1. Microsatellite Instability Detection by Next Generation Sequencing S.J. Salipante, S.M. Scroggins, H.L. Hampel, E.H. Turner, and C.C. Pritchard September 2014 www.clinchem.org/content/60/9/1192.full © Copyright 2014 by the American Association for Clinical Chemistry

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Microsatellite instability (MSI) is a clinically important molecular phenotype used in the evaluation of tumors  Provides information about prognosis, therapeutic choices, familial cancer risk assessment.  MSI is classically associated with colorectal cancers, but is present in other cancer types  Defined by the appearance of new microsatellite alleles that are larger or smaller than those observed in matched germline tissue  Caused by dysregulation of mismatch repair (MMR) system  Mutation in MLH1, MSH2, MSH6, EPCAM, or PMS2 are common in hereditary cancer predisposition conditions  Promoter methylation of MLH1 is common in sporadic disease

3. © Copyright 2009 by the American Association for Clinical Chemistry Current detection methods  PCR detection of instability at informative microsatellite markers (MSI-PCR) is the chief DNA-based method in current clinical use  Small number (typically 5) of mononucleotide microsatellite markers using fluorescently-labeled primers, and products are resolved by capillary electrophoresis  MSI is diagnosed if at least 2 of 5 markers are unstable  Drawbacks:  Requires matched normal tissue  Requires subjective manual interpretation

4. © Copyright 2009 by the American Association for Clinical Chemistry Question  Can next-generation sequencing (NGS) data be used as a clinical diagnostic for MSI?  NGS is becoming increasingly used for molecular oncology  Due to prevalence of epigenetic causes of MSI, one cannot simply look for mutations causing the phenotype

5. © Copyright 2009 by the American Association for Clinical Chemistry Method (mSINGS) 1.Examine mononucleotide microsatellites incidentally captured during pull-down enrichment of DNA  Typically intronic or intergenic DNA 2.Establish “baseline” statistics for the number of fragments observed at each locus for a population of microsatellite stable samples  Even stable specimens will have multiple fragment lengths detectable due to “stutter” artifact – template slippage during PCR  Calculate average and SD for number fragments at each locus 3.Sequence tumor DNA by targeted gene capture 4.Evaluate each locus compared to the baseline  Count number of different fragments  If significantly greater ( > baseline + 3 SD) than baseline number of fragments, score locus as unstable 5.Interpret fraction of unstable loci to infer MSI status

6. © Copyright 2009 by the American Association for Clinical Chemistry Figure 1. Detection of microsatellite instability by MSI-PCR and next-generation DNA sequencing. Representative capillary electrophoresis results from MSI-PCR (left) and “virtual electropherograms” of next-generation DNA sequencing data (right). The length (x-axis) and relative abundance (Y-axis) of variant repeats are plotted. Columns represent individual loci and show paired data for MSI stable (top) and unstable (bottom) specimens. Different microsatellite loci are shown for MSI-PCR and next-generation DNA sequencing, and are not directly comparable. MSI-PCR compared to mSINGS

7. © Copyright 2009 by the American Association for Clinical Chemistry Test Data  Exome capture (44 Mbs, 30,000 genes)  The Cancer Genome Atlas (TCGA) colorectal cancers  2,957 loci included  ColoSeq (1.4 Mbs, 50 whole genes with some flanking sequence)  Colorectal, endometrial, ovarian, breast, and prostate tumors  146 loci included  UW-OncoPlex (0.85 Mbs, 194 gene exons and select introns)  Colon, endometrial, lung, breast, ovarian, melanoma, and other tumors  15 loci included Baseline established for each assay using data from peripheral blood DNA (MSI negative, constitutionally)

8. © Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Fraction of unstable microsatellite loci identified by next-generation sequencing predicts MSI status. The fraction of unstable microsatellite loci (top) and cumulative distribution of that fraction (bottom) are shown for A. Exome capture, B. ColoSeq, and C. UW-OncoPlex targeted gene capture designs. Results are stratified by conventional MSI-PCR status, with the average fraction of unstable loci indicated by a solid horizontal line. The threshold used for interpreting MSI status is indicated by a dashed line. Results

9. © Copyright 2009 by the American Association for Clinical Chemistry Results Table 1. Diagnostic sensitivity and specificity of NGS panels for MSI detection using mSINGS. Results are shown for each of the three assays considered in this study, as well as in aggregate. Only one false positive and one false negative result were encountered (both in the ColoSeq capture design).

10. © Copyright 2009 by the American Association for Clinical Chemistry Figure 3. Quantitative correlation of MSI-PCR and next-generation sequencing results. The fraction of unstable microsatellite markers as determined by next-generation sequencing is plotted with relation to the fraction of unstable markers. Results

11. © Copyright 2009 by the American Association for Clinical Chemistry Conclusions and possible advantages  mSINGS can be used to detect the molecular phenotype of MSI without special gene capture design considerations  mSINGS provides more comprehensive tumor characterization from NGS data  Compared to MSI-PCR  Provides quantitative, digital information for identifying unstable loci  Statistical, rather than qualitative cutoffs for instability  Allows far more microsatellites to be examined than conventional MSI testing  May eliminate the need for matched normal material

12. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Download the free Clinical Chemistry app on iTunes for additional content! Follow us