Digital amplification assays for genetic instability

Inventors

Maar, Dianna

Assignees

Bio Rad Laboratories Inc

Abstract

Methods and compositions for detecting genetic instability using digital amplification assays. The methods may be performed in a set of isolated volumes and generally may involve competitive hybridization of a competitor and a probe/primer with a normal allele and one or more mutant alleles of a microsatellite locus. The competitor may be configured to compete similarly with, or to outcompete, the primer/probe for hybridization with the normal allele. The primer/probe may be configured to outcompete the competitor for hybridization with various mutant alleles of the locus that alter the length of the repetitive sequence by different amounts. Isolated volumes in which the primer/probe outcompetes the competitor may be enumerated, and represent one or more of the mutant alleles. The methods may enable diagnosing microsatellite instability and treating a subject based on the diagnosis.

Core Innovation

The disclosed invention provides a method of detecting one or more mutant alleles that alter a repetitive sequence present in a normal allele of a microsatellite locus by partitioning reagents into isolated volumes. The method forms a set of isolated volumes, each containing a primer pair configured to amplify the normal allele and each mutant allele, and a first probe and a second probe, each having a label. Amplicon is generated using the primer pair and amplification data are collected from the label of each probe.

The core technical concept is competitive hybridization between two labeled probes in the isolated volumes to differentiate normal allele amplicon from mutant allele amplicon. The first probe is configured to compete at a similar efficiency with, or outcompete, the second probe for hybridization with amplicon corresponding to the normal allele, while the second probe is configured to outcompete the first probe for hybridization with amplicon corresponding to each mutant allele. The first probe is configured such that a strand hybridizes with the entire repetitive sequence of the normal allele, whereas a strand of the second probe hybridizes with only a portion of the repetitive sequence of the normal allele.

The approach supports MSI-diagnosis based on mutant allele detection in a partitioned, digital amplification format. Amplification-positive volumes are enumerated using probe-label amplification patterns, and a mutant allele level can be obtained from amplification data and compared to a predetermined threshold. In downstream use, an immunotherapeutic agent may be administered optionally based on the instability result, including a checkpoint inhibitor such as pembrolizumab.

Claims Coverage

Independent claim clm-00001 covers a digital PCR approach in isolated volumes using primer-pair amplification combined with two labeled probes whose competitive hybridization differentiates amplicons for a normal allele versus mutant alleles. The independent claim is centered on a main inventive arrangement of isolated volumes, two labeled probes with defined hybridization coverage over the repetitive sequence of the normal allele, and probe competition/outcompetition behavior that reverses between normal-allele amplicon and mutant-allele amplicon; the dependent claims further add decision logic and constraints on mutant-allele and locus configurations.

Across the claim set, the main inventive concept is differentiation of normal versus mutant microsatellite amplicons by collecting label-specific amplification data from isolated volumes in which two probes exhibit defined competitive hybridization behavior tied to whether the repetitive sequence portion is fully or partially hybridized. Dependent claims further constrain how results are classified using probe-label amplification patterns, add threshold-based decision-making using a mutant allele level, restrict mutant-allele characteristics and/or selected microsatellite loci, and optionally include administering immunotherapeutic agents such as a checkpoint inhibitor including pembrolizumab based on the instability result.

Stated Advantages

Documented Applications