Quantifying the evolution of tumor architecture using serial circulating tumor DNA.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 600-600
Author(s):  
Jason Henry ◽  
Jonathan M. Loree ◽  
John H. Strickler ◽  
Kanwal Pratap Singh Raghav ◽  
Van K. Morris ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Limit Of Detection ◽  
Circulating Tumor Dna ◽  
Tumor Evolution ◽  
Disease Course ◽  
Serial Sampling ◽  
Blood Specimens ◽  
Serial Samples ◽  
Tumor Dna ◽  
Over Time

600 Background: There is limited data regarding changes in the genomic landscape in individual patients over time as serial tissue biopsy has risk and is of uncertain clinical benefit. The advent of circulating tumor DNA (ctDNA) allows for safe and repeated molecular sampling with the potential to investigate evolution of tumor architecture over the disease course. Methods: From 5/15 to 12/17, 116 patients with metastatic CRC had between three to 12 blood specimens taken over the treatment course. Plasma was tested using targeted NGS assay (Guardant360, Guardant Health, 68 gene). To account for variations in the amount of ctDNA in serial samples, a window of evaluable allele frequency was established for each patient as the fold change between the max allele frequency (mAF) and limit of detection for serial samples with the lowest mAF. Mutations not falling within this window were excluded from analysis. Substantial treatment induced selective pressure (SP) was defined as a decrease in the mutant mAF of > 50% in patients with at least an initial mAF of 1%. Results: 116 patients with a total of 317 serial blood samples were evaluable after accounting for ctDNA variations over time. Specimens were collected a median of 12 months apart, with a median of three specimens per patient. Thirteen patients (11%) did not have any changes in mutations on serial sampling, however the remainder of patients gained an average of 1.1 mutations per time point (mut/tp), and lost 1.0 mut/tp. 31% of patients demonstrated evidence of substantial treatment-induced SP. These patients were more likely to demonstrate a change in clonal architecture of the tumor (46% greater rate than those without SP, P = 0.04), predominantly through gain of new clones. In contrast, clonal hematopoiesis alterations that may be induced by chemotherapy, such as JAK2V617F, were neither gained or lost. Conclusions: After correction for variations over time in the total amount of ctDNA in circulation, we identify numerous changes in tumor architecture with serial sampling. For the first time in colorectal cancer we demonstrate that when treatment-induced SP is applied the rate of tumor evolution is increased, demonstrating potential value of monitoring changes in tumor architecture over the disease course.

18 New method of assessing tumor heterogeneity utilizing both circulating tumor DNA and tissue DNA to predict the response to immunotherapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A18-A18
Author(s):  
Jaeyoun Choi ◽  
Myungwoo Nam ◽  
Stanislav Fridland ◽  
Jinyoung Hwang ◽  
Chan Mi Jung ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Immune Response ◽  
Allele Frequency ◽  
Scoring System ◽  
Tumor Heterogeneity ◽  
Metastatic Breast ◽  
Circulating Tumor Dna ◽  
List Type ◽  
Multiple Biopsies ◽  
Tumor Dna

BackgroundTumor heterogeneity assessment may help predict response to immunotherapy. In melanoma mouse models, tumor heterogeneity impaired immune response.1 In addition, among lung cancer patients receiving immunotherapy, the high clonal neoantigen group had favorable survival and outcomes.2 Ideal methods of quantifying tumor heterogeneity are multiple biopsies or autopsy. However, these are not feasible in routine clinical practice. Circulating tumor DNA (ctDNA) is emerging as an alternative. Here, we reviewed the current state of tumor heterogeneity quantification from ctDNA. Furthermore, we propose a new tumor heterogeneity index(THI) based on our own scoring system, utilizing both ctDNA and tissue DNA.MethodsSystematic literature search on Pubmed was conducted up to August 18, 2020. A scoring system and THI were theoretically derived.ResultsTwo studies suggested their own methods of assessing tumor heterogeneity. One suggested clustering mutations with Pyclone,3 and the other suggested using the ratio of allele frequency (AF) to the maximum somatic allele frequency (MSAF).4 According to the former, the mutations in the highest cellular prevalence cluster can be defined as clonal mutations. According to the latter, the mutations with AF/MSAF<10% can be defined as subclonal mutations. To date, there have been no studies on utilizing both ctDNA and tissue DNA simultaneously to quantify tumor heterogeneity. We hypothesize that a mutation found in only one of either ctDNA or tissue DNA has a higher chance of being subclonal.We suggest a scoring system based on the previously mentioned methods to estimate the probability for a mutant allele to be subclonal. Adding up the points that correspond to the conditions results in a subclonality score (table 1). In a given ctDNA, the number of alleles with a subclonality score greater than or equal to 2 divided by the total number of alleles is defined as blood THI (bTHI) (figure 1). We can repeat the same calculation in a given tissue DNA for tissue THI (tTHI) (figure 2). Finally, we define composite THI (cTHI) as the mean of bTHI and tTHI.Abstract 18 Table 1Subclonality scoreAbstract 18 Figure 1Hypothetical distribution of all alleles found in ctDNA bTHI = the number of alleles with a subclonality score greater than or equal to 2/the total number of alleles found in ctDNA = 10/20 =50%Abstract 18 Figure 2Hypothetical distribution of all alleles found in tissue DNA tTHI= the number of alleles with a subclonality score greater than or equal to 2/the total number of alleles found in tissue DNA = 16/40 = 40% cTHI= (bTHI + tTHI)/2 = 45%ConclusionsTumor heterogeneity is becoming an important biomarker for predicting response to immunotherapy. Because autopsy and multiple biopsies are not feasible, utilizing both ctDNA and tissue DNA is the most comprehensive and practical approach. Therefore, we propose cTHI, for the first time, as a quantification measure of tumor heterogeneity.ReferencesWolf Y, Bartok O. UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma. Cell 2019;179:219–235.McGranahan N, Swanton C. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463–1469.Ma F, Guan Y. Assessing tumor heterogeneity using ctDNA to predict and monitor therapeutic response in metastatic breast cancer. Int J Cancer 2020;146:1359–1368.Liu Z, Xie Z. Presence of allele frequency heterogeneity defined by ctDNA profiling predicts unfavorable overall survival of NSCLC. Transl Lung Cancer Res 2019;8:1045–1050.

Analytical and clinical validation of a novel amplicon-based NGS assay for the evaluation of circulating tumor DNA in metastatic colorectal cancer patients

2019 ◽  
Vol 57 (10) ◽  
pp. 1501-1510 ◽  
Author(s):  
Beili Wang ◽  
Shengchao Wu ◽  
Fei Huang ◽  
Minna Shen ◽  
Huiqin Jiang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Circulating Tumor Dna ◽  
Concordance Rate ◽  
Tissue Samples ◽  
Percent Agreement ◽  
Pre Treatment ◽  
Tumor Dna

Abstract Background Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly™, which employs a concatemer-based error correction strategy. Methods Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cell-free DNA (cfDNA) reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. Samples with a mutation abundance below the limit of detection (LOD) were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS. Results The Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency (AF) of 0.2% for 20 ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In the pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly™ and ARMS genotyped, and the positive percent agreement (PPA) and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA were 100% and 99.60%, respectively. Conclusions Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given the high agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.

Serial monitoring of ctDNA to highlight mutation profiles in colorectal cancer.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 641-641 ◽  
Author(s):  
Jonathan M. Loree ◽  
John H. Strickler ◽  
Allan Andresson Lima Pereira ◽  
Michael Lam ◽  
Kanwal Pratap Singh Raghav ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Allele Frequency ◽  
Mutant Allele ◽  
Mapk Pathway ◽  
Circulating Tumor Dna ◽  
Genomic Profile ◽  
Repeated Sampling ◽  
Concentration Changes ◽  
Tumor Dna ◽  
Selection Of

641 Background: Circulating tumor DNA (ctDNA) represents an ideal platform to obtain the most current genomic profile of a patient’s tumor. We aimed to investigate how stable these profiles remain during serial ctDNA assays in metastatic colorectal cancer (mCRC). Methods: In 77 patients (pts) with mCRC and serial Guardant360 assays with a detectable mutation (mt), we compared mt stability by assessing whether variants were gained/lost between serial assays and changes in relative mutant allele frequency (rMAF). rMAF of a mt was defined as (mt allele frequency / mt present at the maximum allele frequency in that assay). rMAF results were normalized to detected ctDNA concentration changes between assays to ensure changes in rMAF were not due to changes in ctDNA concentration. MAPK pathway mutations were defined as RAS, BRAF, EGFR, KIT, or MET mutations. Results: Of 77 pts, 64 (83%) had 2 serial assays and 13 (16.9%) had 3 or 4 assays performed. Serial assays occurred an average of 138 days apart (+/- SD of 111 days). Only 13/77 (17%) pts had no change in the number of mts detected between assays. A new mt was detected in 42/77 (55%) pts, while 43/77 (56%) lost a previously detected mt. Of 52 mts detected in patients with > 2 assays, 16 (31%) were gained and subsequently lost. After controlling for ctDNA concentration, mts were equally likely to have an increasing (129/308 – 42%) or decreasing (150/308 – 49%) allele frequency. Potentially clinically relevant MAPK variants were gained/lost in 29% of patients; though MAPK mts developed in a large number of pts (16/77 – 21%), many pts also lost MAPK mts (9/77 – 12%), showing ongoing subclonal dynamics. Median time between assays did not differ between pts with gain/lost mts or stable mt profiles (P = 0.73), however mt rMAF shift of > 25% was more common if assays were > 90 days apart (OR 4.3, P < 0.0001). Conclusions: Serial ctDNA assays demonstrate ongoing mutational changes in mCRC, with emergence/disappearance of MAPK variants being more common than expansion of a pre-existing clone. Our results suggest repeated sampling may be important to optimize selection of targeted therapies at each regimen alteration.

scholarly journals Advancing quality-control for NGS measurement of actionable mutations in circulating tumor DNA

2021 ◽  
Author(s):  
James C. Willey ◽  
Tom Morrison ◽  
Brad Austermiller ◽  
Erin L. Crawford ◽  
Daniel J. Craig ◽  
...  
Keyword(s):  
Quality Control ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Circulating Tumor Dna ◽  
Primary Objective ◽  
Nucleotide Position ◽  
Targeted Next Generation Sequencing ◽  
Next Generation Sequencing Ngs ◽  
Rate Limit ◽  
Tumor Dna

SUMMARYThe primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next generation sequencing (NGS) method that enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in for each actionable mutation target, suitable for use in NGS following hybrid-capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each sample. True positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

scholarly journals Circulating tumor DNA (ctDNA) detection is associated with shorter progression-free survival in advanced melanoma patients

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Gabriella Taques Marczynski ◽  
Ana Carolina Laus ◽  
Mariana Bisarro dos Reis ◽  
Rui Manuel Reis ◽  
Vinicius de Lima Vazquez
Keyword(s):  
Somatic Mutations ◽  
Limit Of Detection ◽  
Progression Free Survival ◽  
Circulating Tumor Dna ◽  
Advanced Melanoma ◽  
Plasma Samples ◽  
Free Survival ◽  
Melanoma Patients ◽  
Digital Polymerase Chain Reaction ◽  
Tumor Dna

Abstract BRAF, NRAS and TERT mutations occur in more than 2/3 of melanomas. Its detection in patient’s blood, as circulating tumor DNA (ctDNA), represents a possibility for identification and monitoring of metastatic disease. We proposed to standardize a liquid biopsy platform to identify hotspot mutations in BRAF, NRAS and TERT in plasma samples from advanced melanoma patients and investigate whether it was associated to clinical outcome. Firstly, we performed digital polymerase chain reaction using tumor cell lines for validation and determination of limit of detection (LOD) of each assay and screened plasma samples from healthy individuals to determine the limit of blank (LOB). Then, we selected 19 stage III and IV patients and determined the somatic mutations status in tumor tissue and track them in patients’ plasma. We established a specific and sensitive methodology with a LOD ranging from 0.13 to 0.37%, and LOB ranging from of 0 to 5.201 copies/reaction. Somatic mutations occurred in 17/19 (89%) patients, of whom seven (41%) had ctDNA detectable their paired plasma. ctDNA detection was associated with shorter progression free survival (p = 0.01). In conclusion, our data support the use of ctDNA as prognosis biomarker, suggesting that patients with detectable levels have an unfavorable outcome.

Monitoring of circulating tumor DNA in non-small cell lung cancer patients treated with EGFR-inhibitors.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 11535-11535
Author(s):  
Remy B Verheijen ◽  
Tirsa T van Duijl ◽  
Michel M van den Heuvel ◽  
Jos H. Beijnen ◽  
Jan H.M. Schellens ◽  
...  
Keyword(s):  
Progression Free Survival ◽  
Circulating Tumor Dna ◽  
Small Cell ◽  
Driver Mutations ◽  
Small Cell Lung ◽  
T790m Mutation ◽  
Free Survival ◽  
Nsclc Patients ◽  
Tumor Dna ◽  
Over Time

11535 Background: Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib and gefitinib are routinely used in the treatment of non-small cell lung cancer (NSCLC). Monitoring of EGFR mutations in circulating tumor DNA (ctDNA) derived from plasma has been proposed as an alternative for repeated tumor biopsies. Our aim was to investigate the dynamics of ctDNA in a cohort of NSCLC patients and explore the roles of EGFR driver and resistance mutations in predicting disease progression and progression free survival (PFS). Methods: NSCLC patients treated with either erlotinib or gefitinib as first-line anti-EGFR therapy were included. Clinical data was collected retrospectively from medical records. Plasma samples collected as part of routine care were analyzed. First DNA was isolated from plasma using the QIAsymphony SP (Qiagen). Then EGFR driver (L858R and exon 19 deletions) and resistance (T790M) mutations were quantified using the X100 Droplet Digital PCR and analyzed using QuantaSoft software (Bio-Rad). The dynamics of ctDNA mutations over time and the relationship between copy numbers and progression free survival were explored. Results: 68 NSCLC patients and 249 plasma samples (1-13 per patient) were included in the analysis. In 33 patients, the T790M mutation was detected. The median (range) T790M concentration in these samples was of 7.3 (5.1 - 3688.7) copies/mL. In 30 patients, the L858R or exon 19 deletion driver mutations were found in median concentrations of 11.7 (5.1 – 12393.3) and 27.9 (5.9 – 2896.7) copies/mL, respectively. Using local polynomial regression, the copies/mL of EGFR driver mutations increased several weeks prior to progression on standard response evaluation. In Kaplan-Meier analysis, patients with a detectable T790M mutation during the first 8 weeks of treatment had a shorter PFS (7.6 versus 14.4 months, p < 0.01, log-rank test). Conclusions: Early detection of the T790M mutation in plasma ctDNA is related to poor PFS. Furthermore, an increase in the copies/mL of the EGFR driver mutation over time may predict clinical progression.

Non-invasive diagnosis and tracking of tumor evolution by targeted sequencing of circulating tumor DNA in metastatic pancreatic cancer patients.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e15769-e15769
Author(s):  
Thomas Seufferlein ◽  
Andreas W. Berger ◽  
Daniel Schwerdel ◽  
Thomas Jens Ettrich ◽  
Stefan A. Schmidt ◽  
...  
Keyword(s):  
Tumor Tissue ◽  
Early Stage ◽  
Therapy Monitoring ◽  
Circulating Tumor Dna ◽  
Ductal Adenocarcinoma ◽  
Tumor Evolution ◽  
Imaging Data ◽  
Non Invasive ◽  
Outcome Monitoring ◽  
Tumor Dna

e15769 Background: Treatment of stage IV pancreatic ductal adenocarcinoma (PDAC) has made substantial progress over the last years, therapy monitoring still is at an early stage. This could be substantially supported by tools that allow to establish and monitor the molecular setup of the tumor even during treatment. In particular, non-invasive approaches are desirable. Characterization of circulating tumor DNA (ctDNA) may help to achieve this goal. Methods: We analyzed a cohort of 20 patients with histologically confirmed metastatic PDAC (mPDAC) prior to and during palliative treatment including disease progression. ctDNA and corresponding tumor tissue were analyzed by targeted NGS and droplet digital PCR for the 7 most frequently mutated genes in PDAC ( TP53, SMAD4, CDKN2A, KRAS, APC, ATM, FBXW7). Findings were correlated with clinical and imaging data to establish its prognostic and predictive value. Results: ctDNA was analyzed at baseline prior to initiation of the respective line of treatment. Mutations in either of the genes examined were detectable in 15/20 patients (75%). Tissue-blood concordance was 80% in therapy naïve patients. 96% of mutations in ctDNA of therapy naïve patients were in KRAS and/or TP53. The combined mutated allele frequencies (CMAF) of theese 2 genes significantly decreased (p = 0.0173) during therapy and increased at progression (p = 0.0145) across all treatment lines. By sequential ctDNA analyses we detected a change in the mutational landscape compared to the respective baseline ctDNA status in 7/11 patients during 1st line, in 3/7 patients during 2nd line and 2/2 patients during 3rdline treatment. In therapy naïve patients, the decline of the CMAF during therapy significantly correlated with progression-free survival (p = 0.0013). Conclusions: Molecular genotyping of ctDNA in mPDAC patients proved to be feasible and there was a high concordance between tumor tissue and ctDNA. The molecular genotype changed significantly during treatment and changes correlated with outcome. Monitoring of ctDNA may enable to adapt therapeutic strategies to the specific molecular changes present at a certain time during treatment of mPDAC.

Profiling 523 cancer associated genes in circulating tumor DNA of children with CNS tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3023-3023
Author(s):  
Erin R. Bonner ◽  
Robin Harrington ◽  
Biswajit Das ◽  
Paul M. Williams ◽  
Chris Alan Karlovich ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Molecular Subtype ◽  
Circulating Tumor Dna ◽  
Copy Number Variations ◽  
Response To Therapy ◽  
Cns Tumors ◽  
Response To Treatment ◽  
Tumor Evolution ◽  
Cns Tumor ◽  
Tumor Dna

3023 Background: Pediatric central nervous system (CNS) cancers often pose unique challenges including tumor ‘invisibility’, where surgical resection is restricted due to the sensitive tumor location and tissue biopsy is not always feasible. Detecting cancer associated mutations and copy number variations (CNV) at diagnosis is increasingly important, as the WHO classification of pediatric CNS cancers has incorporated molecular signatures with tumor grade. To achieve CNS tumor molecular ‘visibility’, we previously established a liquid biopsy platform for detecting single nucleotide variants in circulating tumor DNA (ctDNA). However, our method was limited by the restricted number of genes that can be monitored and the inability to detect genomic events including CNVs. To address this, we developed a deep sequencing liquid biopsy approach to profile alterations across selected genes. Our platform provides an opportunity for multi-gene monitoring, to assess tumor subclonal evolution and response to treatment in the absence of repeat tissue biopsies. Methods: We tested the performance of our platform using paired tissue, CSF, and plasma/serum from 10 children with diffuse midline glioma (DMG). ctDNA was analyzed using the TruSight Oncology 500 (TSO500) ctDNA targeted panel covering 523 genes. Matched tumor, CSF, and blood were assessed for concordance and sequencing results were compared to digital droplet PCR (ddPCR) detection of H3K27M mutation. Results: The median exons with ³500X coverage was 96% for 7 CSF samples with optimal input (³60ng), 0.01% for 3 CSF samples with < 5ng input, and 74.5% for plasma/serum samples. ctDNA was more readily detectable in CSF, yet concordance between paired tumor, CSF and plasma/serum was observed. DMG associated mutations in genes including H3F3A, HIST1H3B, TP53, and ACVR1 were detected in ctDNA. Of 9 H3K27M mutations identified in tumor, 8 were present in CSF and 3 in plasma/serum, for a positive percent agreement of 89% and 33%, respectively, with the tumor results. Among CSF samples, H3.3K27M was detected in 6/6 cases, and H3.1K27M in 2/3 cases, with variant allele frequencies comparable to ddPCR results. CNVs including PDGFRA/B and MDM4 amplifications were present in CSF and confirmed by analysis of paired tumor. Additional events, including PIK3CA p.E545Q, PPM1D truncation, and KRAS amplification, were detected in CSF but absent from paired tumor, indicating tissue heterogeneity. Strategies to optimize ctDNA detection, including optimization of ctDNA isolation and adjustment of library QC metrics, were identified. Conclusions: This proof-of-concept study demonstrates the feasibility of our high depth, targeted sequencing approach for detecting clinically relevant mutations in ctDNA from children with CNS tumors. This approach may aid in diagnosis of CNS tumor molecular subtype, and monitoring of tumor evolution and response to therapy in serially collected ctDNA.

Serial circulating tumor DNA samples from patients with metastatic breast cancer and BRCA1/2 mutations.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 1025-1025
Author(s):  
Katharine Collier ◽  
David Tallman ◽  
Zachary Weber ◽  
Marcy Haynam ◽  
Elizabeth J. Adams ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Clonal Evolution ◽  
Metastatic Breast ◽  
Circulating Tumor Dna ◽  
Plasma Samples ◽  
Significant Difference ◽  
Platinum Chemotherapy ◽  
Tumor Dna ◽  
Over Time

1025 Background: Analysis of circulating tumor DNA (ctDNA) over time allows non-invasive evaluation of tumor genomic evolution. We characterize changes in tumor fraction (TFx), somatic copy number alterations (SCNAs), and somatic mutations (muts) over time in patients (pts) with BRCA1/2 muts and metastatic breast cancer (mBC) who received a PARP inhibitor (PARPi) or platinum chemotherapy. Specifically, we seek to identify the frequency of BRCA1/2 reversion muts. Methods: Pts with mBC and germline or somatic BRCA1/2 muts were identified on a banking protocol of prospectively-collected serial samples of blood and plasma. Control pts without a BRCA1/2 mut were matched 2:1 by age and hormone receptor (HR) status. Ultra-low-pass whole genome sequencing (ULPWGS) with 0.1x depth was performed on all plasma samples (n = 103) and the ichorCNA algorithm was used to determine TFx and SCNAs. Targeted panel sequencing (TPS) of 402 cancer-related genes was performed at 10,000x depth on plasma samples, and one blood sample per pt. The panel includes BRCA1/2 and 38 other DNA damage repair (DDR) genes. Somatic muts were identified by joint calling with Mutect2 across plasma timepoints with paired pt normal blood. Germline variant calling from TPS on blood with HaplotypeCaller was used to confirm germline muts in BRCA1/2.Results: We identified 10 pts with mBC with a germline (n = 7) or somatic (n = 3) BRCA1 (n = 2) or BRCA2 (n = 8) mut and banked blood and plasma samples at 3-9 timepoints at a median of 8 weeks apart (range 1-43). The control cohort of 20 pts with mBC and wildtype BRCA1/2 was well matched by age and HR status. All pts with BRCA1/2 muts received a PARPi and/or platinum chemotherapy at some point during sample collection. Half of control pts received platinum chemotherapy. Germline BRCA1/2 muts were confirmed in all 7 pts with known germline muts. Among the BRCA1/2 mut cohort, median TFx was 0.04 (range 0-0.57) with 20% of samples having TFx > 0.10. A median of 1.5 (range 0-39) somatic muts per pt were found in DDR genes. Four pts (40%) had secondary non-reversion muts in BRCA1/2. A reversion mut of a germline BRCA2 mut, restoring the open reading frame of BRCA2, was discovered at the last timepoint from 1 pt while receiving carboplatin. A germline BRCA1/2 reversion mut in this cohort occurred in 2.3% of samples, 14.3% of pts. There was no significant difference in the percent of genome with a SCNA between the first and last time point, nor before and after PARPi/platinum. The somatic mut landscape and clonal evolution of TPS using PyClone will be presented. Conclusions: Evaluation of serial ctDNA samples for TFx, SCNAs, and somatic muts from banked plasma and blood from pts with mBC is feasible. The frequency of reversion muts in BRCA1/2 was low, suggesting that either their incidence is low or ctDNA TPS is not sensitive enough to detect them. Secondary non-reversion muts in BRCA1/2 and other somatic DDR muts were more common. SCNAs were stable over time.

scholarly journals A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

2016 ◽  
Vol 62 (8) ◽  
pp. 1129-1139 ◽  
Author(s):  
Sonia Garrigou ◽  
Geraldine Perkins ◽  
Fanny Garlan ◽  
Corinne Normand ◽  
Audrey Didelot ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Genetic Material ◽  
Surrogate Marker ◽  
Digital Pcr ◽  
Tumor Stage ◽  
Circulating Tumor Dna ◽  
Tumor Evolution ◽  
Highly Sensitive ◽  
Tumor Dna

Abstract BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

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