Identification and use of circulating nucleic acid tumor markers

Inventors

Diehn, Maximilian • Alizadeh, Arash Ash • Newman, Aaron M. • Bratman, Scott V

Assignees

Leland Stanford Junior University

Abstract

Methods for creating a selector of mutated genomic regions and for using the selector set to analyze genetic alterations in a cell-free nucleic acid sample are provided. The methods can be used to measure tumor-derived nucleic acids in a blood sample from a subject and thus to monitor the progression of disease in the subject. The methods can also be used for cancer screening, cancer diagnosis, cancer prognosis, and cancer therapy designation.

Core Innovation

The invention provides methods for detecting somatic mutations in a sample of cell-free DNA (cfDNA) from a subject suffering from cancer by using circulating tumor DNA (ctDNA) comprising cancer-associated somatic mutations. The approach includes obtaining a first cfDNA sample, performing hybrid capture with a selector set comprising a plurality of oligonucleotides to enrich for cfDNA corresponding to genomic regions known to contain tumor-specific somatic mutations, and producing a hybrid captured first cfDNA sample.

The hybrid captured cfDNA is sequenced to generate sequencing information, and the sequencing information is analyzed to detect ctDNA in the sample. For selector set production, the invention includes performing a sequencing reaction on a tumor sample and a non-tumor sample from the subject, comparing the sequencing information to identify one or more somatic mutations specific to the sequencing information of the tumor sample, and producing the selector set corresponding to one or more genomic regions comprising the one or more mutations specific to the sequencing information.

The methods are described as capable of detecting a percentage of ctDNA that is less than or equal to 2% of total cfDNA. The document applies to diffuse large B-cell lymphoma (DLBCL) and lymphoma, and describes downstream detection of somatic mutations such as SNVs, indels, rearrangements/breakpoints, and CNVs using background-adjusted and Monte Carlo/digital quantitation approaches.

Claims Coverage

The document includes two independent claims. Both claims cover detecting somatic mutations in cfDNA by hybrid capture using a selector set, followed by sequencing and sequence analysis to detect ctDNA at low abundance (≤2% of total cfDNA), and both define selector-set generation by tumor-versus-non-tumor sequencing comparison; the independent claims differ mainly in the specified cancer type.

Across the two independent claims, the core claim coverage centers on ctDNA detection from cfDNA using hybrid capture with a selector set, followed by sequencing and analysis, with an explicit low-fraction detection capability (≤2%). Selector sets are defined as produced by comparing tumor and non-tumor sequencing from the subject to identify somatic mutations and then selecting genomic regions corresponding to those mutations, with independent claim scope constrained to DLBCL in one claim and lymphoma in the other.

Stated Advantages

Documented Applications