Low Mutant Allele Frequency Research Articles

Abstract 3695: Monitoring response to immunotherapy using cell free DNA fragmentomes

Abstract BACKGROUND: The rapid detection of disease progression in patients receiving immune checkpoint inhibitors (ICIs) is challenging since there are no reliable biomarkers of clinical response. Liquid biopsy assays may address this unmet need by using cell-free DNA (cfDNA) to monitor treatment responses. Current targeted next-generation sequencing cfDNA assays are costly and have limited assay sensitivity for detection of cancer-specific mutations at low mutant allele frequency (MAF). Here, we demonstrate the utility of DELFI Tumor Fraction (DELFI-TF), a tumor- and mutation-independent cfDNA fragmentome approach to monitor treatment response in patients with metastatic non-small cell lung cancer (mNSCLC). METHODS: Overall, 422 longitudinal blood samples were collected from 141 mNSCLC patients of which 109 were treated with immunotherapy and 32 with targeted therapy (TT). Plasma-derived cfDNA was processed with whole genome sequencing at low coverage (~4x). Circulating tumor burden was quantified as the maximum MAF (maxMAF) of tumor-derived variants detected using a 500+gene panel. Matched white blood cells were used to filter out germline and clonal hematopoietic variants. A random forest regression model was trained with maxMAF as the response using fragmentation-derived features. The accuracy of DELFI-TF was assessed using leave one patient out cross-validation in the ICI cohort and holdout validation in the cohort of patients receiving TT. For survival analyses, we used the median baseline scores to define low/high DELFI-TF and maxMAF. RESULTS: In the ICI cohort, DELFI-TF scores were strongly correlated with the maxMAF (r=0.94, p<0.001, Pearson). At baseline, DELFI-TF and maxMAF both differentiate responding from non-responding tumors (p = 0.01 and p = 0.001 for DELFI-TF and maxMAF respectively; Wilcoxon). Patients with high DELFI-TF or maxMAF had significantly shorter progression-free survival (PFS) compared to low DELFI-TF or maxMAF (17.0 vs 4.7 months, p < 0.001; 17.0 vs 4.6 months, p < 0.001; Log-Rank respectively) and similar overall survival (OS) (DELFI-TF high 11.4 vs low 34.1 months, p<0.001; maxMAF high 10.9 vs low 34.1 months, p < 0.001, Log-Rank). Changes in DELFI-TF and maxMAF at the earliest timepoint were highly correlated (n=109; r=0.90; Pearson). In the validation cohort of TT patients, DELFI-TF at baseline outperformed maxMAF in distinguishing patients with PFS and OS benefits (DELFI-TF PFS 36.4 vs 11.5 months, p = 0.03; maxMAF PFS 21.9 vs 15.9 months, p=0.6; Log-Rank) (DELFI-TF OS high 37 months vs low not reached (NR), p < 0.05; Max MAF OS high 37 vs NR, p = 0.1, Log-Rank). The majority of TT patients (81%) had consistent changes with DELFI-TF and maxMAF from baseline to the earliest timepoint. CONCLUSIONS: DELFI-TF is a tumor and mutation independent approach that is cost-efficient with high performance comparable to current ctDNA assays for estimating tumor burden and monitoring response in cancer patients. Citation Format: Bahar Alipanahi, Lavanya Sivapalan, Jamie Medina, Zachary L. Skidmore, Paola Ghanem, Erica Peters, Gavin Pereira, Nisha Rao, Kavya Velliangiri, Alissa Konicki, Stephen Cristiano, Laurel Millberg, Jacob Carey, Keith Lumbard, Noushin Niknafs, Bryan Chesnick, Jennifer Tom, Alessandro Leal, Benjamin Levy, Patrick Forde, Peter Bach, Nicholas C. Dracopoli, Robert B. Scharpf, Victor Velculescu, Lorenzo Rinaldi, Valsamo Anagnostou. Monitoring response to immunotherapy using cell free DNA fragmentomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3695.

Cell-free DNA fragmentation profiling for therapeutic response monitoring in metastatic colorectal cancer.

e15664 Background: Currently available circulating cell-free DNA (cfDNA) assays require deep-targeted sequencing to detect cancer-specific mutations at low mutant allele frequency (MAF) levels in the blood. Recently, we developed a tumor-agnostic, mutation-independent approach that utilizes low-coverage whole genome sequencing called DELFI (DNA evaluation of fragments for early interception) Tumor Fraction (DELFI-TF), a model designed to predict plasma tumor fractions based on genome-wide fragmentation-related features. Here, we report the results of DELFI-TF applied to a prospective cohort of patients with metastatic colorectal cancer (mCRC). Methods: Overall, 692 longitudinal plasma samples collected from 153 initially treatment-naive mCRC patients participating in the phase III CAIRO5 study (NCT02162563) were sequenced at low coverage and used for training and cross-validation. In patients with tumor-tissue-proven RAS/BRAF mutations, the tumor fractions were quantified as the cfDNA MAF of the RAS/BRAF variant measured by droplet digital PCR (ddPCR). Using fragment-sequencing statistics, a Bayesian regression model was trained against the MAF of the tumor-specific driver RAS/BRAF variant in all longitudinal timepoints to generate DELFI-TF scores. Changes in longitudinal DELFI-TF scores during first-line therapy (DELFI-TF slopes) were examined to predict treatment response and survival outcomes. Results: The DELFI-TF scores were strongly correlated with RAS/BRAF cfDNA MAF measured by ddPCR ( Pearson, r= 0.85, p< 0.001). Baseline DELFI-TF correlated with dimensions of liver metastases reported in CT scans ( Pearson, r= 0.49, p< 0.001) as well as clinical response, with pre-treatment levels significantly lower in patients with a later-confirmed partial or complete response ( Wilcoxon, p< 0.05). Patients with low or negative DELFI-TF slopes presented with longer progression-free survival in the overall study population (13.4 months vs 10.4 months, HR = 2.03, 95% CI 1.25 to 3.32, Log-rank p< 0.01) and in patients who experienced durable clinical benefit (16.7 months vs 13.3 months, HR = 2.24, 95% CI 1.1 to 4.55, Log-rank p= 0.023). Patients with low or negative DELFI-TF slopes also experienced significantly longer overall survival (59.4 months vs 29.1 months, HR = 3.05, 95% CI 1.58-5.90, Log-rank p< 0.001). Tissue-informed focal and arm-level copy number changes were detected 4-12 weeks after liver metastases resection. Most patients detected as having a molecular relapse were diagnosed earlier than clinical recurrences identified by conventional imaging. Conclusions: DELFI-TF demonstrates the ability to use cfDNA fragmentomes to estimate cfDNA tumor burden with performance comparable to standard approaches for treatment response monitoring and clinical outcome prediction.

Abstract 5714: Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer

Abstract BACKGROUND: Cell-free circulating tumor DNA (ctDNA) assays have been adopted to monitor therapeutic response in both early- and late-stage cancer. However, tests currently available require deep-targeted sequencing to detect cancer-specific mutations at low mutant allele frequency (MAF) levels in the circulation. Recently, we developed a tumor-agnostic approach called DELFI Tumor Fraction (DELFI-TF), a Bayesian probabilistic model designed to predict plasma tumor fractions based on genome-wide fragmentation-related features. METHODS: Overall, 692 longitudinal plasma samples collected from 153 patients with initially unresectable colorectal cancer (CRC) liver-only metastases participating in the phase III study CAIRO5 (NCT02162563) were split across a training (RAS/BRAF mutant) and a validation cohort (RAS/BRAF wildtype). For all cell-free DNA (cfDNA) samples of patients within the training cohort (n=312), the tumor burden was quantified as the MAF of the tumor-tissue-proven RAS/BRAF variant measured by digital droplet PCR (ddPCR). Using cfDNA fragment-sequencing statistics from low-coverage WGS data, a Bayesian regression model was trained against the MAF of the tumor-specific driver RAS/BRAF variant in all longitudinal timepoints in the training cohort. Changes in DELFI-TF scores during first-line FOLFOX/FOLFIRI and bevacizumab therapy were assessed to predict disease response. A tissue-informed approach was applied to detect minimal residual disease in the subgroup of patients treated with curative intent (complete resection of the primary tumor and liver metastases). RESULTS: In the training cohort, DELFI-TF scores strongly correlated with RAS/BRAF MAF measured by ddPCR (r=0.85, p<0.001). DELFI-TF captured plasma tumor fractions associated with copy number changes when ddPCR failed to detect RAS/BRAF mutations. Before treatment initiation, DELFI-TF correlated with the sum of the largest diameters on CT scans according to RECIST1.1 (r=0.49, p<0.001). Patients with negative and positive changes in DELFI-TF more often exhibited response and progressive disease based on consecutive RECIST assessments, respectively. Median progression-free survival was 9.9 and 12.8 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.007). Overall survival was 28.8 and 58.9 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.001). In patients treated with curative intent, tissue-informed focal and arm-level copy number changes were detected 4-12 weeks after metastasectomy. Most patients presenting molecular relapse were later diagnosed with clinical recurrence by conventional CT imaging. CONCLUSIONS: DELFI-TF demonstrates the ability to use cfDNA fragmentomes to estimate cfDNA tumor burden with a performance comparable to standard approaches for treatment response monitoring and clinical outcome prediction. Citation Format: Bahar Alipanahi, Iris van 't Erve, Keith Lumbard, Laurel K. Millberg, Zach Skidmore, Lorenzo Rinaldi, Jacob Carey, Jennifer Tom, Cornelis J. A. Punt, Nicholas C. Dracopoli, Gerrit A. Meijer, Robert B. Scharpf, Victor E. Velculescu, Remond Fijneman, Alessandro Leal. Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5714.

Abstract 6612: Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia

Abstract The cancer hallmark concept defines biological properties that play a key role in cancer development and progression. The hallmarks reflect generic properties across all cancer types, and are not directly quantifiable nor applicable in cancer diagnostics. Here, we applied a data-driven approach to identify quantifiable hallmarks to be used as a basis for precision cancer medicine (PCM) in Acute Myeloid Leukemia (AML). We applied deep exome, transcriptome, DNA methylation and proteome profiling as well as ex-vivo functional testing of 525 drugs to 118 AML patient samples. Unsupervised multi-omic dimensionality reduction defined 11 independent axes of biological variability that we interpreted to reflect data-driven hallmarks (DDHM) of AML. Each DDHM integrates and ranks different data types and features, pinpointing those molecular features that were most informative for each hallmark. We calculated values for the 11 DDHM for each patient, constructing a DDHM-based precision medicine approach for AML diagnosis and therapy assignment. Most DDHMs were driven by other data types than genomics. For several DDHMs, different cytogenetic and mutational drivers converge on the same hallmarks and specific drug vulnerabilities. We also see how DDHM predictors of poor prognosis and high-risk AML are distinct from those that dictate specific drug response vulnerabilities. DDHM 2 reflected the cell differentiation path leading towards resistance to BH3 mimetics, ACK-inhibitors, and anthracyclines, and sensitivity to MEK inhibitors and TLR8 agonists. DDHM 2 derived a strong impact from protein expression of GATA2, MLLT11, DNMT3B and from RNA expression of WT1 and SOX4 as well as from multiple markers of different monocytic subtypes. DDHM 5 depicted cell cycle regulation and the Megakaryocytic-erythrocyte progenitor cell state that was enriched in AML patients with antecedent hematological disease. Interestingly, DDHM5 was linked to the sensitivity to purine analogs and vinca alkaloids. Moreover, DDHMs 1 and 8 captured clinical risk groups, prognosis, and responsiveness to hypomethylating agents. These DDHMs also revealed the phenotypic and functional biology that distinguished patients with high versus low FLT3-ITD mutant allele frequency. Validation of the DDHMs is realized through profiling and analysis of prospective AML samples, AML cell lines and previously published datasets. In summary, we present a data-driven approach for defining hallmarks in AML. The application of the DDHMs in AML provides a new paradigm for PCM and an opportunity for combinatorial therapeutic targeting. Each patient is characterized by a combination of independent and potentially druggable hallmarks, as opposed to traditional stratification in PCM, where each patient is assigned to one specific subgroup defined by genetic or other biomarkers. Citation Format: Tom Erkers, Nona Struyf, Tojo James, Francesco Marabita, Mattias Vesterlund, Nghia Vu, Cornelia Arnroth, Albin Österroos, Anna Bohlin, Sofia Bengtzén, Matthias Stahl, Rozbeh Jafari, Lukas Orre, Yudi Pawitan, Brinton Seashore-Ludlow, Janne Lehtiö, Sören Lehmann, Päivi Östling, Olli Kallioniemi. Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6612.

Nuclease Enrichment and qPCR Detection of Rare Nucleotide Variants.

The emergence of circulating DNA analysis in blood during the past decade has responded to the need for noninvasive alternatives to classical tissue biopsies. This has coincided with the development of techniques that allow the detection of low-frequency allele variants in clinical samples that typically carry very low amounts of fragmented DNA, such as plasma or FFPE samples. Enrichment of rare variants by nuclease-assisted mutant allele enrichment with overlapping probes (NaME-PrO) enables a more sensitive detection of mutations in tissue biopsy samples alongside standard qPCR detection assays. Such sensitivity is normally achieved by other more complex PCR methods, such as TaqMan qPCR and digital droplet PCR (ddPCR). Here we describe a workflow of mutation-specific nuclease-based enrichment combined with a SYBR Green real-time quantitative PCR detection method that provides comparable results to ddPCR. Using a PIK3CA mutation as an example, this combined workflow enables detection and accurate prediction of initial variant allele fraction in samples with a low mutant allele frequency (<1%) and could be applied flexibly to detect other mutations of interest.

Clinical response to tepotinib according to circulating tumor (ct) DNA biomarkers in patients with advanced NSCLC with high-level MET amplification (METamp) detected by liquid biopsy (LBx).

9121 Background: Tepotinib, a potent, highly selective, oral, MET inhibitor, showed meaningful activity in patients (pts) with NSCLC with high-level METamp by LBx in VISION. Exploratory biomarker analyses are presented herein. Methods: Pts had 0–2 prior therapy lines, high-level METamp by LBx (Guardant360; MET copy number ≥2.5), and no MET exon 14 skipping or EGFR/ ALK alterations. Pts received tepotinib 500 mg once daily (450 mg active moiety). Primary endpoint was objective response by independent review; data cut-off: Aug 20, 2021. Exploratory biomarker analysis included LBx at baseline (BL), on treatment, and end of treatment (EOT). Early molecular response (eMR) was defined as undetectable METamp 6–8 weeks on treatment. Results: 24 pts were enrolled (median age: 63.4 years [yrs]; smokers: 88%; ECOG PS 1: 88%; adenocarcinoma: 67%). Treatment duration was ≥1 yr in five pts and ≥2 yrs in two pts (both ongoing). Overall, objective response rate (ORR) was 41.7% (95% CI: 22.1, 63.4). Treatment-naïve pts (n=7) had an ORR of 71.4% (29.0, 96.3), median (m) DOR was 14.3 months (2.8, not estimable [ne]), and mPFS was 15.6 months (1.4, ne). BL biomarker analyses according to clinical benefit (CR/PR/SD [n=11] vs PD/NE [n=13]) showed association with better outcomes in pts with focal METamp, or without MYCamp or RB1 mutation (Table). MYCamp/ RB1 mutation was detected in 4/7 pts with neuroendocrine/not otherwise specified histology; MYCamp in 2/3 pts with neuroendocrine histology. Low BL ctDNA mutant allele frequency (MAF) was associated with better outcomes. 14 pts had eMRs (ORR 71.4%); persistent METamp (n=4) was associated with lack of clinical response. 2/9 pts with EOT biomarker profiles had emerging resistance mechanisms (MET kinase domain mutations Y1230 and D1228); both had METamp re-emergence. Treatment-related adverse events included edema (composite term; any grade: 46%; Grade 3: 13%) and constipation (any grade: 17%; Grade ≥3: 0%). Conclusions: Tepotinib showed meaningful activity, especially in first line, in the first trial of a MET inhibitor in EGFR WT NSCLC with high-level METamp to enroll based on a convenient LBx assay. BL biomarker analyses indicated focal METamp, MYC/RB1 WT, and low ctDNA MAF were associated with improved outcomes. Serial LBx could monitor molecular response and evaluate resistance. Clinical trial information: NCT02864992. [Table: see text]

Use of the Biocartis Idylla™ Platform for the Detection of Epidermal Growth Factor Receptor, BRAF and KRAS Proto-Oncogene Mutations in Liquid-Based Cytology Specimens from Patients with Non-Small Cell Lung Carcinoma and Pancreatic Adenocarcinoma

The study aimed to demonstrate rapid and effective molecular testing on liquid-based cytology (LBC) samples for EGFR, KRAS and BRAF mutations using the Biocartis Idylla™. Rapid on-site evaluation (ROSE) LBC samples for patients with non-small cell lung carcinoma (NSCLC) or pancreatic ductal adenocarcinoma (PDAC) were tested for EGFR, KRAS and BRAF mutations based on the relevance to tumour subtype. The quantification values (Cq values) and mutation detection status were compared between LBC samples and routine formalin-fixed paraffin-embedded (FFPE) clot samples. ROSE LBC samples (n = 54) showed a higher yield of well-preserved tumour and wild type (WT) DNA, demonstrated by lower quantification cycles, no false positives or false negatives, and a higher sensitivity for low allele frequency mutations when compared with FFPE clot samples. The Biocartis Idylla™ provides highly sensitive, reliable and rapid testing for LBC samples for the detection of EFGR and KRAS mutations. BRAF mutations were not detected in the participant cohort; however, all LBC WT BRAF results correlated with the results from the FFPE clot samples. Access to rapid molecular testing using LBC samples can detect the most frequent driver mutations closer to the time of diagnosis, enabling the selection of the most effective first-line targeted therapy sooner, reducing delays or side effects from suboptimal treatments, patient anxiety and costs to healthcare systems, whilst improving patient outcomes.

Abstract P1-07-01: Real time detection of ESR1 mutation in blood by droplet digital PCR in the PADA-1 trial: Feasibility and cross-validation with NGS

Abstract Background: Mutations of ESR1 (ESR1mut), the gene encoding for Estrogen Receptor (ER) alpha, have been functionally characterized as driving resistance to aromatase inhibitors (AI) given to in ER+ HER2- metastatic breast cancer (mBC). In the randomized multicenter phase 3 PADA-1 trial (NCT03079011), ER+ HER2- mBC patients treated with first line AI+Palbociclib were screened every two months for ESR1mut in blood (bESR1mut), by subjecting cell-free circulating DNA (cfcDNA) to a droplet digital PCR (ddPCR) assay. Upon the onset of a rising (i.e., increasing or appearing) bESR1mut, patients with no concomitant disease progression were randomized between keeping the same regimen (AI-Palbociclib) or switching to Fulvestrant-Palbociclib. We report herein the sample flow of this first-in-its-class trial, from April 2017 to March 2021, and the cross-validation of ddPCR results with NGS. Methods: At each time point, 20 mL of blood were drawn into BCT Streck® tubes. cfcDNA was extracted from 4 mL of plasma. bESR1mut testing was centralized in 2 platforms using the same ddPCR assay, which combines a drop-off ddPCR, targeting the clustered hotspot L536, Y537 and D538 mutations found in exon 8, with an unconventional ddPCR interrogating specifically the E380Q mutation, located in exon 5 (Jeannot et al, Oncogene 2020). Results were expressed as the number of ESR1mut copies detected per mL of plasma together with the Mutant Allele Frequency (MAF). As a control, we submitted 200 ESR1mut+ (by ddPCR) left-over samples to an amplicon-based Next Generation Sequencing (NGS) assay covering all ESR1 exons - with a nominal sensitivity of &amp;gt;0.5% MAF. Results: From 03/2017 to 03/2021, 1,017 MBC patients have been included in 83 centers and 12,552 blood samples have been collected. The median time of delivery to central platforms was 1 day (range: [0-11]). A median volume of 9.5 mL [2-20] of plasma was obtained after 2 centrifugations. bESR1mut results were notified to investigators with a median turnaround time of 13 days [1-813] (32 days in 2017, 9 days in 2019, 8 days in 2021). Among the 12,525 available ddPCR results: N=77 (&amp;lt;1%) were considered as non-contributive (failed); N=11,533 (92%) samples were bESR1mut-; 267 (2%) samples allowed to find a rising bESR1mut (first occurrence of bESR1mut in a given patient under AI-Pal, triggering randomization in the absence of concomitant progression), while 648 (6%) other bESR1mut+ samples were not labeled as rising (e.g. repeated samplings in randomized patients, etc.). Samples with rising bESR1mut had a median MAF of 0.83% [0.11-35] and a median number of 14.5 ESR1mut copies/mL [4-1225]. The 200 bESR1mut+ samples by ddPCR submitted to NGS had a median MAF of 1.39% [0.1-46.6] and a median number of 24 ESR1mut copies/mL [4-6,493]. 168 samples (84%) were confirmed by NGS as displaying an ESR1mut in exon 5 and/or 8, of which 50 samples harbored polyclonal mutations. MAFs retrieved by NGS and ddPCR showed an excellent intraclass correlation coefficient (ICC=0.93; 95%CI[0.85;0.97]). Lack of ESR1mut detection by NGS in 32 samples could be primarily attributed to low MAF (MAF&amp;lt;0.5% by ddPCR, N=14/32; MAF&amp;lt;1%, N=29/32) and/or low coverage (&amp;lt;1,000x) (N=11/32). Conclusion: PADA-1 demonstrated that large scale, real time ESR1mut screening is feasible. The ddPCR assay yielded to valid results in a short turnaround time and at a limited cost. The primary objective of PADA-1, which tested the actionability of bESR1mut, will be reported in another presentation. Funding: Pfizer, INCA PRT-K (Grant nb PRT-K19-110) Citation Format: Céline Callens, François-Clément Bidard, Anais Curto-Taribo, Olfa Trabelsi-Grati, Samia Melaabi, Suzette Delaloge, Anne-Claire Hardy-Bessard, Thomas Bachelot, Florian Clatot, Thibault De La Motte Rouge, Jean-Luc Canon, Laurent Arnould, Fabrice André, Sandrine Marques, Marc-Henri Stern, Jean-Yves Pierga, Anne-Vincent Salomon, Emmanuelle Jeannot, Frederique Berger, Ivan Bieche, Anne Pradines. Real time detection of ESR1 mutation in blood by droplet digital PCR in the PADA-1 trial: Feasibility and cross-validation with NGS [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-01.

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics.

Simple SummaryThe mutational load of BRAFV600E in melanomas has been described as a possible prognostic biomarker but there is no information about the mutant allele frequency (MAF) variability of BRAFV600E within cutaneous melanomas and its potential prognostic implications. Our study suggests that the variation degree of BRAFV600E MAF within primary cutaneous melanoma could act as a determinant in the location of primary melanomas as well as their first metastases and could influence prognostic indicators such as Breslow and mitotic indexes. BRAFV600E MAF variation is also related to the neoplastic cell phenotype and tumor lymphocytic infiltrate of primary tumors. For all these reasons, detection of BRAFV600E MAF variation could be a useful prognostic biomarker. It is worth exploring the role of BRAFV600E MAF variation with regards to the response of melanoma to targeted therapies.Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

Circulating tumor DNA (ctDNA) as a tool to help guide germline testing in patients with solid malignancies.

10602 Background: As the use of circulating tumor DNA (ctDNA) is more widely implemented, incidental identification of pathogenic variants reflecting germline alterations in cancer predisposition genes will occur more frequently. Such mutations are expected to have a high mutant allele frequency (MAF) or occur in genes typically associated with inherited syndromes. When a similar analysis was conducted by our group, we found that MAF of about 30% or greater in BRCA1/ 2 was associated with confirmed putative germline mutations in patients with breast cancer (Jacob et al &amp; Davis et al, SABCS 2020). In this study, we extended this analysis to non-breast malignancies. Methods: Patients with non-breast solid malignancies and ctDNA testing between 2015-2020 were retrospectively identified from Northwestern Medicine. All ctDNA was analyzed using Guardant 360 (Guardant Health, Inc. Redwood City, Ca). Patients with ctDNA samples with mutations at high MAF (&gt;30%) and those with BRCA1/2 mutations at any MAF were identified. We reviewed these charts for referral to genetic counselors and/or CLIA-approved germline testing. Descriptive analysis was reported for these findings. Genetic alterations were classified as pathogenic or of unknown significance based on OncoKB (Chakravarty et al, JCO PO 2017). Results: We identified ctDNA samples of 548 patients with non-breast solid malignancies, of whom 56 had gene mutations occurring at high MAF (&gt;30%). Predominant cancer subtypes were lung (48%), colorectal (21%), pancreatic (7%), ovarian (3.5%), prostate (3.5%), and gastroesophageal (3.5%). The most common gene mutations identified were TP53 (46%), BRCA1/2 (18%), EGFR (18%), APC (13%), and KRAS (9%). 87.5% were pathogenic and 12.5% were of unknown significance. 11 patients (19.6%) had germline testing of whom 6 tested positive. These germline mutations were in BRCA2 (n = 3), EGFR, APC, and TP53. In addition to the 10 patients with BRCA1/2 mutations at high MAF (&gt;30%), we identified 70 patients with BRCA1/2 mutations at low MAF ( &lt; 30%). 54% were pathogenic and 46% were of unknown significance. 11 patients (14%) had germline testing of whom 3 tested positive for BRCA2, all at high MAF. 1 patient with a BRCA2 mutation at low MAF of 1.4% tested positive for a different germline BRCA2 variant. Conclusions: In patients with advanced cancers, ctDNA analysis can reveal variants with MAF &gt;30% that are reflective of a germline mutation. Unfortunately the rate of genetic testing in these patients was low (20%). Future studies with germline testing in patients with high MAF variants would help understand the prevalence of germline variants. This can facilitate developing a more standardized approach for genetic counselor referral to identify families that may benefit from interventions for early detection or prevention of future cancers.

Advances in Detecting Low Prevalence Somatic TERT Promoter Mutations in Papillary Thyroid Carcinoma.

BackgroundTwo recurrent TERT (telomerase reverse transcriptase) promoter mutations, C228T and C250T, have been reported in thyroid carcinomas and were correlated with high-risk clinicopathological features and a worse prognosis. Although far more frequent in the poorly differentiated and undifferentiated thyroid cancer, the TERT promoter mutations play a significant role on PTC recurrence and disease-specific mortality. However, the prevalence varies considerably through studies and it is uncertain if these differences are due to population variation or the methodology used to detect TERT mutations. In this study we aim to compare three different strategies to detect TERT promoter mutations in PTC.MethodsDNA was isolated from formalin-fixed paraffin-embedded (FFPE) specimens from 89 PTC and 40 paired lymph node metastases. The prevalence of the hot spot TERT C228T and C250T mutations was assessed in FFPE samples using TaqMan SNP genotyping assays. Random samples were tested by Sanger Sequencing and droplet digital PCR (ddPCR).ResultsIn general, 16 out of 89 (18%) PTC samples and 14 out of 40 (35%) lymph node metastases harbored TERT promoter mutations by TaqMan assay. Sanger sequencing, performed in random selected samples, failed to detect TERT mutations in four samples that were positive by TaqMan SNP genotyping assay. Remarkably, ddPCR assay allowed detection of TERT promoter mutations in six samples that harbor very low mutant allele frequency (≤ 2%) and were negative by both genotype assay and Sanger Sequencing.ConclusionThis study observed a good concordance among the methodologies used to detect TERT promoter mutations when a high percentage of mutated alleles was present. Sanger analysis demonstrated a limit of detection for mutated alleles. Therefore, the prevalence of TERT promoter mutations in PTC may be higher than previously reported, since most studies have conventionally used Sanger sequencing. The efficient characterization of genetic alterations that are used as preoperative or postoperative diagnostic, risk stratification of the patient and individualized treatment decisions, mainly in highly heterogeneous tumors, require highly sensitive and specific approaches.

Expanded Low Allele Frequency RAS and BRAF V600E Testing in Metastatic Colorectal Cancer as Predictive Biomarkers for Cetuximab in the Randomized CO.17 Trial

Expanded RAS/BRAF mutations have not been assessed as predictive for single-agent cetuximab in metastatic colorectal cancer (mCRC), and low mutant allele frequency (MAF) mutations are of unclear significance. We aimed to establish cetuximab efficacy in optimally selected patients using highly sensitive beads, emulsion, amplification, and magnetics (BEAMing) analysis, capable of detecting alterations below standard clinical assays. CO.17 trial compared cetuximab versus best supportive care (BSC) in RAS/BRAF-unselected mCRC. We performed RAS/BRAF analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for KRAS/NRAS (codons 12/13/59/61/117/146) and BRAF V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included. KRAS, NRAS, and BRAF mutations were present in 53%, 4%, and 3% of tumors, respectively. Cetuximab improved overall survival [OS; HR, 0.51; 95% confidence interval (CI), 0.32-0.81; P = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; P < 0.0001) compared with BSC in RAS/BRAF wild-type patients. Cetuximab did not improve OS/PFS for KRAS-, NRAS-, or BRAF-mutated tumors, and tests of interaction confirmed expanded KRAS (P = 0.0002) and NRAS (P = 0.006) as predictive, while BRAF mutations were not (P = 0.089). BEAMing identified 14% more tumors as RAS mutant than Sanger sequencing, and cetuximab lacked activity in these patients. Mutations at MAF < 5% were noted in 6 of 242 patients (2%). One patient with a KRAS A59T mutation (MAF = 2%) responded to cetuximab. More NRAS than KRAS mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; P = 0.0038). We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal RAS/BRAF alterations are uncommon and remain of indeterminate significance.

QOL-53. GENOME ASSOCIATIONS WITH NEUROCOGNITIVE OUTCOMES, CEREBRAL MICROBLEEDS (CMBS), AND BRAIN VOLUME AND WHITE MATTER (WM) CHANGES IN PEDIATRIC BRAIN TUMOR SURVIVORS

OBJECTIVETo identify genetic predictors of neurocognition, CMBs, brain volume, and WM changes in pediatric brain tumor survivors.METHODSPatients were selected from an existing cohort (RadART) if they had: 1) at least one neurocognitive evaluation using computer-based CogState; 2) available DNA; 3) standard imaging. Candidate gene or genome-wide genotyping was performed on all patients. CMBs were identified using a semi-automated algorithm developed in MATLAB. Volume of T2/FLAIR WM signal abnormality was measured using a semi-automated method based on a convolutional neural network. Brain volume and cortical thickness were measured using FreeSurfer volumetric analysis. Logistic and linear regression were done to compare phenotypes with candidate genotypes. Genome-wide efficient mixed-model analysis was done to compare neurocognition and CMBs. Gene set analysis was done using https://fuma.ctglab.nl/.RESULTSAPOE4 was a candidate variant associated with non-lobar, larger volume CMBs (p<0.05). At the GWAS-level (n=225), specific genes trended with visual memory, psychomotor function, and CMB count (p<5x10-8). Using gene set analyses, there were gene set trends seen with CMB count and psychomotor function. Small sample size and low mutant allele frequency limited reliability of these findings. Preliminary volumetric analysis show reduced volume within the right parietal, medial occipital and inferior temporal lobes with increased cortical thickness in the left occipital and medial parietal lobe in patients carrying the ApoE4 allele. WM signal assessments are ongoing.CONCLUSIONGenetic markers may be associated with neurocognition, CMBs, brain volume and WM changes in pediatric brain tumor survivors; however, larger cohorts are needed to confirm specific gene relevance.

NCOG-71. GENOME ASSOCIATIONS WITH NEUROCOGNITIVE OUTCOMES, CEREBRAL MICROBLEEDS (CMBS), AND BRAIN VOLUME AND WHITE MATTER (WM) CHANGES IN PEDIATRIC BRAIN TUMOR SURVIVORS

Abstract OBJECTIVE To identify genetic predictors of neurocognition, CMBs, brain volume, and WM changes in pediatric brain tumor survivors. METHODS Patients were selected from an existing cohort (RadART) if they had: 1) at least one neurocognitive evaluation using computer-based CogState; 2) available DNA; 3) standard imaging. Candidate gene or genome-wide genotyping was performed on all patients. CMBs were identified using a semi-automated algorithm developed in MATLAB. Volume of T2/FLAIR WM signal abnormality was measured using a semi-automated method based on a convolutional neural network. Brain volume and cortical thickness were measured using FreeSurfer volumetric analysis. Logistic and linear regression were done to compare phenotypes with candidate genotypes. Genome-wide efficient mixed-model analysis was done to compare neurocognition and CMBs. Gene set analysis was done using https://fuma.ctglab.nl/. RESULTS APOE4 was a candidate variant associated with non-lobar, larger volume CMBs (p&amp;lt; 0.05). At the GWAS-level (n=225), specific genes trended with visual memory, psychomotor function, and CMB count (p&amp;lt; 5x10-8). Using gene set analyses, there were gene set trends seen with CMB count and psychomotor function. Small sample size and low mutant allele frequency limited reliability of these findings. Preliminary volumetric analysis show reduced volume within the right parietal, medial occipital and inferior temporal lobes with increased cortical thickness in the left occipital and medial parietal lobe in patients carrying the ApoE4 allele. WM signal assessments are ongoing. CONCLUSION Genetic markers may be associated with neurocognition, CMBs, brain volume and WM changes in pediatric brain tumor survivors; however, larger cohorts are needed to confirm specific gene relevance.

Fragment Enrichment of Circulating Tumor DNA With Low-Frequency Mutations.

Human blood contains cell-free DNA (cfDNA), with circulating tumor-derived DNAs (ctDNAs) widely used in cancer diagnosis and treatment. However, it is still difficult to efficiently and accurately identify and distinguish specific ctDNAs from normal cfDNA in cancer patient blood samples. In this study, ctDNA fragment length distribution analysis showed that ctDNA fragments are frequently shorter than the normal cfDNAs, which is consistent with previous findings. Interestingly, the ctDNA fragment length was found to be partially associated with the mutant allele frequency, with a low mutant allele frequency (< ~0.6%) associated with a longer ctDNA fragment length when compared to normal cfDNAs. The findings of this study contribute to improving the detection of low-frequency tumor mutations.

SinoDuplex: An Improved Duplex Sequencing Approach to Detect Low-frequency Variants in Plasma cfDNA Samples

Accurate detection of low frequency mutations from plasma cell-free DNA in blood using targeted next generation sequencing technology has shown promising benefits in clinical settings. Duplex sequencing technology is the most commonly used approach in liquid biopsies. Unique molecular identifiers are attached to each double-stranded DNA template, followed by production of low-error consensus sequences to detect low frequency variants. However, high sequencing costs have hindered application of this approach in clinical practice. Here, we have developed an improved duplex sequencing approach called SinoDuplex, which utilizes a pool of adapters containing pre-defined barcode sequences to generate far fewer barcode combinations than with random sequences, and implemented a novel computational analysis algorithm to generate duplex consensus sequences more precisely. SinoDuplex increased the output of duplex sequencing technology, making it more cost-effective. We evaluated our approach using reference standard samples and cell-free DNA samples from lung cancer patients. Our results showed that SinoDuplex has high sensitivity and specificity in detecting very low allele frequency mutations. The source code for SinoDuplex is freely available at https://github.com/SinOncology/sinoduplex.

Evaluation of Commercial Circulating Tumor DNA Test in Metastatic Prostate Cancer.

PURPOSECirculating tumor DNA (ctDNA) sequencing provides a minimally invasive method for tumor molecular stratification. Commercial ctDNA sequencing is increasingly used in the clinic, but its accuracy in metastatic prostate cancer is untested. We compared the commercial Guardant360 ctDNA test against an academic sequencing approach for profiling metastatic prostate cancer.PATIENTS AND METHODSPlasma cell-free DNA was collected between September 2016 and April 2018 from 24 patients with clinically progressive metastatic prostate cancer representing a range of clinical scenarios. Each sample was analyzed using Guardant360 and a research panel encompassing 73 prostate cancer genes. Concordance of somatic mutation and copy number calls was evaluated between the two approaches.RESULTSTargeted sequencing independently confirmed 94% of somatic mutations identified by Guardant360 at an allele fraction greater than 1%. AR amplifications and mutations were detected with high concordance in 14 patients, with only three discordant subclonal mutations at an allele fraction lower than 0.5%. Many somatic mutations identified by Guardant360 at an allele fraction lower than 1% seemed to represent subclonal passenger events or non–prostate-derived clones. Most of the non-AR gene amplifications reported by Guardant360 represented single copy gains. The research approach detected several clinically relevant DNA repair gene alterations not reported by Guardant360, including four germline truncating BRCA2/ATM mutations, two somatic ATM stop gain mutations, one BRCA2 biallelic deletion, 11 BRCA2 stop gain reversal mutations in a patient treated with olaparib, and a hypermutator phenotype in a patient sample with 42 mutations per megabase.CONCLUSIONGuardant360 accurately identifies somatic ctDNA mutations in patients with metastatic prostate cancer, but low allele frequency mutations should be interpreted with caution. Test utility in metastatic prostate cancer is currently limited by the lack of reporting on actionable deletions, rearrangements, and germline mutations.

Whole-Organ Genomic Characterization of Mucosal Field Effects Initiating Bladder Carcinogenesis

We used whole-organ mapping to study the locoregional molecular changes in a human bladder containing multifocal cancer. Widespread DNA methylation changes were identified in the entire mucosa, representing the initial field effect. The field effect was associated with subclonal low-allele frequency mutations and a small number of DNA copy alterations. A founder mutation in the RNA splicing gene, ACIN1, was identified in normal mucosa and expanded clonally with an additional 21 mutations in progression to carcinoma. The patterns of mutations and copy number changes in carcinoma in situ and foci of carcinoma were almost identical, confirming their clonal origins. The pathways affected by the DNA copy alterations and mutations, including the Kras pathway, were preceded by the field changes in DNA methylation, suggesting that they reinforced mechanisms that had already been initiated by methylation. The results demonstrate that DNA methylation can serve as the initiator of bladder carcinogenesis.

Molecular and Computational Methods for the Detection of Microsatellite Instability in Cancer.

Microsatellite instability (MSI) is a genomic alteration in which microsatellites, usually of one to four nucleotide repeats, accumulate mutations corresponding to deletions/insertions of a few nucleotides. The MSI phenotype has been extensively characterized in colorectal cancer and is due to a deficiency of the DNA mismatch repair system. MSI has recently been shown to be present in most types of cancer with variable frequencies (from <1 to 30%). It correlates positively to survival outcome and predicts the response to immune checkpoint blockade therapy. The different methods developed for MSI detection in cancer require taking into consideration two critical parameters which influence method performance. First, the microsatellite markers used should be chosen carefully to ensure they are highly sensitive and specific for MSI detection. Second, the analytical method used should be highly resolute to allow clear identification of MSI and of the mutant allele genotype, and should present the lowest limit of detection possible for application in samples with low mutant allele frequency. In this review, we describe all the different molecular and computational methods developed to date for the detection of MSI in cancer, how they have evolved and improved over the years, and their advantages and drawbacks.

Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy.

BACKGROUND: Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS: To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency. RESULTS: We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM. CONCLUSION: Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies.