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US-20260125769-A1 - METHODS AND SYSTEMS FOR ADJUSTING TUMOR MUTATIONAL BURDEN BY TUMOR FRACTION AND COVERAGE

US20260125769A1US 20260125769 A1US20260125769 A1US 20260125769A1US-20260125769-A1

Abstract

Provided herein are methods for detecting tumor mutational burden (TMB) in subjects. In one aspect, the methods include determining observed mutational counts from sequence information obtained from nucleic acids in samples from the subjects and determining a tumor fraction and/or a coverage of the nucleic acids to generate sequencing parameters. The methods also include determining an expected mutational fraction and/or an expected distribution of the expected mutational fraction given the sequencing parameters to generate an expected result, and adjusting the observed mutational count given the expected result to generate an adjusted result, thereby detecting the TMB in the subject. Other aspects are directed to methods of selecting customized therapies for treating cancer in subjects, and methods of treating cancer in subjects. Yet other aspects include related systems and computer readable media used to detect TMB in subjects.

Inventors

  • Katie Julia QUINN
  • Elena HELMAN
  • Darya Chudova

Assignees

  • GUARDANT HEALTH, INC.

Dates

Publication Date
20260507
Application Date
20260105

Claims (20)

  1. 1 . A method for detecting a tumor mutational burden (TMB) in a subject, comprising: (a) extracting cell-free nucleic acid (cfNA) molecules comprising cell-free DNA from a plasma fraction from a subject; (b) extracting nucleic acid molecules from cells from a buffy coat fraction from the subject; (c) determining an observed mutational count from sequence information obtained from sequencing the cfNA molecules from the plasma fraction or amplification products thereof; (d) sequencing the nucleic acid molecules from the buffy coat fraction to detect one or more clonal hematopoiesis of indeterminate potential (CHIP) mutations originating from white blood cells in the buffy coat fraction; (e) correcting the observed mutational count to provide a corrected mutational count, the correction comprising excluding the one or more CHIP mutations; and (f) using the corrected mutational count to generate a plasma TMB score indicative of the TMB in the subject.
  2. 2 . The method of claim 1 , wherein the plasma fraction and the buffy coat fraction are produced from the same blood sample from the subject.
  3. 3 . The method of claim 1 , wherein the cfNA molecules comprise cell-free RNA.
  4. 4 . The method of claim 1 , wherein the sequencing in (c) comprises sequencing exons from a plurality of different genes in the cfNA or amplification products thereof.
  5. 5 . The method of claim 1 , wherein the sequencing in (c) comprises whole exome sequencing.
  6. 6 . The method of claim 1 , wherein the sequencing in (c) comprises whole transcriptome sequencing.
  7. 7 . The method of claim 1 , wherein the sequencing in (c) is performed at a read depth of about 1,000 to about 50,000 reads per locus.
  8. 8 . The method of claim 1 , wherein the observed mutational count comprises single nucleotide variants (SNVs), insertions or deletions (indels), copy number variants (CNVs), fusions, transversions, translocations, frame shifts, duplications, repeat expansions, and/or epigenetic variants.
  9. 9 . The method of claim 1 , wherein the observed mutational count comprises synonymous mutations, nonsynonymous mutations, and/or non-coding mutations.
  10. 10 . The method of claim 1 , wherein the observed mutational count used in (e) to generate the plasma TMB score excludes germline mutations, driver mutations, passenger mutations, and/or resistance mutations.
  11. 11 . The method of claim 1 , wherein the plasma fraction has a tumor fraction below about 1% of all cfNA molecules in the plasma fraction.
  12. 12 . The method of claim 1 , wherein the plasma fraction has a coverage between 10 and 50,000 cfNA molecules at a given nucleotide position in the cfNA molecules in the plasma fraction.
  13. 13 . The method of claim 1 , further comprising assessing single nucleotide variants (SNVs), microsatellite instability (MSI), and fusions in the plasma fraction.
  14. 14 . The method of claim 1 , further comprising determining human leukocyte antigen (HLA) loss, HLA sequencing, immune repertoire sequencing, and/or determining methylation profiles of the cfNA.
  15. 15 . The method of claim 1 , further comprising correcting the observed mutational count for tumor shedding.
  16. 16 . The method of claim 1 , further comprising correcting the observed mutational count for coverage at a given nucleotide position in the cfNA molecules in the plasma fraction.
  17. 17 . The method of claim 1 , further comprising classifying the subject as TMB-high from the TMB score exceeding a threshold TMB score .
  18. 18 . The method of claim 17 , wherein the threshold TMB score is at least 15 mutations per Megabase.
  19. 19 . The method of claim 1 , further comprising enriching the extracted cfNA molecules from the plasma fraction for nucleic acid molecules corresponding at least 500 genes.
  20. 20 . The method of claim 1 , wherein the extracted cfNA molecules from the plasma fraction comprise at least 20,000 different genomic regions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 19/095,831, filed Mar. 31, 2025, which is a continuation of U.S. patent application Ser. No. 17/387,830, filed Jul. 28, 2021, (now U.S. Pat. No. 12,291,751), which is a continuation of U.S. patent application Ser. No. 16/917,582, filed Jun. 30, 2020, (now U.S. Pat. No. 11,118,234), which is a continuation of International Patent Application No. PCT/US2019/042882, filed Jul. 22, 2019, which claims the benefit of, and relies on the filing dates of, U.S. provisional patent application Nos. 62/702,280, filed Jul. 23, 2018, 62/741,770, filed Oct. 5, 2018, 62/782,894, filed Dec. 20, 2018, and 62/824,246, filed Mar. 26, 2019, the entire disclosures of which are incorporated herein by reference. BACKGROUND A tumor is an abnormal growth of cells. DNA is often released into bodily fluids when, for example, normal and/or cancer cells die, as cell-free DNA and/or circulating tumor DNA. A tumor can be benign or malignant. A malignant tumor is often referred to as a cancer. Cancer is a major cause of disease worldwide. Each year, tens of millions of people are diagnosed with cancer around the world, and more than half eventually die from it. In many countries, cancer ranks as the second most common cause of death following cardiovascular diseases. Early detection is associated with improved outcomes for many cancers. Cancer is usually caused by the accumulation of mutations within an individual's normal cells, at least some of which result in improperly regulated cell division. Such mutations commonly include single nucleotide variations (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations, and inversions. The number of mutations within a cancer can be an indicator of the cancer's susceptibility to immunotherapy. Cancers are often detected by biopsies of tumors followed by analysis of cell pathologies, biomarkers or DNA extracted from cells. But more recently it has been proposed that cancers can also be detected from cell-free nucleic acids (e.g., circulating nucleic acid, circulating tumor nucleic acid, exosomes, nucleic acids from apoptotic cells and/or necrotic cells) in body fluids, such as blood or urine (see, e.g., Siravegna et al., Nature Reviews, 14:531-548 (2017)). Such tests have the advantage that they are non-invasive, can be performed without identifying suspected cancer cells to biopsy and sample nucleic acids from all parts of a cancer. However, such tests are complicated by the fact that the amount of nucleic acids released into body fluids is low and variable as is recovery of nucleic acids from such fluids in analyzable form. These sources of variation can obscure predictive value of comparing tumor mutation burden (TMB) among samples. TMB is a measurement of the mutations carried by tumor cells in a tumor genome. TMB is a type of biomarker that can be used to evaluate whether a subject diagnosed with, or suspected of having signs of, a cancer will benefit from a specific type of cancer therapy, such as Immuno-Oncology (I-O) therapy. SUMMARY OF INVENTION This application discloses methods, computer readable media, and systems that are useful in determining and analyzing tumor mutational burden (TMB) in patient samples and which help guide cancer treatment decisions. Traditionally, TMB obtained by counting the rate of mutations is frequently inaccurate when the tumor fraction (e.g., mutant allele fraction (MAF)) and/or coverage is low, because the assay sensitivity for calling mutations is reduced. Accordingly, in certain aspects, observed TMBs are adjusted in view of various measures of assay sensitivity, such as tumor fraction (which sets the MAFs of mutations in a given sample), coverage, and/or the like. In the absence of such adjustment, for example, samples that are TMB-High, but which have low tumor fraction and/or low coverage will typically be erroneously reported as TMB-Low. Such an outcome may have significant consequences downstream for patients when making treatment decisions based on such results. Accordingly, prior to the implementation of the adjustment methods and related aspects disclosed herein, mean mutation count among control samples typically depended on max-MAF and coverage. After implementation of these methods and related aspects, mean mutation count among control or comparator samples is essentially independent of both max-MAF and coverage. Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and descripti