Personalized ctDNA disease monitoring via representative DNA sequencing
Inventors
Alexander, Nelson R. • Litchfield, Kevin Richard • Stanislaw, Stacey • Turajlic, Samra
Assignees
Royal Marsden NHS Foundation Trust • The Francis Crick Institute Ltd • Ventana Medical Systems Inc
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Publication Number
US-12692546-B2
Publication Date
2026-07-28
Expiration Date
Abstract
Disclosed herein is a method of deriving a plurality of genetic variants from a homogenized input sample. Also disclosed herein are methods of identifying a plurality of genetic variants in a sample comprising: homogenizing one or more input samples to provide a homogenized sample; preparing genomic material isolated from the homogenized input sample for sequencing; and identifying the plurality of genetic variants within sequencing data derived after sequencing the prepared genomic material.
Core Innovation
The invention provides a method for tumor subpopulation mutation profiling using Representative Sequencing (Rep-Seq) in which a fixed tumor sample from a patient diagnosed with cancer is homogenized to provide a homogenized sample. The fixed tumor sample is residual tumor tissue, and any aliquot removed from the homogenized sample comprises a plurality of genetic mutations at a proportion at which they exist within the residual tumor tissue. Genomic material derived from the homogenized sample is sequenced to obtain a sequencing data set for the homogenized sample.
The method identifies the plurality of genetic mutations within the obtained sequencing data set, including one or more common genetic mutations present in all cells in the homogenized sample. It also identifies one or more specific genetic mutations present in one or more subpopulations of cells within the homogenized sample but not in all cells in the homogenized sample. Based on these identified common and specific mutations, the method prepares a ctDNA monitoring panel specific for the patient diagnosed with cancer.
Using the prepared ctDNA monitoring panel, the method repeatedly determines in one or more ctDNA samples obtained from the patient which of the identified specific genetic mutations present in the one or more subpopulations has recurred or is resistant to therapy. The workflow supports distinguishing clonal and subclonal genetic variants derived from the homogenized residual tumor tissue and translating them into a patient-specific ctDNA monitoring panel for repeated monitoring.
Claims Coverage
The claim coverage includes six inventive features. It covers Rep-Seq based tumor mutation profiling, clonal versus subpopulation-specific mutation identification, patient-specific ctDNA monitoring panel preparation, repeated ctDNA-based determination of recurrence or therapy resistance, computation of clonal structure, and determination of a therapeutic strategy based on subpopulation-specific mutations.
Homogenize residual tumor tissue and sequence derived genomic material
Homogenizing a fixed tumor sample from a patient diagnosed with cancer to provide a homogenized sample, wherein the fixed tumor sample is residual tumor tissue, and sequencing genomic material derived from the homogenized sample to obtain a sequencing data set for the homogenized sample.
Identify clonal and subpopulation-specific genetic mutations
Identifying the plurality of genetic mutations within the obtained sequencing data set, identifying one or more common genetic mutations that are present in all cells in the homogenized sample, and identifying one or more specific genetic mutations present in one or more subpopulations of cells within the homogenized sample but not in all cells in the homogenized sample.
Prepare a patient-specific ctDNA monitoring panel using clonal and subclonal mutations
Preparing a ctDNA monitoring panel specific for the patient diagnosed with cancer and based on the identified one or more common genetic mutations present in all cells in the homogenized sample and the identified one or more specific genetic mutations present in the one or more subpopulations of cells within the homogenized sample but not in all cells in the homogenized sample.
Repeated ctDNA determination of recurrence or therapy resistance using the panel
Using the prepared ctDNA monitoring panel, repeatedly determining in one or more ctDNA samples obtained from the patient diagnosed with cancer which of the identified one or more specific genetic mutations present in the one or more subpopulations of cells has recurred or is resistant to therapy.
Compute clonal structure from mutation estimates and mutational clusters
Computing a clonal structure from identified genetic mutations by estimating cancer cell fractions for each mutation and grouping those estimates into mutational clusters.
Determine a therapeutic strategy based on subpopulation-specific mutations
Determining a therapeutic strategy based on identifying specific genetic mutations that are present in one or more subpopulations of cells within a homogenized sample but not in all cells of the sample.
The claim coverage is directed to homogenizing residual tumor tissue, sequencing it, distinguishing common clonal mutations from subpopulation-specific mutations, constructing a patient-specific ctDNA monitoring panel from both mutation classes, and repeatedly using that panel to determine which subpopulation-specific mutations recur or are resistant to therapy. Dependent claim coverage also includes computing clonal structure and determining a therapeutic strategy based on subpopulation-specific mutations.
Stated Advantages
Improved mutation detection, improved clonal/subclonal classification accuracy, and higher reproducibility versus single/multi-region approaches.
Representative sampling advantages indicated by audit and TCGA analysis that current biopsies sample only a small fraction of tumor mass, while Rep-Seq pools residual tumor material.
Documented Applications
Personalized ctDNA monitoring for therapy response and determining whether specific subpopulation genetic mutations have recurred or are resistant to therapy.
Tumor mutation profiling to support identifying clonal versus subclonal genetic variants and monitoring cancer presence/relapse and minimal residual disease detection, including post-surgical primary/metastatic contexts.
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