Detection Of Genomic Alterations Research Articles

Liquid profiling for patients with advanced cancer is ready for clinical integration

SummaryMolecular profiling of circulating tumor DNA (ctDNA) to guide treatment decisions has found its way into routine management of patients with advanced cancer. This represents a pivotal advancement in precision oncology, offering a non-invasive and fast-tracked method to detecting clinically relevant biomarkers. With the backing of international oncology guidelines, ctDNA analysis is now a standard approach to consider in molecular diagnostics. Despite the promise of ctDNA in refining treatment strategies through the detection of genomic alterations and treatment-relevant biomarkers with high concordance to tissue biopsies, challenges persist. These include the interpretation of discordances due to tumor heterogeneity, sampling biases, and technical limitations, alongside the differentiation of tumor-derived mutations from clonal hematopoiesis. The current consensus supports the utility of comprehensive genomic profiling (CGP) panels for a broad spectrum of actionable targets, while acknowledging the limitations and advocating for a balanced application of “tissue-first” and “plasma-first” approaches tailored to individual patient scenarios. The essential role of molecular tumor boards (MTBs) is in navigating the complexities of ctDNA data interpretation, thereby ensuring the effective incorporation of liquid biopsy into personalized cancer treatment regimens.

Comprehensive Genomic Studies on the Cell Blocks of Pancreatic Cancer.

Pancreatic cancer is one of the deadliest malignancies, characterized by late-stage diagnosis and limited treatment options. Comprehensive genomic profiling plays an important role in understanding the molecular mechanisms underlying the disease and identifying potential therapeutic targets. Cell blocks (CBs), derived from EUS-FNA, have become valuable resources for diagnosis and genomic analysis. We examine the molecular profile of pancreatic ductal adenocarcinoma (PDAC) using specimens obtained from CB EUS-FNA, across a large gene panel, within the framework of next-generation sequencing (NGS). Our findings revealed that over half (55%) of PDAC CB cases provided adequate nucleic acid for next-generation sequencing, with tumor cell percentages averaging above 30%. Despite challenges such as low DNA quantification and degraded DNA, sequencing reads showed satisfactory quality control statistics, demonstrating the detection of genomic alterations. Most cases (84.6%) harbored at least one gene variant, including clinically significant gene mutation variants such as KRAS, TP53, and CDKN2A. Even at minimal concentrations, as long as the extracted DNA is of high quality, performing comprehensive molecular profiling on PDAC samples from cell blocks has remained feasible. This strategy has yielded valuable information about the diagnosis, genetic landscape, and potential therapeutic targets, aligning closely with a precision cytopathology approach.

Abstract 2291: Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation

Abstract Background: Current genomic and epigenomic profiling of cancer tissue DNA or cfDNA (cell-free DNA) in liquid biopsy relies upon separate, time- and sample-consuming technologies for somatic variant detection or methylation analysis. Here we describe workflow and performance of the Agilent Bravo automated liquid handling platform with the Agilent Avida targeted enrichment solution for next generation sequencing of somatic variants and methylation profiling. This solution can effectively analyze low-input tumor DNA or cfDNA samples. The Avida Duo workflow enables highly sensitive detection of single nucleotide variant (SNV), insertions and deletions (INDEL), copy number variation (CNV) and DNA methylation profiles from a single sample, without any sample splitting. Methods and Results: Panels, reagents, and automated workflows for Avida DNA, Avida Methyl, and Avida Duo Methyl (combined DNA & methylation) kits were developed to accommodate up to 96 samples on the Bravo NGS workstation. The automated solution supports independent single-day workflows for somatic variants or methylation sample preparation. The Avida Duo analysis combines both workflows without sample splitting, streamlining the process and reducing sample consumption. Leveraging a focused cancer hotspot panel, we demonstrate excellent reproducibility and low allele frequency (SNV at sub 1%) variant detection in cfDNA samples and reference standards with as little as 10ng DNA input. We exhibit similar performance with a larger ~300 kb target region tumor profiling panel. Finally, we demonstrate DNA methylation detection in the standalone or combined (“Duo”) workflows across differentially methylated regions (DMRs) with a panel of ~3400 targets. Conclusions: Automation of the Avida targeted enrichment solution with the Bravo NGS workstation enables a sensitive, high-throughput, end-to-end analysis for detection of genomic alterations and DNA methylation changes from a single low-input cfDNA or tumor DNA sample. Citation Format: Ashraf Wahba, Tony Ho, Sarah Johns, Aswati Aravind, Heng Wang, Neelima V. Mehendale, Gilbert P. Amparo, Khine Win, Manuel Gomez, Grace Zhao, Douglas N. Roberts. Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation [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 2291.

Fragmentomics of cell-free DNA from targeted panels in genitourinary malignancies.

199 Background: The detection of genomic alterations in cancer is critical for identifying clinically actionable alterations for treatment decisions. Tumor samples historically have been required, but obtaining tissue for molecular profiling is not always feasible, especially in the metastatic setting. The isolation and analysis of cell-free DNA (cfDNA) including circulating tumor DNA (ctDNA) via blood-based “liquid” biopsies offers non-invasive sampling of the tumor. Recently, fragmentation patterns of cfDNA (i.e. “fragmentomics”) have emerged as a method for inferring epigenomic and transcriptomic information. However, these analyses use whole-genome sequencing, which lacks the necessary depth to cost-effectively assess genomic alterations, limiting the application of these techniques clinically. Methods: We developed a novel cfDNA fragmentomics machine learning approach for standard targeted cancer gene panels in order to identify genitourinary (GU) and non-GU cancers from two independent metastatic cancer cohorts: a published cohort from GRAIL (prostate cancer, non-GU cancers, normal samples, N=198) and an expanded institutional cohort from the University of Wisconsin (prostate adenocarcinoma, neuroendocrine prostate cancer (NEPC), renal cell carcinoma (RCC), bladder cancer, non-GU cancers, normal samples, n = 431). Results: In the GRAIL cohort, 10-fold cross-validation AUCs were 0.987 for identifying prostate cancer, 1.00 for normal samples, and ranged from 0.922-0.958 for the non-GU cancers. In the UW cohort, 10-fold cross-validation AUCs were 0.950 for bladder cancer, 0.982 for prostate cancer, 0.993 for NEPC, 0.925 for RCC, and 0.980 for normal samples, and ranged from 0.874-0.954 for non-GU cancers. Our assay can sensitively detect and accurately distinguish GU and non-GU malignancies despite a median ctDNA fraction of only 0.076 in the GRAIL cohort, and a median ctDNA fraction of only 0.022 in the UW cohort. Conclusions: Our enhanced machine learning approach for examining fragmentomics in standard cancer gene cfDNA panels unlocks the potential for these established assays to be utilized to answer critical clinical questions beyond somatic variant identification. The excellent performance of our innovative approach even in samples with low ctDNA fractions suggest potential applications such as multi-cancer early detection and minimal residual disease monitoring. This framework could dramatically expand the potential of already existing clinically used assays and minimizes barriers for continued biomarker development.

Retrieving single-cells and cell clusters from cytology direct smear slides for genomic evaluation

Abstract Introduction/Objective The key role of cytopathologists is to generate diagnoses based on cytologically stained samples. However, diagnoses increasingly need genomic data in addition to morphologic information. By current methods, morphologically diagnostic samples may be inadequate to perform comprehensive ancillary testing. Therefore, there is a great need for a workflow that can reliably identify and retrieve target cells based on their morphological features. This workflow should also enable highly sensitive molecular profiling from individual cells or small clusters of cells, allowing accurate detection of genomic alterations like copy number variations (CNVs) and single nucleotide variations (SNVs). Methods/Case Report Previously, we established a workflow that combines the RareCyte (Seattle, WA) cell-retrieval platform with primary template-directed amplification (PTA) to generate whole genome libraries from Propidium iodide (PI) stained effusion tumor single-cells or cell clusters and detected copy number variation. In this study, we modified and expanded the assay to identify and retrieve malignant or suspicious cells from Diff-Quik direct smear slides from patient pleural effusion specimens. The samples were further subjected to PTA amplification and library preparation for future copy number variation (CNV) evaluation. In parallel, patient cells from PI-stained smear slides were also obtained and processed to serve as controls. Results (if a Case Study enter NA) Direct smear slides were made from patient pleural fluid (N=5). In contrast to the previous PI staining, which only highlighted nuclei under fluorescence microscopy, Diff-Quik staining allowed nuclear and cytoplasmic morphology evaluation to identify target cells via light microscope. The XYZ coordinates of each target cell or cluster were further documented in the RareCyte Imager software to facilitate cell retrieval. We have successfully collected 39 single-cell and 18 bulk-cell samples from 5 different cases. PTA amplification has been completed and DNA yields of amplified libraries are promising. Conclusion We demonstrated that our modified cell retrieval platform can reliably obtain both single-cell or cluster samples based on morphology from cytologic smear slides made from patient effusion specimens. These collected samples were shown to be compatible with downstream amplification and library preparation enabling CNV evaluation and future deep sequencing for diagnostic, prognostic, and predictive pathogenic mutation identification.

PACT: a pipeline for analysis of circulating tumor DNA.

Detection of genomic alterations in circulating tumor DNA (ctDNA) is currently used for active clinical monitoring of cancer progression and treatment response. While methods for analysis of small mutations are more developed, strategies for detecting structural variants (SVs) in ctDNA are limited. Additionally, reproducibly calling small-scale mutations, copy number alterations, and SVs in ctDNA is challenging due to the lack to unified tools for these different classes of variants. We developed a unified pipeline for the analysis of ctDNA [Pipeline for the Analysis of ctDNA (PACT)] that accurately detects SVs and consistently outperformed similar tools when applied to simulated, cell line, and clinical data. We provide PACT in the form of a Common Workflow Language pipeline which can be run by popular workflow management systems in high-performance computing environments. PACT is freely available at https://github.com/ChrisMaherLab/PACT.

Single‐tube two‐pronged approach using both cell‐free DNA and RNA for multimodal biomarker tests at the time of biopsy

AbstractThe emergence of noninvasive liquid biopsy procedures as an alternative to surgical biopsies has fueled an intensive research effort and investment into the detection of cell‐free disease biomarkers. By pairing next‐generation sequencing (NGS) and RT‐qPCR technologies on cell‐free DNA (cfDNA) and RNA (cfRNA), respectively, we have established and validated a high‐throughput and quick‐turnaround workflow for highly sensitive detection of genomic alterations using a single tube of blood. This emphasizes the importance of our cfDNA/cfRNA recovery and enrichment knowhow to maximize the chance to capture relevant rare biomarkers. On the cfDNA‐NGS axis, sensitivity and specificity of 95% and 98% were recorded for somatic variants of ≥1% VAF, with high concordance observed between the in‐house and orthogonal assays. Overall accuracy for the cfRNA and RT‐qPCR portion of the workflow was >95%. Real‐world data of patients underwent the two‐pronged testing scheme revealed dramatic beneficial outcomes guided by genomic‐matched treatments. When implemented at the time of biopsy to expedite treatment decision‐making in the earliest possible way, the single‐tube two‐pronged approach can effectively reduce time to treatment initiation by about 70%. The cfDNA–cfRNA paired test is suitable for implementation in routine clinical decision‐making to maximize the benefit without delay.

Analysis of Plasma Circulating Tumor DNA in Borderline Resectable Pancreatic Cancer Treated with Neoadjuvant Modified FOLFIRINOX: Clinical Relevance of DNA Damage Repair Gene Alteration Detection.

There are no reliable biomarkers to guide treatment for patients with borderline resectable pancreatic cancer (BRPC) in the neoadjuvant setting. We used plasma circulating tumor DNA (ctDNA) sequencing to search biomarkers for patients with BRPC receiving neoadjuvant mFOLFIRINOX in our phase 2 clinical trial (NCT02749136). Among the 44 patients enrolled in the trial, patients with plasma ctDNA sequencing at baseline or post-operation were included in this analysis. Plasma cell-free DNA isolation and sequencing were performed using the Guardant 360 assay. Detection of genomic alterations, including DNA damage repair (DDR) genes, were examined for correlations with survival. Among the 44 patients, 28 patients had ctDNA sequencing data qualified for the analysis and were included in this study. Among the 25 patients with baseline plasma ctDNA data, 10 patients (40%) had alterations of DDR genes detected at baseline, inclu-ding ATM, BRCA1, BRCA2 and MLH1, and showed significantly better progression-free survival than those without such DDR gene alterations detected (median, 26.6 vs. 13.5 months; log-rank p=0.004). Patients with somatic KRAS mutations detected at baseline (n=6) had significantly worse overall survival (median, 8.5 months vs. not applicable; log-rank p=0.003) than those without. Among 13 patients with post-operative plasma ctDNA data, eight patients (61.5%) had detectable somatic alterations. Detection of DDR gene mutations from plasma ctDNA at baseline was associated with better survival outcomes of pati-ents with borderline resectable pancreatic ductal adenocarcinoma treated with neoadjuvant mFOLFIRINOX and may be a prognostic biomarker.

Practical Applications of Chromosomal Microarray in Prenatal Diagnosis

AbstractG-banded karyotyping is the most common approach for the detection of genomic alterations. However, this is unable to detect genomic changes of less than 5 Mb. The ability of fluorescence in situ hybridization (FISH) to detect cryptic chromosomal rearrangements exceeds the resolution of routine karyotype. However, conventional FISH is for targeted regions only, whereas the chromosomal microarray is a whole-genome copy number evaluation technique with a resolution of 10 to 20 kb. In this article, we discuss the application of chromosomal microarray 750 K to 384 consecutive prenatal diagnosis cases. Overall diagnostic yield is 15.36%, and chromosomal microarray accounts for a 3.6% additional detection rate. We suggest applying this technique in routine prenatal diagnosis as a first-tier test in prenatal diagnosis along with a backup culture in all cases.

Tumor Fraction Correlates With Detection of Actionable Variants Across > 23,000 Circulating Tumor DNA Samples.

Profiling of circulating tumor DNA (ctDNA) is increasingly adopted in the management of solid tumors, concurrent with increased availability of more comprehensive ctDNA panels. However, variable ctDNA shed can result in variable assay sensitivity. We studied the relationship between ctDNA tumor fraction (TF) and detection of actionable alterations across cancer types. A total of 23,482 liquid biopsies (LBx) submitted between September 2020 and October 2021 were sequenced using a hybrid capture panel that reports genomic alterations (GAs) and genomic biomarkers across 324 cancer-related genes. The primary end points were the prevalence of targetable GAs by cancer type and detection in relationship to ctDNA TF. Sensitivity of detection in LBx was assessed in 1,289 patients with available tissue results. 94% (n = 22,130) of LBx had detectable ctDNA, with a median TF of 2.2%. LBx profiling detected GAs in National Comprehensive Cancer Network category 1 genes in 37% of lung, 30% of prostate, 36% of breast, and 51% of colon cancer cases. Potential germline GAs flagged on clinical reports were detected in genes including BRCA1/2, PALB2, CHEK2, and ATM. Polyclonal mutations in genes associated with resistance such as AR, ESR1, RB1, and NF1 were detected. The sensitivity of LBx to detect driver alterations identified in tissue biopsy from the same patient ranged from 58% to 86% but was consistently at or near 100% in cases with TF ≥ 10%. Elevated ctDNA shed is associated with both high sensitivity and negative predictive value for detection of actionable GAs. The presence of elevated TF suggests adequate tumor profiling and may reduce the value of subsequent reflex to confirmatory tissue testing in patients with negative LBx results.

SY22-4 Current and future paradigms of liquid biopsy for cancer care

Liquid biopsy, especially circulating tumor DNA (ctDNA) has demonstrated its capability in the detection of genomic alterations. Our large-scale study comparing ctDNA-genotyping versus tissue-genotyping studies for trial enrollment demonstrated that ctDNA genotyping has the advantage of shorter turnaround times and improved patient enrollment compared to tissue biopsy for clinical trials, without compromising treatment efficacy. Based on the utility of ctDNA genotyping, we are conducting an umbrella, basket project targeting rare fractions of patients with advanced solid malignancies.

Changes in circulating tumor DNA (ctDNA) and outcomes in solid tumors treated with immune checkpoint inhibitors (ICIs).

2544 Background: Quantification of ctDNA levels can be a reliable prognostic tool in several malignancies. More recently, detection of genomic alterations in ctDNA have been validated as a predictive biomarker to guide treatment planning. Dynamic changes in ctDNA levels over time and in response to treatment may also provide prognostic information. However, less is known about the value of changes to ctDNA levels in response to immune checkpoint inhibitors (ICIs). Methods: We searched MEDLINE (host:PubMed) and reviewed trials exploring outcomes of patients with advanced solid tumors receiving ICIs in which outcomes were reported based on changes to ctDNA levels. ctDNA clearance was defined as reported in individual trials. Typically, this was defined as either >50% reduction or a reduction to undetectable levels. We extracted progression free survival (PFS) and/or overall survival (OS) values, related 95% Confidence intervals (CI) and/or p-values. Data were then included in a meta-analysis utilizing the generic inverse variance and random effects model. Variation in effect size was examined using random effects meta-regression analysis. Results: A total of 17 trials were included in the meta-analysis; ctDNA levels were detectable in all participants in all studies prior to initiation of ICIs. Method of detection included next generation sequencing and/or droplet digital polymerase chain reaction assays. Overall, low to undetectable ctDNA levels, measured 6-16 weeks after starting treatment was associated with significantly better PFS, (HR 0.20 95% CI, 0.14-0.28; p<0.001). Similarly, OS was superior in patients with substantially reduced or undetectable ctDNA levels after receiving ICIs, (HR 0.18, 95% CI, 0.12-0.26; p<0.001. The results were consistent across all disease sites, lines of treatment, level of change (undetectable vs. >50% reduction) and whether treatment exposure comprised single or multiple ICIs (see Table). Conclusions: In unselected advanced solid tumors, a substantial fall in ctDNA levels in response to ICIs is associated with substantial improvements in both PFS and OS. ctDNA change is an early response biomarker which may allow for de-escalation of cross-sectional imaging in patients receiving ICIs, or support treatment de-escalation strategies. Further research is needed to quantify variations in sensitivity between the available NGS assays, as well as differences discovery range between assay platforms. [Table: see text]

Mutational landscape of low-grade serous carcinoma of the ovary.

5578 Background: Tumor genomic profiling is a critical component of precision oncology allowing the detection of genomic alterations (GA) that are potential therapeutic targets. We present an analysis of comprehensive genomic profiling (CGP) of a large series of low grade serous ovarian carcinoma patients assayed in a nationwide cancer network. Methods: 40 Pts with advanced low grade serous ovarian carcinoma (LGSOC) underwent hybrid-capture based CGP for up to 324 cancer-related genes on archival tumor tissue or 62 genes on circulating tumor DNA ordered during clinical care for treatment decision-making between 01-2013 thru 06-2021. Clinically relevant genomic alterations (CRGA) were defined as associated with targeted therapies or mechanism-driven clinical trials. The treatment histories for these patients were obtained with IRB-approved retrospective review. Results: Median age was 44 years (range, 25-65), 68% were White and 23% were Black. Genomic alterations (GA) were identified in 80% (32/40) of LGSOC, of which 22 (55%) had a clinically relevant genomic alteration (CRGA). There were no apparent racial differences between mutation frequencies. The table below details the frequency of the most common of these clinically relevant mutations. Additionally, 1 patient had an ESR1 activating mutation commonly seen in acquired hormone therapy resistance in breast cancer; and single cases of the following mutations were also noted: TSC1, PTCH1, PTEN, FGFR1, and AKT2. Conclusions: In a large series of Low Grade Serous Ovarian Cancer patients assayed with CGP, 55% of pts had mutations potentially targeted by precision medicine therapy. The most common target is KRAS, seen in 33% of patients, these are potentially treatable with FAK and MEK inhibitors. Another 10% of patients had either a BRCA or ATM mutations making PARP inhibitor therapy an additional targeted treatment option. While mutations in the RAS pathway are most common in LGSOC they are not the only actionable target. Furthermore, this approach might also help identify baseline or acquired resistance to the hormone therapies often used to treat this cancer. The results of this study demonstrate the significant heterogeneity of genomic alterations in this rare cancer and highlight the importance of genomic profiling in this cancer which has a marked propensity for recurrence. [Table: see text]

Early detection of cancer using cell-free DNA (cfDNA) size analysis on a multiplexed amplicon-based next-generation sequencing (NGS) platform.

3041 Background: Tumor-derived cfDNA fragments are observed to be shorter in length than normal cfDNA. This size (length) difference can be analyzed as a tumor-specific signal. Whole genome or probe hybridization-based NGS methods can capture cfDNA fragments of native sizes. However, amplicon-based NGS assays are not directly amenable to cfDNA size analysis due to predetermination of amplicon sizes by design. Here we present a method to extract relative distribution of cfDNA fragment lengths from a targeted amplicon-based assay and show its utility in cancer detection. Methods: The LiquidHALLMARK cfDNA assay is an amplicon-based NGS test for the sensitive detection of genomic alterations in 80 genes. Although panel design is optimized for cfDNA with average amplicon length of ̃150 bp, the consecutive tiling design of amplicons for genes with contiguously targeted regions, e.g. BRCA permits the formation of longer amplicons (> 150 bp) from physically subsequent primer pairs, provided longer template cfDNA molecules are present. Cancer samples (n = 281), clinically tested by LiquidHALLMARK during Sep 2020-Sep 2021, and healthy samples (n = 28) were included for analysis. For each sample, fragment lengths were inferred from sequencing alignment files, and binned into “short” (0-150 bp) and “long” (151-500 bp) groups. A relative “size ratio” of the total number of short vs. long fragments per sample was calculated, and examined with clinical features, plasma cfDNA concentration (cfDNA/ml) and highest mutation allele frequency (AF%), in a model to predict cancer. Results: Calculated size ratios (relative abundance of short fragments) were higher in cancer than normal samples (median 47.6 vs. 31.2, p < 0.001). Cholangiocarcinoma and colorectal cancer samples had the highest size ratios (medians: 62.9 and 60.9, respectively) in agreement with a genome-wide NGS study that profiled cfDNA sizes. Size ratios were higher in metastatic (n = 143) compared to early stage (n = 30) lung cancers (p = 0.0039), indicating a stage-dependent accumulation of shorter cfDNA fragments. Size ratio was correlated with cfDNA/ml ( r= 0.63, p < 0.01) and AF% ( r= 0.42, p < 0.01). In 20-fold cross-validation of a logistic regression model trained to predict cancer, average area under curve (AUC) was 0.82 using size ratio, 0.86 using cfDNA/ml, and increased to 0.95 with the two features combined. Conclusions: Our analysis shows that it is feasible to derive meaningful cfDNA fragment size information from amplicon-based NGS data. Importantly, relative fragment size distributions observed in cancer and healthy plasma samples by this method are concordant with alternate target capture methods. Fragment size ratios derived from relatively small, targeted amplicon panels are a novel feature that, combined with other molecular and clinical features, can enhance non-invasive methods of cancer detection.

Therapeutic implications of mutations in high grade serous ovarian carcinoma when evaluated by race.

e17590 Background: Individualizing oncologic treatment by using precision oncology techniques that allow for detection of genomic alterations (GA) has become critical. We present an analysis of comprehensive genomic profiling (CGP) of a large series of high grade serous ovarian carcinoma (HGSOC) patients assayed in a nationwide cancer network. Racial disparities in genomic alterations may assist in better targeting therapies, predicting drug resistance, and potential germline mutations. Methods: 312 patients with HGSOC underwent CGP with hybrid capture on tumor tissue for treatment decision-making between 01-2013 to 06-2021. Clinically relevant genomic alterations (CRGA) were reported as targetable marker. Statistical analysis performed with Fisher Exact test. Results: Median age was 55 years (range, 23-81), 71.2% were White, 16.4% were Black, 3.9% were Hispanic, and 1.3% were Asian. GA were identified in 98.7% (308/312) of these patients. TP53 mutations were present in 286 of 312 patients (91.7%) and interestingly, while not statistically significant (p = 0.06) was present in 98% of black women and 89.2% of white women. BRCA1 mutation was present in 14.4% of white women and in 17.6% of black women. BRCA2 mutation was present in 9.9% of white women and 9.8% of black women. Several interesting mutations were identified, see table below. Conclusions: In this 312-patient series of women with HGSOC, we note several prominent mutations in the tumors. While not statistically significant, there is a trend of larger number of poorer prognostic mutations in black women including p53, KRAS and CCNE1. Some studies have found the amplification of CCNE1 and mutation of homologous recombination repair genes to be mutually exclusive in HGSOC which may compel us to find other therapeutic interventions than the PARP inhibitors for these patients.[Table: see text]

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay.

Sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.

Clinical and pathological features associated with circulating tumor DNA content in real-world patients with metastatic prostate cancer.

BackgroundLiquid biopsy is a powerful tool that can enable treatment decisions for metastatic prostate cancer patients with difficult‐to‐biopsy tumors. However, the detection of genomic alterations via liquid biopsy is limited by the fraction (tumor fraction [TF]) of circulating tumor DNA (ctDNA) within the total cell‐free DNA content. While prior work has preliminarily correlated TF with clinical features of prostate cancer, we sought to validate and provide additional resolution, such that a clinical practitioner might anticipate the probability of successful liquid biopsy profiling leveraging commonly assessed clinical and laboratory features.MethodsA total of 813 liquid biopsy specimens were assessable, with 545 associated with a PSA prostate specific antigen measurement, collected in standard‐of‐care settings across approximately 280 US academic or community‐based cancer clinics from September 2018 to July 2021. Deidentified data were captured into a real‐world clinico‐genomic database (CGDB). Comprehensive genomic profiling (CGP) was performed on extracted cell‐free DNA from liquid biopsy samples.ResultsIn multivariable models, higher PSA level, lower hemoglobin, lower albumin, higher alkaline phosphatase (all p < 0.001), and collection of liquid biopsy blood draw within 60 days of new treatment initiation (p = 0.002) were the most strongly associated features with higher TF. At PSA levels of <5 ng/ml, 43% of patients had a TF of <1% indicating an increased likelihood of unevaluable results. Conversely, at PSA levels of >5 ng/ml, 78% of patients had a TF of at least 1% and 46% had a TF of ≥10%, suggesting improved sensitivity for detection of targetable alterations.ConclusionsUniversal genomic profiling of prostate cancers will require complementary use of liquid biopsy and tumor tissue profiling for suitable patients. The likelihood of adequate ctDNA shedding into plasma is one consideration when deciding whether to pursue CGP via liquid biopsy versus tumor profiling. Our real‐world data suggest that PSA < 5 ng/ml is associated with lower ctDNA yield on liquid biopsy, potentially increasing the incidence of negative results or a need for confirmation with tissue testing.

Early dynamics of circulating tumor DNA predict clinical response to immune checkpoint inhibitors in metastatic renal cell carcinoma.

ObjectivesDetection of genomic alterations in circulating tumor deoxyribonucleic acid of peripheral blood can guide the selection of systemic therapy in cancer patients. The predictive significance of circulating tumor deoxyribonucleic acid in metastatic renal cell carcinoma remains unclear, especially for patients treated with immune checkpoint inhibitors.MethodsIn this study, we collected plasma samples before and 1 month after commencing nivolumab monotherapy or nivolumab plus ipilimumab therapy from 14 metastatic renal cell carcinoma patients. We performed circulating tumor deoxyribonucleic acid genomic profiling in plasma cell‐free deoxyribonucleic acid by next‐generation sequencing using a commercially available pan‐cancer panel (Guardant360 CDx). Additionally, we also performed whole exome sequencing of tumor tissues and compared the concordance of genomic profiles with circulating tumor deoxyribonucleic acid.ResultsNine patients had circulating tumor deoxyribonucleic acid in pretreatment plasma samples with a total of 20 mutations (15 single nucleotide variants, three insertions/deletions, and two copy number amplification). VHL (30.0%) was the most frequently mutated gene, followed by TP53 (20.0%), and 45.0% of circulating tumor deoxyribonucleic acid mutations were concordant with somatic mutations in tumor tissues. Patients with decreasing circulating tumor deoxyribonucleic acid mutant allele frequency had better progression free survival when compared to those with increasing mutant allele frequency (P = 0.0441).ConclusionsOur findings revealed that early circulating tumor deoxyribonucleic acid dynamics can serve as a predictive biomarker for response to immune checkpoint inhibitors in metastatic renal cell carcinoma patients.

Abstract P2-01-18: Orthogonal assessment of PIK3CA and ESR1 mutation detection in longitudinal cfDNA samples from endocrine-resistant HR+/HER2- advanced breast cancer patients using dPCR and NGS-based SafeSEQ technology

Abstract Background: Indentification of actionable or therapy resistance-associated mutations may guide sequential treatments in Hormone Receptor-positive and HER2-negative (HR+/HER2-) Advanced Breast Cancer (ABC) patients with resistance to prior endocrine therapies. Molecular profiling from cell-free DNA (cfDNA) is progressively permeating the clinical setting as an alternative or adjunct to tissue biopsies. Digital PCR (dPCR) assays permit the detection of genomic alterations with high sensitivity but are associated with cumbersome workflows and limited genomic coverage. NGS-based cfDNA assays are being increasingly adopted, as they can offer broader coverage while maintaining competitive sensitivity. Methods:Mutation testing was performed on plasma samples obtained from patients with HR+/HER2- ABC who had recurred or progressed after receiving an aromatase inhibitor. Samples were collected at the start of new treatment, during therapy, and at progression. dPCR tests were carried out using QuantStudio3D Digital PCR at the Molecular Oncology Laboratory at Clínico San Carlos Hospital. dPCR targets included PIK3CA E542K, E545K, and H1047R, and ESR1 Y537S and D538G. Samples[CdS1] were also tested with the SafeSEQ Breast Cancer Panel to assess clinically relevant genomic regions across PIK3CA, ESR1, AKT1, TP53, KRAS, and ERBB2. SafeSEQ testing was performed in Sysmex Inostics’ CLIA-certified, CAP-accredited laboratory. Results: Mutational data obtained from both dPCR and SafeSEQ testing were available for concordance analysis in 107 samples from 50 patients for PIK3CA, and 86 samples from 41 patients for ESR1. Combined results showed PIK3CA mutations in 47 samples (43.9%), with H1047R (14%), E545K (11.2%), and E542K (9.3%) being the most frequent mutations detected. ESR1 mutations were present in 35 samples (32.7%), where D538G (19.6%) and Y537S (9.3%) were most commonly observed. Among samples with no mutations detected by dPCR, the expanded coverage of SafeSEQ enabled the detection of clinically relevant mutations in PIK3CA and ESR1 in 10 (9.3%) and 3 (2.8%) samples, respectively. The concordance rates for dPCR and SafeSEQ were 93.1% and 88.9% for PIK3CA and ESR1, with positive percent agreements (PPA) of 93.8% and 84.6%, and negative percent agreements (NPA) of 92.8% and 90.9% for PIK3CA and ESR1, respectively. A strong correlation was observed between MAF levels (Spearman's ρ = 0.87 [95% CI 0.79-0.93], p&amp;lt;0.001) as well as number of mutant molecules per mL of plasma (ρ = 0.79 [95% CI 0.66-0.88], p&amp;lt;0.001). When samples with MAFs&amp;lt;0.1% were evaluated, lower concordance rate was detected (38.5%; N=13; 46.2% detected by SafeSEQ and 92.3% by dPCR). Finally, no significant difference between assays was found when comparing concordance rates among samples at different timepoints. Regarding longitudinal ctDNA data, 18 and 16 patients showed mutations in PIK3CA and/or ESR1 in plasma at least in one timepoint. Monitoring results were concordant in 50%/56.3% of patients for PIK3CA/ESR1 (during therapy and/or at progression time, 5.6%/18.8% of patients showed decrease or clearance of ctDNA and 38.9%/25% showed increase of ctDNA).Conclusion:In this cohort of AI-progressing HR+/HER2- ABC patients, dPCR and SafeSEQ methods showed high overall agreement for the detection of mutations in PIK3CA and ESR1, key genes for clinical decision-making and enrollment in clinical trials. The expanded coverage of SafeSEQ makes it a powerful tool to inform patient management considering the growing pool of clinically relevant genomic alterations in this type of patients. Efforts are ongoing to expand the analysis to a larger cohort and incorporate long-term outcomes and multivariate adjustment. Supported by AstraZeneca. Citation Format: Jesus Fuentes Antras, Vanesa García-Barberán, Fernando Moreno, Hillary Sloane, Igor López-Cade, Alicia De Luna, Alejandro Pascual, Carmen Ramirez-Ruda, Victor Lorca, Paloma Flores, Pedro Perez-Segura, Alberto Ocaña, Jennifer Preston, Hannah Quinn, Frederick Jones, Jose Angel Garcia-Saenz. Orthogonal assessment of PIK3CA and ESR1 mutation detection in longitudinal cfDNA samples from endocrine-resistant HR+/HER2- advanced breast cancer patients using dPCR and NGS-based SafeSEQ technology [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 P2-01-18.

Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine.

The ability to identify genetic alterations in cancers is essential for precision medicine; however, surgical approaches to obtain brain tumor tissue are invasive. Profiling circulating tumor DNA (ctDNA) in liquid biopsies has emerged as a promising approach to avoid invasive procedures. Here, we systematically evaluated the feasibility of profiling pediatric brain tumors using ctDNA obtained from plasma, cerebrospinal fluid (CSF), and urine. We prospectively collected 564 specimens (257 blood, 240 urine, and 67 CSF samples) from 258 patients across all histopathologies. We performed ultra-low-pass whole-genome sequencing (ULP-WGS) to assess copy number variations and estimate tumor fraction and developed a pediatric CNS tumor hybrid capture panel for deep sequencing of specific mutations and fusions. ULP-WGS detected copy number alterations in 9/46 (20%) CSF, 3/230 (1.3%) plasma, and 0/153 urine samples. Sequencing detected alterations in 3/10 (30%) CSF, 2/74 (2.7%) plasma, and 0/2 urine samples. The only positive results were in high-grade tumors. However, most samples had insufficient somatic mutations (median 1, range 0-39) discoverable by the sequencing panel to provide sufficient power to detect tumor fractions of greater than 0.1%. Children with brain tumors harbor very low levels of ctDNA in blood, CSF, and urine, with CSF having the most DNA detectable. Molecular profiling is feasible in a small subset of high-grade tumors. The level of clonal aberrations per genome is low in most of the tumors, posing a challenge for detection using whole-genome or even targeted sequencing methods. Substantial challenges therefore remain to genetically characterize pediatric brain tumors from liquid biopsies.