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JP-7855659-B2 - Cancer detection and monitoring methods using personalized detection of circulating tumor DNA

JP7855659B2JP 7855659 B2JP7855659 B2JP 7855659B2JP-7855659-B2

Inventors

  • ジマーマン，バーンハード
  • サラリ，ラーヘル
  • スウィンナートン，ライアン
  • ウー，シン－タ
  • セチ，ヒマンシュ

Assignees

  • ナテラ， インコーポレイテッド

Dates

Publication Date
20260508
Application Date
20241015
Priority Date
20180414

Claims (16)

  1. A method for monitoring and detecting minimal residual disease in colorectal cancer, Whole exome sequencing is performed on tumor samples from patients diagnosed with colorectal cancer to identify somatic mutations associated with the colorectal cancer, and a set of at least eight or sixteen patient-specific single nucleotide variant (SNV) loci is selected based on the somatic mutations identified in the tumor samples. A set of amplicons is created by performing a multiple amplification reaction on nucleic acids isolated from the patient's blood sample or a fraction thereof, wherein each amplicon in the set of amplicons extends to the patient-specific SNV locus of the patient-specific set of SNV loci associated with colorectal cancer. High-throughput sequencing is performed to determine the sequence of at least one segment of each amplicon in the set of amplicons spread across patient-specific SNV loci, wherein the high-throughput sequencing is performed with a read depth of at least 100,000 per locus, and the detection of two or more patient-specific SNVs exceeding a confidence threshold of 0.97 from the set of at least eight or sixteen patient-specific SNV loci is an indicator of the presence of circulating tumor DNA (ctDNA) in the blood sample and minimal residual disease in the patient. Methods that include...
  2. A method for monitoring and detecting minimal residual disease in colorectal cancer, Creating a set of amplicons by performing multiple amplification reactions on nucleic acids isolated from blood samples or fractions thereof from patients diagnosed with colorectal cancer, wherein each amplicon in the set of amplicons extends to at least one SNV locus from a set of at least eight or sixteen patient-specific single nucleotide variant (SNV) loci associated with colorectal cancer, selected based on somatic mutations associated with colorectal cancer identified in the patient's tumor sample. High-throughput sequencing is performed to determine the sequence of at least one segment of each amplicon in the set of amplicons spread across patient-specific SNV loci, wherein the high-throughput sequencing is performed with a read depth of at least 100,000 per locus, and the detection of two or more patient-specific SNVs exceeding a confidence threshold of 0.97 from the set of at least eight or sixteen patient-specific SNV loci is an indicator of the presence of circulating tumor DNA (ctDNA) in the blood sample and minimal residual disease in the patient. Methods that include...
  3. The method according to claim 1 or 2, wherein the method detects two or more patient-specific SNVs in a patient with early recurrence or metastasis of cancer at least 10.2 months prior to clinical recurrence or metastasis of cancer detectable by CT imaging.
  4. The method according to any one of claims 1 to 3, wherein the method detects two or more patient-specific SNVs in at least 85% of patients with early recurrence or metastasis of cancer.
  5. The method according to any one of claims 1 to 4, wherein the method does not detect two or more patient-specific SNVs in at least 95% of patients without early recurrence or metastasis of cancer.
  6. A method for monitoring and detecting minimal residual disease in colorectal cancer, Creating a set of amplicons by performing multiple amplification reactions on nucleic acids isolated from blood samples or fractions thereof from patients treated for colorectal cancer, wherein each amplicon in the set of amplicons extends to at least one SNV locus from a set of at least eight or sixteen patient-specific single nucleotide variant (SNV) loci associated with colorectal cancer, selected based on somatic mutations associated with colorectal cancer identified in the patient's tumor sample. High-throughput sequencing is performed to determine the sequence of at least one segment of each amplicon in the set of amplicons spread across patient-specific SNV loci, wherein the high-throughput sequencing is performed with a read depth of at least 100,000 per locus, and the detection of two or more patient-specific SNVs exceeding a confidence threshold of 0.97 from the set of at least eight or sixteen patient-specific SNV loci is an indicator of the presence of circulating tumor DNA (ctDNA) in the blood sample and minimal residual disease in the patient. Methods that include...
  7. The method according to claim 6, wherein the method has at least 95% specificity in detecting minimal residual disease in the patient.
  8. The method according to claim 6 or 7, wherein at least five patient-specific SNVs are detected from the blood sample.
  9. The method according to any one of claims 6 to 8, wherein the treatment is neoadjuvant therapy or adjuvant therapy.
  10. The method according to any one of claims 6 to 9, wherein the patient is receiving chemotherapy, radiotherapy, or adjuvant therapy before the blood sample is taken from the patient.
  11. The method according to any one of claims 6 to 10, further comprising determining the variant allele frequency for each of the patient-specific SNVs detected from the blood sample.
  12. The method according to claim 11, further comprising selecting a treatment that targets the clonal SNV, wherein a variant allele frequency exceeding 1% or exceeding 5% is an indicator of clonal SNV in the colorectal cancer.
  13. The method according to any one of claims 1 to 12, further comprising: forming an amplification reaction mixture by combining polymerase, nucleotide triphosphates, nucleic acid fragments from a nucleic acid library prepared from the blood sample, and a set of target-specific primers that each bind within 150 base pairs of the SNV gene locus; and subjecting the amplification reaction mixture to amplification conditions to create a set of amplicons.
  14. The method according to any one of claims 1 to 13, wherein the efficiency and error rate per cycle are determined for each amplification of the multiple amplification reaction of the set of SNV loci, and the efficiency and error rate are used to determine whether patient-specific SNVs are present in the sample.
  15. The aforementioned multiple amplification reaction is a PCR reaction performed using a set of primers. (i) The annealing temperature is 1 to 15°C higher than the melting point of at least 50% of the set of primers. (ii) The length of the annealing step during the PCR reaction is 15 to 120 minutes. (iii) The concentration of each primer in the set of primers in the amplification reaction mixture is 1 to 10 nM, and/or (iv) The set of primers is designed to minimize primer dimer formation, according to any one of claims 1 to 14.
  16. The method according to any one of claims 1 to 15, wherein the patient-specific set of SNV loci includes 64 or more somatic mutations identified in the tumor sample.

Description

Cross-reference of Related Applications This application claims priority to U.S. Provisional Application No. 62/657,727 filed on 14 April 2018, U.S. Provisional Application No. 62/669,330 filed on 9 May 2018, U.S. Provisional Application No. 62/693,843 filed on 3 July 2018, U.S. Provisional Application No. 62/715,143 filed on 6 August 2018, U.S. Provisional Application No. 62/746,210 filed on 16 October 2018, U.S. Provisional Application No. 62/777,973 filed on 11 December 2018, and U.S. Provisional Application No. 62/804,566 filed on 12 February 2019. Each of these applications cited above is incorporated herein by reference in its entirety. The detection of early cancer recurrence or metastasis has traditionally relied on imaging and tissue biopsy. While tumor tissue biopsy is invasive and carries a risk of potentially contributing to metastasis or surgical complications, imaging-based detection is not sufficiently sensitive to detecting early recurrence or metastasis. A better, less invasive method is needed to detect cancer recurrence or metastasis. The patent or application file must include at least one color drawing. A copy of the published patent or patent application containing the color drawing will be provided by the Office upon request and payment of the necessary fees. The embodiments disclosed herein will be further described with reference to the accompanying drawings, and similar structures will be referred to by similar figures throughout some of the drawings. The drawings shown are not necessarily to scale, but rather are generally emphasized when illustrating the principles of the embodiments disclosed herein. This is a workflow diagram.Upper panel: Number of SNVs per sample; Lower panel: Working assay sorted by driver category.Measured cfDNA concentration. Each data point refers to a plasma sample.This sample demonstrates a good correlation between previously determined tissue VAF measurements (x-axis) and the current measurements using mPCR-NGS (y-axis). Each sample is represented by a separate rectangle, and the VAF data points are color-coded according to the tissue sub-section.This sample shows a weak correlation between previously determined tissue VAF measurements (x-axis) and the current measurements using mPCR-NGS (y-axis). Each sample is represented by a separate rectangle, and the VAF data points are color-coded according to the tissue sub-section.Depth of the read histogram as a function of the obtained calls. Top: This assay did not detect the expected plasma SNV. Bottom: This assay detected the expected plasma SNV.The number of SNVs detected in plasma by histological type.SNV detection in plasma (left) and sample (right) according to tumor stage.Plasma VAF as a function of tumor stage and SNV clonality.The number of SNVs detected in plasma from each sample as a function of the amount of DNA input.Plasma VAF as a function of mean tumor VAF. Mean tumor VAF was calculated across all tumor sub-subThe clonal ratio (red vs. blue) and mutant variant allele frequency (MutVAF) for each detected SNV are shown. All SNVs detected from each sample are placed in a single column, and the sample is classified by tumor stage (pTNM stage). Samples in which no SNVs were detected are included. The clonal ratio is defined as the ratio between the number of tumor segments in which SNVs were observed and the total number of segments analyzed from that tumor.The clonal status (blue for clones, red for subclones) and mutant variant allele frequency (MutVAF) of each detected SNV are shown. All SNVs detected from each sample are placed in a single column, and the sample is classified by tumor stage (pTNM stage). Samples in which no SNVs were detected are included. Clonal status was determined by PyCloneCluster using whole exome sequencing data from tumor tissue.The clonal status (blue for clones, red for subclones) and mutant variant allele frequency (MutVAF) of each detected SNV are shown. The upper panel shows only clonal SNVs, and the lower panel shows only subclonal SNVs. All SNVs detected from each sample are placed in a single column, and the sample is classified by tumor stage (pTNM stage). Samples in which no SNVs were detected are included. Clonal status was determined by PyCloneCluster using whole exome sequencing data from tumor tissue.This shows the number of SNVs detected in plasma as a function of histological type and tumor size. Histological type and tumor stage were determined by pathology reports. Each data point is color-coded by size, with red indicating the largest tumor size and blue indicating the smallest tumor size.This is a table of cfDNA analysis results showing DNA concentration, genomic copy equivalents in library preparations, plasma hemolysis grade, and cDNA profile for all samples.This is a table showing the SNV detected in plasma for each sample.This is a table of further SNVs detected in plasma.This is an example of a detection assay for plasma samples during relapse