Method for determining if a tumor has a mutation in a microsatellite
Inventors
Assignees
Leland Stanford Junior University
Publication Number
US-12344900-B2
Publication Date
2025-07-01
Expiration Date
2040-12-17
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Abstract
A method for determining if a tumor has a mutation in a microsatellite is provided. In some embodiments, the method may comprise: (a) isolating genomic DNA from a tumor sample and a non-tumor sample from the same patient to produce: i. a sample of tumor DNA and ii. a sample of non-tumor DNA, respectively, (b) without pre-amplifying the tumor or non-tumor DNA, sequencing a plurality of microsatellite loci from both the tumor and non-tumor DNA using OS-seq to provide sequence reads, wherein the sequenced microsatellite loci comprise mononucleotide, dinucleotide, trinucleotide and tetranucleotide microsatellites loci, and (c) comparing the results.
Core Innovation
The invention provides a method for determining if a tumor has a mutation in a microsatellite by isolating genomic DNA from both tumor and non-tumor samples of the same patient, sequencing a plurality of microsatellite loci without pre-amplification using oligonucleotide-selective sequencing (OS-seq), and comparing the allele profiles to identify somatic mutations. The method sequences diverse microsatellite loci comprising mononucleotide, dinucleotide, trinucleotide, and tetranucleotide repeats, thereby expanding beyond traditional assays that focus almost exclusively on mono- and dinucleotide repeats.
The background details the problem that existing methods for detecting microsatellite instability (MSI), such as PCR amplification-based assays and immunohistochemistry, are limited by amplification artifacts and focus primarily on mono- and dinucleotide repeats, often missing complex features like tetranucleotide repeats and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). Additionally, next-generation sequencing methods are prone to PCR amplification stutter errors that compromise sensitivity especially when tumor cellularity is low. Thus, current methods may fail to detect the full spectrum of MSI and related genomic instability, impeding accurate tumor classification and impacting clinical decision-making, particularly in the context of cancer immunotherapy.
To address these challenges, the invention employs a PCR-free targeted sequencing approach that captures and sequences microsatellite loci on a solid support, reducing amplification artifacts and enabling ultra-deep coverage to sensitively detect somatic microsatellite allele shifts. It quantifies MSI intensity across an expanded set of microsatellite classes and simultaneously assesses chromosomal instability (CIN) by analyzing copy number alterations in cancer driver genes. This integrated analysis delineates mutational heterogeneity, subclonal diversity, and mixed genomic instability states in tumors, such as co-occurrence of MSI and CIN, with implications for tumor classification and immunotherapy response prediction.
Claims Coverage
The patent includes fourteen claims with one independent claim that describes a method for determining tumor microsatellite mutations by sequencing without pre-amplification and subsequent analysis, along with various dependent claims refining the method and its applications.
Determining tumor microsatellite mutations by PCR-free sequencing and Euclidean distance analysis
Isolating genomic DNA from tumor and matched non-tumor samples; sequencing a plurality of microsatellite loci comprising mono-, di-, tri- and tetranucleotide repeats without pre-amplification; analyzing allele profiles by determining read count distributions across multiple alleles and calculating Euclidean distance between tumor and non-tumor profiles; comparing this distance to a threshold to designate mutation presence.
Using OS-seq for targeted capture, copying, and sequencing of microsatellite loci
Performing step (b) with oligonucleotide-selective sequencing (OS-seq) that captures microsatellite loci on a solid support using immobilized primers, copies the loci by primer extension, and sequences the extension products without buffer-phase amplification to reduce artifacts.
Quantifying microsatellite instability across multiple loci
Repeating allele profile comparison and mutation designation for all sequenced microsatellite loci to determine the total number of loci with mutations, thereby providing a quantitative measure of microsatellite instability in the tumor.
Identifying patients with elevated microsatellite instability and administering immunotherapy
Using the quantitative MSI measure to identify patients whose tumors exhibit MSI levels above a threshold and administering immunotherapy treatments accordingly.
Immune checkpoint inhibitor-based immunotherapy
Using immune checkpoint inhibitors such as anti-CTLA-4, anti-PD1, anti-PD-L1, anti-TIM-3, anti-VISTA, anti-LAG-3, anti-IDO or anti-KIR antibodies in therapy to patients identified with elevated MSI.
Sequencing at least 100 microsatellite loci for improved detection
Including a sufficiently large number of microsatellite loci (at least 100) from both tumor and non-tumor DNA to enhance the sensitivity and specificity of MSI detection.
Simultaneous sequencing of unique non-microsatellite loci for chromosomal instability assessment
Sequencing a plurality of unique non-microsatellite loci from all chromosomes without pre-amplification; analyzing read count ratios between tumor and normal for these loci; comparing ratios to thresholds to designate chromosomal instability presence based on copy number alterations.
Ratio calculations normalized and log2 transformed
Using normalized log2 ratios of read counts for analyzing copy number changes indicative of chromosomal instability.
Paired-end sequencing read usage
Performing sequencing as paired-end and using read 2 sequences for comparing between tumor and normal; using read 1 sequences to assign microsatellite allele read counts by binning paired-end reads based on read 2 sequences with the first 40 bases as identifiers.
Targeting multiple unique non-microsatellite loci including cancer genes
Including at least 50 genes among the unique non-microsatellite loci to comprehensively assess copy number alterations relevant to cancer.
Analyzing allele profiles considering multiple alleles per microsatellite locus
Determining read count distribution across n alleles (where n ranges from 4 to 20) at each microsatellite locus to improve sensitivity and specificity of mutation detection.
The claims cover a method for PCR-free targeted sequencing of diverse microsatellite loci from tumor and matched normal samples, analysis of allele profiles using Euclidean distance to detect somatic microsatellite mutations, quantitative assessment of MSI, simultaneous characterization of chromosomal instability via copy number analysis, and application of these results to guide immunotherapy treatment decisions.
Stated Advantages
The method provides highly sensitive detection of MSI and other genetic aberrations with minimal amplification error by eliminating PCR amplification steps.
Simultaneous profiling of all classes of microsatellites, including mono-, di-, tri-, and tetranucleotide repeats, enables more comprehensive and quantitative MSI assessment compared to conventional methods.
Accurate measurement of copy number alterations alongside MSI facilitates integrated genomic instability characterization, revealing mixed instability states such as co-occurrence of MSI and chromosomal instability.
High sequencing depth and minimal amplification artifacts allow detection of subclonal tumor populations with low allelic fraction mutations, improving detection sensitivity.
The sequencing approach enables robust, multiplexed targeting of hundreds of microsatellites and cancer genes in a single assay.
The method's quantitative outputs can inform patient stratification for immunotherapy, potentially improving prediction of therapeutic response.
Documented Applications
Detection and quantitation of microsatellite instability (MSI) in tumor samples for colorectal and other cancers.
Classification of colorectal cancers based on MSI and chromosomal instability (CIN) status.
Identification of tumors with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST).
Subclonal analysis and assessment of tumor genetic heterogeneity and clonal architecture.
Guiding therapeutic decisions for immunotherapy, particularly treatment with immune checkpoint inhibitors, based on MSI status.
Simultaneous detection of somatic mutations and copy number alterations in cancer driver genes to characterize tumor genomic profiles.
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