2 
                 Hot Exon Analysis 
               
               
                   
                 CCND1 
                 Hot Exon Analysis 
               
               
                   
                 CCND2 
                 Hot Exon Analysis 
               
               
                   
                 CCND3 
                 Hot Exon Analysis 
               
               
                   
                 CD274 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 CDK4 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 CDK6 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 CDKN2A 
                 Hot Exon Analysis 
               
               
                   
                 CTNNB1 
                 Hot Exon Analysis 
               
               
                   
                 DNMT3A 
                 Hot Exon Analysis 
               
               
                   
                 EGFR 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 ERBB2 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 ESR1 
                 Hot Exon Analysis 
               
               
                   
                 EZH2 
                 Hot Exon Analysis 
               
               
                   
                 FGFR1 
                 Hot Exon Analysis 
               
               
                   
                 FGFR2 
                 Hot Exon Analysis 
               
               
                   
                 FGFR3 
                 Hot Exon Analysis 
               
               
                   
                 FLT3 
                 Hot Exon Analysis 
               
               
                   
                 GNAS 
                 Hot Exon Analysis 
               
               
                   
                 HRAS 
                 Hot Exon Analysis 
               
               
                   
                 IDH1 
                 Hot Exon Analysis 
               
               
                   
                 IDH2 
                 Hot Exon Analysis 
               
               
                   
                 JAK2 
                 Hot Exon Analysis 
               
               
                   
                 KIT 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 KRAS 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 MAP2K1 
                 Kinase Domain 
               
               
                   
                 MET 
                 Hot Exon Analysis + Adjacent Exon  
               
               
                   
                   
                 14 Introns 
               
               
                   
                 MTOR 
                 Hot Exon Analysis 
               
               
                   
                 MYC 
                 Hot Exon Analysis 
               
               
                   
                 MYCN 
                 Hot Exon Analysis 
               
               
                   
                 NPM1 
                 Hot Exon Analysis 
               
               
                   
                 NRAS 
                 Hot Exon Analysis 
               
               
                   
                 NTRK1 
                 Hot Exon Analysis 
               
               
                   
                 NTRK2 
                 Hot Exon Analysis 
               
               
                   
                 NTRK3 
                 Hot Exon Analysis 
               
               
                   
                 PALB2 
                 Hot Exon Analysis 
               
               
                   
                 PIK3CA 
                 Hot Exon Analysis 
               
               
                   
                 PIK3CB 
                 Hot Exon Analysis 
               
               
                   
                 PIK3R1 
                 Hot Exon Analysis 
               
               
                   
                 POLD1 
                 Exonuclease Domain 
               
               
                   
                 POLE 
                 Exonuclease Domain 
               
               
                   
                 PTCH1 
                 Hot Exon Analysis 
               
               
                   
                 PTEN 
                 Hot Exon Analysis 
               
               
                   
                 RB1 
                 Hot Exon Analysis 
               
               
                   
                 RET 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 RNF43 
                 Hot Exon Analysis 
               
               
                   
                 ROS1 
                 Kinase and Catalytic Domain 
               
               
                   
                 TERT 
                 Hot Exon Analysis + Promoter 
               
               
                   
                 TP53 
                 Full RefSeq/CCDS Coding Sequence 
               
               
                   
                 TSC1 
                 Hot Exon Analysis 
               
               
                   
                 TSC2 
                 Hot Exon Analysis 
               
               
                   
                 VHL 
                 Hot Exon Analysis 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Summary of Next Generation Sequencing Statistics for Healthy Donor 
                   
               
               
                 Samples, Contrived Samples, and Clinical Plasma Samples 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 Clinical 
                   
                   
               
               
                   
                   
                   
                   
                 Bases 
                   
                   
                 Trial 
               
               
                   
                   
                   
                   
                 Mapped to 
                 Average 
                 Average 
                 Tissue 
                 Plasma 
                 Plasma 
               
               
                   
                   
                   
                 Bases 
                 Targeted 
                 Total 
                 Distinct 
                 Enrollment 
                 MSI 
                 TMB 
               
               
                 Case 
                 Sample 
                 Bases 
                 Mapped to 
                 Regions of 
                 Coverage 
                 Coverage 
                 MSI 
                 Analysis 
                 Analysis 
               
               
                 ID 
                 Type 
                 Sequenced 
                 Genome 
                 Interest 
                 (Fold) 
                 (Fold) 
                 Status 
                 Result 
                 Result 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 HD1 
                 Healthy 
                 4,610,602,000 
                 4,591,372,800 
                 2,313,780,895 
                 23,135 
                 1,326 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD2 
                 Healthy 
                 8,891,644,000 
                 8,866,148,100 
                 4,437,521,303 
                 44,386 
                 2,567 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD3 
                 Healthy 
                 5,591,552,000 
                 5,569,655,300 
                 2,532,932,984 
                 25,273 
                 1,413 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD4 
                 Healthy 
                 5,573,545,400 
                 5,543,102,100 
                 2,255,758,545 
                 22,481 
                 1,654 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD5 
                 Healthy 
                 5,207,559,600 
                 5,185,499,400 
                 2,470,481,571 
                 24,671 
                 1,860 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD6 
                 Healthy 
                 6,388,732,200 
                 6,377,549,900 
                 3,432,543,524 
                 34,319 
                 3,762 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD7 
                 Healthy 
                 4,734,677,000 
                 4,712,020,800 
                 2,345,085,749 
                 23,450 
                 1,514 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD8 
                 Healthy 
                 5,302,549,600 
                 5,278,776,000 
                 2,691,437,847 
                 26,923 
                 1,141 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD9 
                 Healthy 
                 7,465,978,000 
                 7,443,127,900 
                 3,937,377,476 
                 39,278 
                 2,632 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD10 
                 Healthy 
                 6,074,039,400 
                 6,052,256,300 
                 3,176,126,200 
                 31,723 
                 1,707 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD11 
                 Healthy 
                 6,213,183,600 
                 6,193,263,500 
                 3,215,135,348 
                 31,924 
                 1,629 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD12 
                 Healthy 
                 7,312,985,200 
                 7,287,955,400 
                 3,361,626,922 
                 33,392 
                 2,219 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD13 
                 Healthy 
                 6,510,483,400 
                 6,494,893,800 
                 2,976,435,079 
                 29,539 
                 4,803 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD14 
                 Healthy 
                 8,627,645,800 
                 8,610,309,100 
                 4,370,055,158 
                 43,159 
                 4,240 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD15 
                 Healthy 
                 8,091,438,800 
                 8,070,832,700 
                 4,064,773,281 
                 40,137 
                 2,755 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD16 
                 Healthy 
                 8,479,048,000 
                 8,460,878,600 
                 4,274,823,876 
                 42,218 
                 2,387 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD17 
                 Healthy 
                 9,956,617,400 
                 9,928,487,500 
                 4,056,620,966 
                 40,013 
                 6,204 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD18 
                 Healthy 
                 8,764,661,800 
                 8,741,658,300 
                 4,365,489,881 
                 43,257 
                 1,659 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD19 
                 Healthy 
                 7,889,783,000 
                 7,869,480,500 
                 3,564,990,160 
                 35,371 
                 3,264 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD20 
                 Healthy 
                 7,633,405,000 
                 7,615,920,000 
                 3,881,573,734 
                 38,491 
                 1,890 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD21 
                 Healthy 
                 7,861,255,200 
                 7,840,463,800 
                 3,898,962,179 
                 38,636 
                 1,558 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD22 
                 Healthy 
                 4,781,596,200 
                 4,700,767,800 
                 2,047,246,059 
                 20,023 
                 914 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD23 
                 Healthy 
                 6,681,047,200 
                 6,637,094,200 
                 3,496,324,530 
                 34,651 
                 1,777 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD24 
                 Healthy 
                 7,177,461,600 
                 7,153,542,900 
                 3,634,434,110 
                 36,048 
                 1,926 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD25 
                 Healthy 
                 7,434,671,400 
                 7,407,050,700 
                 3,898,804,784 
                 38,653 
                 2,302 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD26 
                 Healthy 
                 7,429,101,000 
                 7,401,652,100 
                 3,673,202,567 
                 36,392 
                 2,038 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD27 
                 Healthy 
                 8,503,220,200 
                 8,481,220,900 
                 4,481,836,913 
                 44,189 
                 4,007 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD28 
                 Healthy 
                 7,913,436,400 
                 7,891,591,400 
                 3,999,331,489 
                 39,604 
                 6,476 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD29 
                 Healthy 
                 4,614,537,000 
                 4,554,941,500 
                 2,105,579,210 
                 20,597 
                 697 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD30 
                 Healthy 
                 7,492,256,600 
                 7,465,117,200 
                 3,476,188,532 
                 34,328 
                 2,857 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD31 
                 Healthy 
                 8,328,282,600 
                 8,286,892,200 
                 4,210,419,884 
                 41,650 
                 3,095 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD32 
                 Healthy 
                 7,016,633,400 
                 6,995,998,500 
                 3,531,038,365 
                 34,933 
                 1,236 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD33 
                 Healthy 
                 8,194,639,600 
                 8,172,001,600 
                 4,176,117,225 
                 41,166 
                 2,952 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD34 
                 Healthy 
                 6,007,170,600 
                 5,988,709,000 
                 2,841,711,258 
                 28,277 
                 3,526 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD35 
                 Healthy 
                 7,712,474,800 
                 7,687,926,200 
                 3,538,858,830 
                 34,870 
                 3,962 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD36 
                 Healthy 
                 6,447,382,600 
                 6,427,425,700 
                 3,393,662,901 
                 33,415 
                 2,859 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD37 
                 Healthy 
                 8,134,672,200 
                 8,105,317,200 
                 4,054,212,131 
                 39,967 
                 1,544 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD38 
                 Healthy 
                 5,535,483,200 
                 5,524,427,300 
                 2,816,615,357 
                 28,054 
                 1,983 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD39 
                 Healthy 
                 7,564,324,200 
                 7,546,630,900 
                 3,764,230,400 
                 37,490 
                 4,300 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD40 
                 Healthy 
                 8,036,286,000 
                 8,014,954,300 
                 4,048,484,998 
                 40,197 
                 3,096 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD41 
                 Healthy 
                 7,640,735,400 
                 7,622,537,500 
                 3,929,173,586 
                 39,049 
                 1,971 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD42 
                 Healthy 
                 6,677,376,600 
                 6,656,214,200 
                 2,797,826,119 
                 27,836 
                 1,938 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD43 
                 Healthy 
                 8,409,420,800 
                 8,391,690,200 
                 4,451,721,807 
                 43,978 
                 3,316 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD44 
                 Healthy 
                 8,467,700,000 
                 8,440,226,300 
                 3,675,196,602 
                 36,497 
                 5,083 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD45 
                 Healthy 
                 7,197,353,200 
                 7,170,267,700 
                 3,698,926,248 
                 36,497 
                 1,831 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD46 
                 Healthy 
                 8,318,236,800 
                 8,281,776,900 
                 4,148,545,120 
                 40,815 
                 1,773 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD47 
                 Healthy 
                 9,006,412,400 
                 8,978,934,000 
                 4,760,007,319 
                 46,907 
                 2,891 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD48 
                 Healthy 
                 7,344,659,400 
                 7,321,998,700 
                 3,843,864,822 
                 37,828 
                 1,883 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD49 
                 Healthy 
                 8,288,914,400 
                 8,270,435,900 
                 4,445,831,613 
                 43,940 
                 3,272 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD50 
                 Healthy 
                 8,639,110,000 
                 8,615,224,600 
                 4,502,933,702 
                 44,423 
                 2,244 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD51 
                 Healthy 
                 7,575,511,200 
                 7,555,273,900 
                 3,939,210,286 
                 39,053 
                 3,320 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD52 
                 Healthy 
                 8,427,667,800 
                 8,400,593,500 
                 4,420,970,865 
                 43,296 
                 3,497 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD53 
                 Healthy 
                 8,542,647,000 
                 8,516,087,000 
                 4,385,330,122 
                 42,944 
                 3,771 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD54 
                 Healthy 
                 8,453,014,000 
                 8,428,500,700 
                 4,387,819,781 
                 43,296 
                 2,325 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD55 
                 Healthy 
                 9,298,955,400 
                 9,271,088,500 
                 4,819,496,438 
                 47,546 
                 2,341 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD56 
                 Healthy 
                 8,478,268,200 
                 8,444,312,700 
                 4,094,638,378 
                 40,360 
                 2,050 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD57 
                 Healthy 
                 8,199,783,400 
                 8,170,058,600 
                 3,957,778,287 
                 39,001 
                 2,457 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD58 
                 Healthy 
                 9,346,566,000 
                 9,314,731,900 
                 4,924,333,229 
                 48,509 
                 1,727 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD59 
                 Healthy 
                 8,919,385,800 
                 8,892,662,500 
                 4,390,523,968 
                 43,293 
                 3,513 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD60 
                 Healthy 
                 8,389,446,000 
                 8,370,187,100 
                 4,369,738,805 
                 43,148 
                 1,477 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD61 
                 Healthy 
                 9,905,663,600 
                 9,881,313,200 
                 4,974,727,427 
                 49,048 
                 6,168 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD62 
                 Healthy 
                 9,174,224,000 
                 9,148,992,500 
                 4,535,168,624 
                 44,655 
                 2,138 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD63 
                 Healthy 
                 8,084,463,600 
                 8,057,658,800 
                 3,968,831,440 
                 38,915 
                 1,992 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD64 
                 Healthy 
                 8,983,082,400 
                 8,950,678,700 
                 3,826,555,016 
                 37,464 
                 3,223 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD65 
                 Healthy 
                 7,442,509,800 
                 7,422,697,300 
                 3,854,553,775 
                 38,261 
                 2,341 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD66 
                 Healthy 
                 8,337,674,200 
                 8,316,432,900 
                 4,007,211,501 
                 39,529 
                 2,369 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD67 
                 Healthy 
                 7,154,104,000 
                 7,129,207,200 
                 3,440,461,741 
                 34,040 
                 2,871 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD68 
                 Healthy 
                 8,659,740,200 
                 8,618,184,400 
                 4,184,609,095 
                 41,321 
                 3,957 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD69 
                 Healthy 
                 7,771,232,400 
                 7,753,568,600 
                 3,681,096,130 
                 36,485 
                 3,124 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD70 
                 Healthy 
                 4,405,077,200 
                 4,384,751,000 
                 1,552,688,622 
                 15,295 
                 2,094 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD71 
                 Healthy 
                 5,920,713,000 
                 5,898,405,200 
                 2,633,560,458 
                 26,073 
                 1,061 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD72 
                 Healthy 
                 7,579,429,200 
                 7,554,654,800 
                 2,748,860,562 
                 27,222 
                 2,043 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD73 
                 Healthy 
                 8,631,626,800 
                 8,607,231,300 
                 3,343,564,755 
                 33,121 
                 3,291 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD74 
                 Healthy 
                 6,949,033,000 
                 6,931,273,900 
                 3,127,174,082 
                 30,958 
                 3,031 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD75 
                 Healthy 
                 5,875,099,600 
                 5,864,389,100 
                 2,962,088,150 
                 29,390 
                 4,277 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD76 
                 Healthy 
                 6,626,185,400 
                 6,609,772,800 
                 3,084,072,011 
                 30,517 
                 2,136 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD77 
                 Healthy 
                 11,291,302,400 
                 11,238,394,500 
                 5,110,554,826 
                 49,923 
                 3,453 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD78 
                 Healthy 
                 5,515,433,800 
                 5,483,434,100 
                 1,965,775,908 
                 19,120 
                 834 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD79 
                 Healthy 
                 6,954,396,400 
                 6,931,242,800 
                 3,311,490,206 
                 32,327 
                 3,145 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD80 
                 Healthy 
                 6,152,936,200 
                 6,131,263,700 
                 2,720,849,245 
                 26,546 
                 2,270 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD81 
                 Healthy 
                 8,733,434,600 
                 8,702,900,400 
                 4,271,410,537 
                 41,795 
                 3,890 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD82 
                 Healthy 
                 6,720,050,800 
                 6,692,163,400 
                 2,871,213,549 
                 28,127 
                 2,200 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD83 
                 Healthy 
                 7,729,687,400 
                 7,705,631,600 
                 3,769,457,577 
                 37,031 
                 3,098 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD84 
                 Healthy 
                 8,665,550,000 
                 8,633,041,400 
                 4,135,285,473 
                 40,550 
                 2,542 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD85 
                 Healthy 
                 7,972,481,400 
                 7,950,462,100 
                 3,776,002,282 
                 37,290 
                 3,000 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD86 
                 Healthy 
                 8,250,349,800 
                 8,215,560,400 
                 4,149,026,011 
                 40,906 
                 2,274 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD87 
                 Healthy 
                 7,218,789,600 
                 7,194,779,100 
                 3,266,906,096 
                 32,493 
                 3,137 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD88 
                 Healthy 
                 6,682,720,200 
                 6,654,240,600 
                 3,392,475,194 
                 33,738 
                 2,498 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD89 
                 Healthy 
                 6,871,691,000 
                 6,856,541,800 
                 3,521,282,340 
                 34,894 
                 2,744 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD90 
                 Healthy 
                 8,772,448,000 
                 8,749,280,600 
                 4,178,273,953 
                 41,258 
                 1,494 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD91 
                 Healthy 
                 7,480,832,800 
                 7,457,217,500 
                 3,595,805,873 
                 35,471 
                 2,266 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD92 
                 Healthy 
                 5,975,083,600 
                 5,958,618,100 
                 2,873,989,002 
                 28,615 
                 1,701 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD93 
                 Healthy 
                 5,375,821,400 
                 5,360,555,500 
                 2,567,619,902 
                 25,583 
                 2,090 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD94 
                 Healthy 
                 6,280,445,200 
                 6,260,287,600 
                 3,139,767,399 
                 31,191 
                 2,533 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD95 
                 Healthy 
                 8,135,958,600 
                 8,115,624,700 
                 4,130,225,448 
                 40,731 
                 2,317 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD96 
                 Healthy 
                 7,017,152,200 
                 7,000,775,900 
                 3,355,455,453 
                 33,091 
                 2,169 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD97 
                 Healthy 
                 7,423,045,000 
                 7,401,835,700 
                 3,612,561,174 
                 35,619 
                 1,489 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD98 
                 Healthy 
                 7,575,649,400 
                 7,542,405,300 
                 3,306,732,212 
                 32,637 
                 1,985 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD99 
                 Healthy 
                 8,101,683,000 
                 8,073,383,300 
                 3,916,838,207 
                 38,584 
                 2,250 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD100 
                 Healthy 
                 8,227,634,200 
                 8,195,376,800 
                 3,707,557,242 
                 36,571 
                 1,908 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD101 
                 Healthy 
                 7,409,985,800 
                 7,378,039,500 
                 2,943,037,267 
                 29,002 
                 1,815 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD102 
                 Healthy 
                 7,813,906,600 
                 7,786,978,900 
                 3,615,038,253 
                 35,836 
                 3,086 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD103 
                 Healthy 
                 7,127,926,200 
                 7,092,785,200 
                 2,681,989,940 
                 26,428 
                 1,720 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD104 
                 Healthy 
                 7,010,324,000 
                 6,980,650,200 
                 2,688,769,169 
                 26,520 
                 1,612 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD105 
                 Healthy 
                 7,822,779,200 
                 7,785,449,900 
                 3,212,888,783 
                 31,319 
                 1,715 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD106 
                 Healthy 
                 7,364,897,200 
                 7,339,416,100 
                 3,586,900,299 
                 35,320 
                 2,452 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD107 
                 Healthy 
                 8,493,402,800 
                 8,458,319,500 
                 3,260,053,476 
                 32,076 
                 3,556 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD108 
                 Healthy 
                 9,834,233,000 
                 9,805,201,200 
                 4,706,975,042 
                 46,015 
                 2,353 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD109 
                 Healthy 
                 6,747,679,000 
                 6,733,999,600 
                 3,110,916,970 
                 30,972 
                 1,938 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD110 
                 Healthy 
                 7,069,059,200 
                 7,054,426,600 
                 3,473,558,000 
                 34,223 
                 2,862 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD111 
                 Healthy 
                 10,374,032,800 
                 10,334,762,500 
                 5,226,977,407 
                 51,127 
                 3,737 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD112 
                 Healthy 
                 9,373,668,400 
                 9,330,427,800 
                 3,826,086,189 
                 37,392 
                 2,960 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD113 
                 Healthy 
                 6,510,073,600 
                 6,495,529,700 
                 3,083,434,125 
                 30,646 
                 2,714 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD114 
                 Healthy 
                 5,788,275,000 
                 5,775,686,700 
                 2,790,114,913 
                 27,752 
                 1,891 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD115 
                 Healthy 
                 5,628,781,800 
                 5,608,788,400 
                 2,324,130,331 
                 23,040 
                 1,336 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD116 
                 Healthy 
                 6,622,736,800 
                 6,595,711,900 
                 2,853,684,669 
                 28,019 
                 1,732 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD117 
                 Healthy 
                 8,235,416,200 
                 8,206,562,800 
                 4,066,198,888 
                 40,037 
                 2,147 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD118 
                 Healthy 
                 8,142,539,800 
                 8,113,673,000 
                 3,498,426,122 
                 34,518 
                 2,319 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD119 
                 Healthy 
                 6,567,610,600 
                 6,552,480,100 
                 3,520,404,897 
                 35,102 
                 1,423 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD120 
                 Healthy 
                 8,172,503,000 
                 8,146,438,000 
                 3,738,973,301 
                 36,834 
                 2,391 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD121 
                 Healthy 
                 7,086,855,800 
                 7,066,717,300 
                 3,531,225,022 
                 35,183 
                 2,010 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD122 
                 Healthy 
                 6,632,081,800 
                 6,613,761,500 
                 2,824,890,605 
                 28,087 
                 4,498 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD123 
                 Healthy 
                 8,716,718,200 
                 8,692,336,500 
                 4,249,954,641 
                 42,108 
                 2,897 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD124 
                 Healthy 
                 5,846,065,600 
                 5,827,023,000 
                 2,398,968,620 
                 23,846 
                 1,985 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD125 
                 Healthy 
                 5,987,677,000 
                 5,975,740,400 
                 3,037,726,557 
                 30,200 
                 1,896 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD126 
                 Healthy 
                 6,450,910,600 
                 6,433,946,800 
                 2,901,586,776 
                 28,839 
                 1,732 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD127 
                 Healthy 
                 6,521,277,600 
                 6,505,071,200 
                 3,237,555,270 
                 32,254 
                 1,946 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD128 
                 Healthy 
                 5,183,805,800 
                 5,174,096,900 
                 2,624,001,866 
                 26,114 
                 1,626 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD129 
                 Healthy 
                 6,060,923,800 
                 6,032,344,900 
                 3,294,503,839 
                 33,028 
                 2,041 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD130 
                 Healthy 
                 6,931,215,400 
                 6,664,206,100 
                 2,861,090,396 
                 27,128 
                 2,616 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD131 
                 Healthy 
                 6,881,530,800 
                 6,868,642,100 
                 3,207,081,669 
                 31,800 
                 5,415 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD132 
                 Healthy 
                 8,447,741,200 
                 8,422,297,100 
                 4,171,378,734 
                 41,085 
                 2,511 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD133 
                 Healthy 
                 6,647,519,000 
                 6,618,288,100 
                 3,015,315,572 
                 29,699 
                 974 
                 NA 
                 MSI 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD134 
                 Healthy 
                 9,017,435,200 
                 8,992,906,300 
                 4,677,984,882 
                 46,146 
                 3,595 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD135 
                 Healthy 
                 6,184,647,200 
                 6,158,774,700 
                 2,847,813,354 
                 28,060 
                 1,659 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD136 
                 Healthy 
                 7,819,615,400 
                 7,794,596,100 
                 3,895,863,689 
                 38,457 
                 2,214 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD137 
                 Healthy 
                 9,297,185,200 
                 9,266,997,400 
                 4,986,929,926 
                 49,160 
                 3,316 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD138 
                 Healthy 
                 6,088,725,600 
                 6,071,004,400 
                 2,871,318,994 
                 28,468 
                 1,376 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD139 
                 Healthy 
                 7,078,148,600 
                 7,064,739,600 
                 3,681,471,294 
                 36,397 
                 3,204 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD140 
                 Healthy 
                 7,991,284,600 
                 7,973,783,500 
                 3,998,658,283 
                 39,667 
                 3,312 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD141 
                 Healthy 
                 8,078,032,000 
                 8,054,876,800 
                 4,060,189,135 
                 40,087 
                 1,762 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD142 
                 Healthy 
                 7,768,653,400 
                 7,744,059,500 
                 3,415,300,515 
                 33,884 
                 1,990 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD143 
                 Healthy 
                 6,099,199,600 
                 6,080,885,000 
                 2,775,841,946 
                 27,543 
                 3,106 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD144 
                 Healthy 
                 7,710,555,200 
                 7,694,753,100 
                 3,714,118,122 
                 36,768 
                 3,794 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD145 
                 Healthy 
                 7,799,141,400 
                 7,769,239,200 
                 3,941,900,921 
                 38,944 
                 2,524 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD146 
                 Healthy 
                 6,726,282,600 
                 6,712,047,100 
                 3,413,345,451 
                 33,896 
                 2,071 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD147 
                 Healthy 
                 7,976,941,800 
                 7,958,488,700 
                 3,953,916,025 
                 39,148 
                 3,075 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD148 
                 Healthy 
                 6,773,777,600 
                 6,756,376,400 
                 3,398,770,425 
                 33,685 
                 1,878 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD149 
                 Healthy 
                 7,241,584,800 
                 7,214,148,600 
                 3,197,797,413 
                 31,671 
                 1,957 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD150 
                 Healthy 
                 8,772,019,000 
                 8,744,087,400 
                 4,198,896,205 
                 41,505 
                 2,404 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD151 
                 Healthy 
                 9,597,923,600 
                 9,554,309,500 
                 4,304,212,463 
                 42,013 
                 1,918 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD152 
                 Healthy 
                 9,766,675,200 
                 9,730,121,800 
                 4,232,551,267 
                 41,381 
                 2,131 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD153 
                 Healthy 
                 7,964,424,400 
                 7,902,830,700 
                 3,958,568,012 
                 38,872 
                 2,430 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD154 
                 Healthy 
                 8,703,468,000 
                 8,679,744,500 
                 4,526,278,703 
                 44,722 
                 5,251 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD155 
                 Healthy 
                 7,877,226,800 
                 7,859,052,900 
                 3,985,810,986 
                 39,418 
                 3,773 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD156 
                 Healthy 
                 7,747,095,200 
                 7,729,059,800 
                 3,978,272,416 
                 39,396 
                 3,948 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD157 
                 Healthy 
                 7,689,538,200 
                 7,670,324,700 
                 3,678,385,491 
                 36,403 
                 2,146 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD158 
                 Healthy 
                 6,036,060,400 
                 6,021,050,700 
                 2,871,615,508 
                 28,452 
                 1,793 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD159 
                 Healthy 
                 9,284,331,400 
                 9,261,722,300 
                 4,572,013,112 
                 45,219 
                 4,489 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD160 
                 Healthy 
                 8,039,083,200 
                 8,017,829,900 
                 3,668,403,298 
                 36,433 
                 2,958 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD161 
                 Healthy 
                 6,337,931,000 
                 6,315,879,600 
                 2,863,889,709 
                 28,364 
                 1,286 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD162 
                 Healthy 
                 10,292,765,800 
                 10,260,537,400 
                 5,291,273,703 
                 51,768 
                 3,546 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 HD163 
                 Healthy 
                 9,258,149,800 
                 9,233,715,900 
                 4,485,768,075 
                 43,995 
                 4,143 
                 NA 
                 MSS 
                 TMB- 
               
               
                   
                 Donor 
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CL1 
                 LS180 
                 7,612,745,000 
                 7,589,215,100 
                 3,392,459,288 
                 33,523 
                 2,083 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL2 
                 LS411N 
                 7,678,713,000 
                 7,654,819,800 
                 3,291,800,936 
                 32,532 
                 2,149 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL3 
                 SNU- 
                 6,256,132,400 
                 6,240,909,800 
                 2,807,306,207 
                 27,761 
                 2,420 
                 MSI 
                 MSI 
                 N/A 
               
               
                   
                 C2B 
               
               
                 CL4 
                 RKO 
                 7,066,840,000 
                 7,048,897,500 
                 3,177,373,078 
                 31,421 
                 2,085 
                 MSI 
                 MSS 
                 N/A 
               
               
                 CL5 
                 SNU- 
                 7,669,517,600 
                 7,650,812,200 
                 3,439,833,485 
                 34,079 
                 3,069 
                 MSI 
                 MSI 
                 N/A 
               
               
                   
                 C2A 
               
               
                 CL6 
                 LS180 
                 8,691,502,000 
                 8,658,803,000 
                 3,445,624,572 
                 33,838 
                 2,426 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL7 
                 LS180 
                 8,535,101,200 
                 8,503,984,000 
                 3,893,865,285 
                 38,211 
                 2,595 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL8 
                 LS180 
                 8,083,764,400 
                 8,056,986,800 
                 3,780,724,828 
                 37,152 
                 2,455 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL9 
                 LS180 
                 7,904,478,600 
                 7,881,702,700 
                 3,696,511,241 
                 36,324 
                 2,407 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CLIO 
                 LS180 
                 7,764,828,000 
                 7,737,044,900 
                 3,531,063,394 
                 34,455 
                 2,138 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL11 
                 LS411N 
                 8,245,419,000 
                 8,222,207,200 
                 3,748,315,492 
                 36,967 
                 2,471 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL12 
                 LS411N 
                 6,575,842,800 
                 6,554,550,700 
                 3,030,898,415 
                 29,795 
                 2,430 
                 MSI 
                 MSS 
                 N/A 
               
               
                 CL13 
                 LS411N 
                 8,271,559,000 
                 8,245,273,600 
                 3,762,761,032 
                 36,919 
                 2,295 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL14 
                 LS411N 
                 7,934,153,000 
                 7,905,178,000 
                 3,458,080,463 
                 33,948 
                 2,451 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL15 
                 LS411N 
                 7,108,328,800 
                 7,085,747,100 
                 3,057,622,227 
                 30,157 
                 2,159 
                 MSI 
                 MSI 
                 N/A 
               
               
                 CL16 
                 SNU- 
                 8,456,505,800 
                 8,424,591,600 
                 3,925,699,391 
                 38,462 
                 2,482 
                 MSI 
                 MSI 
                 N/A 
               
               
                   
                 C2B 
               
               
                 CL17 
                 SNU- 
                 7,577,529,000 
                 7,556,499,800 
                 3,380,433,809 
                 33,424 
                 2,261 
                 MSI 
                 MSI 
                 N/A 
               
               
                   
                 C2B 
               
               
                 CL18 
                 SNU- 
                 6,993,859,200 
                 6,976,543,600 
                 3,225,795,617 
                 31,918 
                 2,171 
                 MSI 
                 MSI 
                 N/A 
               
               
                   
                 C2B 
               
               
                 CL19 
                 SNU- 
                 5,882,123,600 
                 5,860,372,800 
                 2,447,923,970 
                 24,221 
                 2,066 
                 MSI 
                 MSI 
                 N/A 
               
               
                   
                 C2B 
               
               
                 CL20 
                 SNU- 
                 7,878,616,400 
                 7,858,594,100 
                 3,506,238,369 
                 34,685 
                 2,058 
                 MSI 
                 MSI 
                 N/A 
               
               
                   
                 C2B 
               
               
                 CS94P1 
                 Clinical 
                 9,263,762,400 
                 9,244,770,800 
                 3,825,312,992 
                 37,868 
                 8,416 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS94P2 
                 Clinical 
                 8,813,423,000 
                 8,792,480,600 
                 3,978,488,566 
                 39,021 
                 8,506 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS94P3 
                 Clinical 
                 8,964,792,200 
                 8,937,833,100 
                 3,676,739,247 
                 36,159 
                 9,963 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS95P1 
                 Clinical 
                 7,636,898,200 
                 7,570,902,200 
                 2,114,468,194 
                 20,804 
                 2,175 
                 MSI 
                 MSS 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS95P2 
                 Clinical 
                 8,719,884,400 
                 8,686,639,300 
                 3,776,909,959 
                 37,371 
                 3,279 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS95P3 
                 Clinical 
                 7,946,606,600 
                 7,923,725,300 
                 3,681,799,356 
                 36,417 
                 3,069 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS96P1 
                 Clinical 
                 8,340,755,000 
                 8,311,711,700 
                 3,710,084,604 
                 36,690 
                 5,686 
                 MSS 
                 MSS 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS96P2 
                 Clinical 
                 6,198,454,200 
                 6,168,225,100 
                 2,565,799,692 
                 24,735 
                 5,781 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS96P3 
                 Clinical 
                 5,912,813,200 
                 5,893,031,300 
                 2,980,746,401 
                 28,844 
                 7,161 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS97P1 
                 Clinical 
                 7,017,701,200 
                 6,998,604,600 
                 3,287,599,575 
                 31,839 
                 8,100 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 High 
               
               
                 CS97P2 
                 Clinical 
                 7,308,707,000 
                 7,285,576,000 
                 3,660,108,635 
                 35,033 
                 8,563 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS97P3 
                 Clinical 
                 5,469,610,600 
                 5,445,096,000 
                 2,704,635,549 
                 25,902 
                 4,807 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS97P4 
                 Clinical 
                 6,624,844,800 
                 6,602,615,600 
                 3,295,259,498 
                 31,752 
                 5,692 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS97P5 
                 Clinical 
                 7,934,394,400 
                 7,916,551,300 
                 3,787,601,674 
                 36,642 
                 7,400 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS97P6 
                 Clinical 
                 5,527,711,600 
                 5,504,889,500 
                 2,466,586,812 
                 23,568 
                 3,104 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS98P1 
                 Clinical 
                 6,412,760,400 
                 6,389,582,100 
                 3,056,873,694 
                 29,246 
                 6,535 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 High 
               
               
                 CS98P2 
                 Clinical 
                 6,672,529,200 
                 6,656,140,900 
                 3,244,885,418 
                 31,287 
                 6,232 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS98P3 
                 Clinical 
                 7,239,611,200 
                 7,210,354,900 
                 3,337,128,346 
                 31,960 
                 4,571 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS98P4 
                 Clinical 
                 4,884,469,600 
                 4,870,410,300 
                 2,398,774,907 
                 23,146 
                 3,886 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS98P5 
                 Clinical 
                 6,684,455,800 
                 6,629,107,600 
                 3,043,981,443 
                 29,758 
                 2,048 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS99P1 
                 Clinical 
                 7,515,207,800 
                 7,492,829,500 
                 3,558,158,353 
                 35,107 
                 3,567 
                 MSS 
                 MSS 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS99P2 
                 Clinical 
                 7,295,781,200 
                 7,266,983,100 
                 3,137,279,510 
                 30,900 
                 2,698 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS99P3 
                 Clinical 
                 8,069,010,600 
                 8,015,635,800 
                 3,679,851,982 
                 36,047 
                 4,776 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS99P4 
                 Clinical 
                 7,293,700,400 
                 7,259,226,900 
                 3,264,415,728 
                 32,140 
                 2,399 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS00P1 
                 Clinical 
                 6,374,270,600 
                 6,354,057,400 
                 3,037,772,591 
                 29,333 
                 4,464 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 High 
               
               
                 CS00P2 
                 Clinical 
                 7,800,574,000 
                 7,772,352,900 
                 3,639,270,013 
                 35,789 
                 7,769 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS00P3 
                 Clinical 
                 8,999,308,800 
                 8,975,347,800 
                 4,386,806,111 
                 43,097 
                 7,970 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS00P4 
                 Clinical 
                 8,380,704,400 
                 8,356,079,400 
                 4,080,252,921 
                 40,115 
                 6,470 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS00P5 
                 Clinical 
                 9,582,201,400 
                 9,546,328,100 
                 3,353,260,614 
                 32,916 
                 6,017 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS00P6 
                 Clinical 
                 10,156,844,200 
                 10,115,758,100 
                 4,837,193,865 
                 47,487 
                 3,644 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS01P1 
                 Clinical 
                 8,967,808,600 
                 8,936,498,200 
                 2,764,042,296 
                 27,189 
                 7,682 
                 MSS 
                 MSS 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS01P2 
                 Clinical 
                 7,912,113,000 
                 7,890,663,500 
                 4,022,370,530 
                 38,855 
                 8,822 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS01P3 
                 Clinical 
                 6,484,354,600 
                 6,455,565,800 
                 3,188,722,876 
                 30,729 
                 7,517 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS02P1 
                 Clinical 
                 4,189,797,200 
                 4,152,277,300 
                 1,904,218,928 
                 18,244 
                 1,223 
                 MSI 
                 MSS 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS02P2 
                 Clinical 
                 10,780,428,800 
                 10,746,068,700 
                 5,446,642,208 
                 52,607 
                 4,747 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS03P1 
                 Clinical 
                 7,050,276,200 
                 7,025,996,100 
                 3,272,196,914 
                 31,585 
                 4,411 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 High 
               
               
                 CS03P2 
                 Clinical 
                 7,863,350,800 
                 7,834,129,600 
                 3,881,768,067 
                 37,547 
                 4,004 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS03P3 
                 Clinical 
                 5,886,551,400 
                 5,855,839,700 
                 2,592,954,330 
                 25,054 
                 1,821 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS03P4 
                 Clinical 
                 5,120,290,200 
                 5,089,916,800 
                 2,462,851,445 
                 23,841 
                 1,370 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS04P1 
                 Clinical 
                 7,761,417,200 
                 7,737,522,100 
                 3,680,626,639 
                 35,734 
                 5,451 
                 MSS 
                 MSS 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS04P2 
                 Clinical 
                 7,248,720,000 
                 7,230,958,400 
                 3,711,116,296 
                 36,019 
                 5,988 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS04P3 
                 Clinical 
                 6,981,545,200 
                 6,963,312,600 
                 3,392,271,571 
                 32,934 
                 6,670 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS04P4 
                 Clinical 
                 8,074,351,200 
                 8,052,943,600 
                 3,852,185,218 
                 37,331 
                 8,083 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS04P5 
                 Clinical 
                 5,970,210,800 
                 5,949,218,300 
                 2,795,371,329 
                 27,047 
                 8,641 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS05P1 
                 Clinical 
                 5,968,039,800 
                 5,946,488,700 
                 2,729,734,351 
                 26,410 
                 5,574 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS05P2 
                 Clinical 
                 6,623,933,000 
                 6,600,539,800 
                 3,226,910,099 
                 31,277 
                 6,687 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS05P3 
                 Clinical 
                 4,496,120,400 
                 4,477,585,500 
                 2,194,338,785 
                 21,245 
                 3,297 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS06P1 
                 Clinical 
                 8,211,159,400 
                 8,186,988,800 
                 4,156,128,019 
                 40,389 
                 8,002 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 High 
               
               
                 CS06P2 
                 Clinical 
                 6,178,650,200 
                 6,150,639,400 
                 2,894,449,740 
                 27,945 
                 6,038 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS06P3 
                 Clinical 
                 6,478,543,800 
                 6,455,283,900 
                 3,163,299,171 
                 30,602 
                 4,990 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS06P4 
                 Clinical 
                 6,548,847,200 
                 6,526,980,300 
                 3,098,470,879 
                 30,056 
                 6,184 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS06P5 
                 Clinical 
                 5,595,054,000 
                 5,528,052,700 
                 2,505,771,405 
                 23,684 
                 3,530 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS07P1 
                 Clinical 
                 10,952,067,600 
                 10,913,583,200 
                 2,792,942,847 
                 27,492 
                 8,179 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 High 
               
               
                 CS07P2 
                 Clinical 
                 10,529,570,200 
                 10,492,696,000 
                 4,154,390,112 
                 40,862 
                 7,298 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS07P3 
                 Clinical 
                 9,716,580,000 
                 9,688,288,100 
                 4,626,575,627 
                 45,358 
                 9,111 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS08P1 
                 Clinical 
                 6,015,494,400 
                 5,979,103,500 
                 2,862,781,196 
                 27,006 
                 7,909 
                 MSI 
                 MSI 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS08P2 
                 Clinical 
                 6,132,402,200 
                 6,089,687,400 
                 3,043,241,991 
                 28,664 
                 9,537 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS08P3 
                 Clinical 
                 6,909,139,800 
                 6,867,393,100 
                 3,360,042,873 
                 31,839 
                 7,216 
                 Timepoint 
                 MSI 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                 CS09P1 
                 Clinical 
                 5,711,066,800 
                 5,673,140,500 
                 2,598,635,892 
                 24,508 
                 4,781 
                 MSI 
                 MSS 
                 TMB- 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Low 
               
               
                 CS09P2 
                 Clinical 
                 6,038,788,400 
                 6,017,962,600 
                 2,867,990,049 
                 27,725 
                 6,346 
                 Timepoint 
                 MSS 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Sample 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Comparison of Microsatellite Status Determined through  
               
               
                 Healthy Donor, Contrived, and Clinical Plasma Analyses 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Healthy Donors and 
                 Expected Status 
               
            
           
           
               
               
               
               
            
               
                   
                 Contrived Sample Analysis 
                 MSI-H 
                 MSS 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 58 Gene Targeted Panel 
                 MSI 
                 18 
                 1 
               
               
                   
                   
                 MSS 
                 2 
                 162 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                   
                 Tissue MSI Status 
               
            
           
           
               
               
               
               
            
               
                   
                 Clinical Plasma Analysis 
                 MSI-H 
                 MSS 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 58 Gene Targeted Panel 
                 MSI 
                 9 
                 0 
               
               
                   
                   
                 MSS 
                 3 
                 4 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Summary of Clinical Information for 16 Patients Evaluated for Response to Immune Checkpoint Blockade 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Tissue 
                   
                   
                   
                 Lynch 
                   
                 Time 
                   
                   
                   
               
               
                   
                 Enrollment 
                   
                   
                 Metastases 
                 Syndrome 
                   
                 to Best 
                 Time 
                 Time 
                 Duration of 
               
               
                 Case 
                 MSI 
                 Tumor 
                 Stage 
                 Detected At 
                 (Medical 
                 Best 
                 Response 
                 to ORR 
                 to CR 
                 Response 
               
               
                 ID 
                 Status 
                 Type 
                 (On Study) 
                 Baseline 
                 History) 
                 Reponse 
                 (Months) 
                 (Months) 
                 (Months) 
                 (Months) 
               
               
                   
               
               
                 CS94 
                 MSI-H 
                 Ampulla of 
                 IV 
                 Y 
                 Lynch 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                   
                 Vater 
                   
                   
                 syndrome 
               
               
                 CS95 
                 MSI-H 
                 Small 
                 IV 
                 Y 
                 Lynch 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                   
                 Intestine 
                   
                   
                 syndrome 
               
               
                 CS96 
                 MSS 
                 Colorectal 
                 IV 
                 Y 
                 No 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                 CS97 
                 MSI-H 
                 Colorectal 
                 IV 
                 Y 
                 Lynch 
                 CR 
                 20.2 
                 6.5 
                 20.2 
                 34.7 
               
               
                   
                   
                   
                   
                   
                 syndrome 
               
               
                 CS98 
                 MSI-H 
                 Colorectal 
                 IV 
                 Y 
                 Lynch 
                 CR 
                 16.9 
                 12.4  
                 16.9 
                 36.2 
               
               
                   
                   
                   
                   
                   
                 syndrome 
               
               
                 CS99 
                 MSS 
                 Colorectal 
                 IV 
                 Y 
                 No 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                 CS00 
                 MSI-H 
                 Ampulla of 
                 IV 
                 Y 
                 Lynch 
                 CR 
                 17.1 
                 2.4 
                 17.0 
                 45.4 
               
               
                   
                   
                 Vater 
                   
                   
                 syndrome 
               
               
                 CS01 
                 MSS 
                 Colorectal 
                 IV 
                 Y 
                 No 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                 CS02 
                 MSI-H 
                 Small 
                 IV 
                 Y 
                 No 
                 PR 
                  2.6 
                 2.6 
                 N/A 
                  4.8 
               
               
                   
                   
                 Intestine 
               
               
                 CS03 
                 MSI-H 
                 Colorectal 
                 IV 
                 Y 
                 Lynch 
                 CR 
                 15.2 
                 2.9 
                 15.2 
                 39.1 
               
               
                   
                   
                   
                   
                   
                 syndrome 
               
               
                 CS04 
                 MSS 
                 Colorectal 
                 IV 
                 Y 
                 No 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                 CS05 
                 MSI-H 
                 Colorectal 
                 IV 
                 Y 
                 Lynch 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 syndrome 
               
               
                 CS06 
                 MSI-H 
                 Colorectal 
                 IV 
                 Y 
                 Lynch 
                 PR 
                  2.6 
                 2.6 
                 N/A 
                 13.6 
               
               
                   
                   
                   
                   
                   
                 syndrome 
               
               
                 CS07 
                 MSI-H 
                 Ampulla of 
                 IV 
                 Y 
                 Lynch 
                 NE 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                   
                 Vater 
                   
                   
                 syndrome 
               
               
                 CS08 
                 MSI-H 
                 Colorectal 
                 IV 
                 Y 
                 No 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                 CS09 
                 MSI-H 
                 Colorectal 
                 IV 
                 Y 
                 Unknown 
                 PD 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 Two 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Consecutive 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Timepoints 
               
               
                   
                   
                   
                   
                   
                   
                   
                 with &gt;80% 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Reduction in 
               
               
                   
                 Progression 
                   
                   
                   
                   
                   
                 Baseline 
               
               
                   
                 Free 
                 Overall 
                 Last 
                   
                   
                 Protein 
                 Protein 
               
               
                 Case 
                 Survival 
                 Survival 
                 Dose 
                 Censored 
                 Censored 
                 Biomarkers 
                 Biomarker 
               
               
                 ID 
                 (Months) 
                 (Months) 
                 (Months) 
                 (Progression) 
                 (Overall) 
                 Evaluated 
                 Levels 
               
               
                   
               
               
                 CS94 
                 3.0 
                 3.6 
                 3.0 
                 1 
                 1 
                 CEA 
                 No 
               
               
                 CS95 
                 2.8 
                 20.7 
                 5.6 
                 1 
                 1 
                 CEA 
                 N/A - Baseline Normal 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Reference Range 
               
               
                 CS96 
                 2.8 
                 5.0 
                 2.4 
                 1 
                 1 
                 CEA 
                 No 
               
               
                 CS97 
                 41.2 
                 48.8 
                 10.6 
                 0 
                 0 
                 CEA 
                 Yes 
               
               
                 CS98 
                 48.6 
                 48.8 
                 23.8 
                 0 
                 0 
                 CEA 
                 Yes 
               
               
                 CS99 
                 2.8 
                 8.8 
                 2.3 
                 1 
                 1 
                 CEA 
                 No 
               
               
                 CS00 
                 47.8 
                 47.8 
                 23.9 
                 0 
                 0 
                 CEA; 
                 CEA: N/A - Baseline Normal 
               
               
                   
                   
                   
                   
                   
                   
                 CA19-9 
                 Reference Range 
               
               
                   
                   
                   
                   
                   
                   
                   
                 CA19-9: Yes 
               
               
                 CS01 
                 1.7 
                 4.9 
                 1.8 
                 1 
                 1 
                 CEA 
                 No 
               
               
                 CS02 
                 5.5 
                 43.9 
                 23.6 
                 1 
                 0 
                 CEA; 
                 CEA: N/A - Baseline Normal 
               
               
                   
                   
                   
                   
                   
                   
                 CA19-9 
                 Reference Range 
               
               
                   
                   
                   
                   
                   
                   
                   
                 CA19-9: N/A - Baseline 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Normal Reference Range 
               
               
                 CS03 
                 42.0 
                 42.0 
                 23.8 
                 0 
                 0 
                 CEA 
                 N/A - Baseline Normal 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Reference Range 
               
               
                 CS04 
                 2.9 
                 7.6 
                 3.8 
                 1 
                 1 
                 CEA 
                 No 
               
               
                 CS05 
                 2.9 
                 15.9 
                 4.8 
                 1 
                 1 
                 CEA 
                 No 
               
               
                 CS06 
                 16.2 
                 40.0 
                 23.7 
                 1 
                 0 
                 CEA 
                 No 
               
               
                 CS07 
                 2.4 
                 2.4 
                 1.4 
                 1 
                 1 
                 CEA 
                 No 
               
               
                 CS08 
                 3.0 
                 7.6 
                 3.4 
                 1 
                 1 
                 CEA 
                 N/A - Baseline Normal 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Reference Range 
               
               
                 CS09 
                 1.4 
                 6.9 
                 4.5 
                 1 
                 1 
                 CEA 
                 No 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Plasma 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Exome 
                 Mutation 
               
               
                   
                 Total 
                 Plasma 
                 Plasma 
                 Plasma 
                 Plasma 
                 Plasma 
                 Plasma 
                 Mutation 
                 Load 
               
               
                   
                 Plasma 
                 Time 
                 Time 
                 Time 
                 Time 
                 Time 
                 Time 
                 Load 
                 (mutations/ 
               
               
                 Case 
                 Samples 
                 Point 1 
                 Point 2 
                 Point 3 
                 Point 4 
                 Point 5 
                 Point 6 
                 (mutations/ 
                 Mbp 
               
               
                 ID 
                 Evaluated 
                 (Months) 
                 (Months) 
                 (Months) 
                 (Months) 
                 (Months) 
                 (Months) 
                 Mbp) 
                 Sequenced) 
               
               
                   
               
               
                 CS94 
                 3 
                 0.1 
                 0.5 
                 3.0 
                 N/A 
                 N/A 
                 N/A 
                 23.1 
                 40.6 
               
               
                 CS95 
                 3 
                 0.0 
                 0.5 
                 6.5 
                 N/A 
                 N/A 
                 N/A 
                 64.0 
                 10.2 
               
               
                 CS96 
                 3 
                 0.0 
                 0.5 
                 2.8 
                 N/A 
                 N/A 
                 N/A 
                 0.1 
                 10.2 
               
               
                 CS97 
                 6 
                 0.0 
                 0.5 
                 2.8 
                 10.6 
                 12.5 
                 22.8 
                 70.2 
                 111.7 
               
               
                 CS98 
                 5 
                 0.0 
                 0.5 
                 4.8 
                 14.0 
                 28.7 
                 N/A 
                 120.5 
                 203.2 
               
               
                 CS99 
                 4 
                 0.0 
                 0.5 
                 0.9 
                  2.8 
                 N/A 
                 N/A 
                 N/A 
                 10.2 
               
               
                 CS00 
                 6 
                 0.0 
                 0.6 
                 2.9 
                  4.4 
                 11.7 
                 25.9 
                 139.2 
                 152.4 
               
               
                 CS01 
                 3 
                 0.0 
                 0.5 
                 0.9 
                 N/A 
                 N/A 
                 N/A 
                 2.3 
                 20.3 
               
               
                 CS02 
                 2 
                 0.0 
                 0.6 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 40.8 
                 0.0 
               
               
                 CS03 
                 4 
                 0.0 
                 0.6 
                 12.8  
                 27.3 
                 N/A 
                 N/A 
                 28.2 
                 50.8 
               
               
                 CS04 
                 5 
                 0.0 
                 0.6 
                 1.3 
                  2.9 
                  4.5 
                 N/A 
                 0.8 
                 20.3 
               
               
                 CS05 
                 3 
                 0.0 
                 0.5 
                 4.8 
                 N/A 
                 N/A 
                 N/A 
                 39.8 
                 10.2 
               
               
                 CS06 
                 5 
                 0.0 
                 0.5 
                 5.1 
                 11.1 
                 23.7 
                 N/A 
                 11.0 
                 91.4 
               
               
                 CS07 
                 3 
                 0.0 
                 0.4 
                 0.9 
                 N/A 
                 N/A 
                 N/A 
                 68.7 
                 233.6 
               
               
                 CS08 
                 3 
                 0.0 
                 0.7 
                 3.0 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 40.6 
               
               
                 CS09 
                 2 
                 0.0 
                 0.7 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 20.3 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Two 
                   
                 Two 
               
               
                   
                   
                 Time 
                   
                   
                 Consecutive 
                   
                 Consecutive 
               
               
                   
                   
                 Difference 
                   
                 Baseline 
                 Timepoints 
                   
                 Timepoints 
               
               
                   
                   
                 Between 
                   
                 Plasma 
                 with &gt;90% 
                   
                 with 0% 
               
               
                   
                   
                 Tissue and 
                 Average 
                 Tumor 
                 Reduction 
                 Baseline 
                 Residual 
               
               
                   
                   
                 Plasma 
                 ctDNA 
                 Mutation 
                 in TMB 
                 Plasma 
                 MSI 
               
               
                   
                 Case 
                 Collection 
                 Level at 
                 Burden 
                 Levels on 
                 MSI 
                 Alleles on 
               
               
                   
                 ID 
                 (Months) 
                 Baseline 
                 Status 
                 Treatment 
                 Status 
                 Treatment 
               
               
                   
                   
               
               
                   
                 CS94 
                 10.6 
                 1.3% 
                 TMB-Low 
                 No 
                 MSI-H 
                 No 
               
               
                   
                 CS95 
                 54.0 
                 0.4% 
                 TMB-Low 
                 No 
                 MSS 
                 N/A 
               
               
                   
                 CS96 
                 25.3 
                 2.3% 
                 TMB-Low 
                 No 
                 MSS 
                 N/A 
               
               
                   
                 CS97 
                 4.9 
                 7.1% 
                 TMB-High 
                 Yes 
                 MSI-H 
                 Yes 
               
               
                   
                 CS98 
                 30.3 
                 5.5% 
                 TMB-High 
                 Yes 
                 MSI-H 
                 Yes 
               
               
                   
                 CS99 
                 N/A 
                 15.0% 
                 TMB-Low 
                 No 
                 MSS 
                 N/A 
               
               
                   
                 CS00 
                 13.7 
                 2.3% 
                 TMB-High 
                 Yes 
                 MSI-H 
                 Yes 
               
               
                   
                 CS01 
                 76.2 
                 0.8% 
                 TMB-Low 
                 No 
                 MSS 
                 N/A 
               
               
                   
                 CS02 
                 0.0 
                 0.0% 
                 TMB-Low 
                 N/A 
                 MSS 
                 N/A 
               
               
                   
                 CS03 
                 18.5 
                 0.5% 
                 TMB-High 
                 Yes 
                 MSI-H 
                 Yes 
               
               
                   
                 CS04 
                 48.8 
                 2.3% 
                 TMB-Low 
                 No 
                 MSS 
                 N/A 
               
               
                   
                 CS05 
                 6.2 
                 0.7% 
                 TMB-Low 
                 No 
                 MSI-H 
                 No 
               
               
                   
                 CS06 
                 16.3 
                 4.6% 
                 TMB-High 
                 No 
                 MSI-H 
                 No 
               
               
                   
                 CS07 
                 16.3 
                 7.9% 
                 TMB-High 
                 No 
                 MSI-H 
                 No 
               
               
                   
                 CS08 
                 N/A 
                 7.2% 
                 TMB-Low 
                 No 
                 MSI-H 
                 No 
               
               
                   
                 CS09 
                 N/A 
                 1.1% 
                 TMB-Low 
                 No 
                 MSS 
                 N/A 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Comparison of Tumor Mutation Burden and Microsatellite Status  
               
               
                 for Patients Evaluated for Response to Immune Checkpoint Blockade 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Tissue 
                 Plasma 
               
            
           
           
               
               
               
               
               
            
               
                 Response 
                 MSI-H 
                 MSS 
                 MSI 
                 MSS 
               
               
                   
               
               
                 Complete Response 
                 4 
                 0 
                 4 
                 0 
               
               
                 Partial Response 
                 2 
                 0 
                 1 
                 1 
               
               
                 Progressive Disease 
                 5 
                 4 
                 3 
                 6 
               
               
                 Not Evaluable 
                 1 
                 0 
                 1 
                 0 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Tissue 
                 Plasma 
               
            
           
           
               
               
               
               
               
            
               
                 Response 
                 TMB-High 
                 TMB-Low 
                 TMB-High 
                 TMB-Low 
               
               
                   
               
               
                 Complete Response 
                 4 
                 0 
                 4 
                 0 
               
               
                 Partial Response 
                 2 
                 0 
                 1 
                 1 
               
               
                 Progressive Disease 
                 3 
                 3 
                 0 
                 9 
               
               
                 Not Evaluable 
                 1 
                 0 
                 1 
                 0 
               
               
                   
               
               
                 TMB-H is classified as ≥10 mutations/Mbp sequenced fortissue and ≥50.8 mutations/Mbp sequenced for plasma 
               
            
           
         
       
     
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     Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, that have been made throughout this disclosure are hereby incorporated herein by reference in their entirety for all purposes. 
     Although the present invention has been described with reference to specific details of certain embodiments thereof in the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.