||1R33CA222344-01 Interpret this number
||University Of Washington
||Advanced Development and Validation of Genome-Scale Molecular Diagnostics for Microsatelliteinstability Using Targeted Molecular Counting Methods
Microsatellite Instability (MSI), the spontaneous loss or gain of nucleotides from repetitive DNA tracts, is a
molecular phenotype in oncology that is associated with genomic hypermutability and mutations in DNA repair
enzymes. Although originally described in colorectal tumors, MSI has lately been regarded with newfound
importance for cancers in general, owing to the recent discoveries that MSI-affected tumors are susceptible to
immune checkpoint inhibitor immunotherapies, and that MSI is a generalized tumor phenotype found across
virtually all cancer types. Despite its emerging importance as a powerful pan-cancer therapeutic marker,
existing diagnostics for MSI are limited in throughput, sensitivity, specificity, quantitation, prognostic
capabilities, and generality across tumor types, among other important features. There is consequently a need
for clinical diagnostics with improved performance and enhanced diagnostic capabilities. We have recently
developed a new experimental paradigm (smMIP capture) that can overcome these limitations. In our
approach, each copy of a target sequence that is present in a sample is molecularly tagged during the first
cycle of a multiplex capture reaction with a unique random sequence. After amplification, target amplicons and
their corresponding molecular tags are subjected to massively parallel sequencing. During analysis, the
molecular tags are used to associate sequence reads sharing a common origin. Through oversampling, reads
bearing the same molecular tag error-correct one another to yield an independent haploid consensus for each
progenitor molecule, effectively bypassing length-altering slippage mutations which occur at repetitive
microsatellite tracts (?stutter? artifact) and enabling sensitive quantitation of even low prevalence microsatellite
mutations. Among other benefits, the approach is scalable to large numbers of genomic targets and is
sensitive to at least 1 variant in a background of 10,000 unmutated templates. Here we propose the advanced
development and validation of smMIP capture as a clinical diagnostic for MSI. In our first Aim, we will develop
a multiplexed panel to broadly target ~1,000 highly informative microsatellite loci, as well as protocols and
analytic methods for its use and interpretation. In our second Aim, we will define clinical performance
characteristics of the assay, and will validate it against existing, clinical standard-of-care MSI diagnostics. In
our third Aim, we will apply the smMIP panel prospectively in a clinical trial cohort, and assess its potential
impact on clinical care and correlation with patient outcomes compared to standard testing. This work will
provide information and deliverables with direct, transformative benefit cancer to patients, by expanding the
scope of MSI testing across tumor types, and by providing better stratification for immunotherapy treatment,
prognostic estimation, and quantitative data on mutational burden that is biologically-meaningful for
understanding patient outcomes and identifying MSI subtypes.
Accurate Pan-Cancer Molecular Diagnosis of Microsatellite Instability by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing.
, Smith N.
, Penewit K.
, Hempelmann J.
, Konnick E.Q.
, Hause R.J.
, Pritchard C.C.
, Salipante S.J.
Clinical Chemistry, 2018 Jun; 64(6), p. 950-958.