Mutations In Cell-free DNA Research Articles

Evaluating Bioinformatics Processing of Somatic Variant Detection in cfDNA Using Targeted Sequencing with UMIs.

Circulating tumor DNA (ctDNA) is a promising cancer biomarker, but accurately detecting tumor mutations in cell-free DNA (cfDNA) is challenging due to their low frequency and sequencing errors. Our study benchmarked Mutect2, VarScan2, shearwater, and DREAMS-vc using deep targeted sequencing of cfDNA with Unique Molecular Identifiers (UMIs) from 111 colorectal cancer patients. Performance was assessed at both the mutation level (distinguish tumor variants from errors) and the sample level (detect if an individual has cancer). Additionally, we investigated the effects of various UMI grouping and consensus strategies. The shearwater-AND variant calling method demonstrated the highest precision in detecting tumor-derived mutations from plasma, and reached the highest ROC-AUC of 0.984 for sample classification in tumor-informed cfDNA analyses. DREAMS-vc exhibited the highest ROC-AUC of 0.808 for sample classification in tumor-agnostic studies. We also found that sequencing depth differences in PBMCs could lead to false positives, particularly with VarScan2 and Mutect2, which was addressed by downsampling to equivalent mean depths. Additionally, network-based UMI grouping methods outperformed those using identical UMIs when all reads were retained. Our findings emphasize that the optimal variant caller depends on the study context-whether focused on mutation or sample classification, and whether conducted under tumor-informed or tumor-agnostic conditions.

Highly sensitive and accurate detection of ALK-TKI resistance mutations by oligoribonucleotide interference-PCR (ORNi-PCR)-based methods

BackgroundPatients with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) are treated with ALK tyrosine kinase inhibitors (TKIs). Although most patients benefit from ALK-TKIs, the development of resistance mutations is common and results in NSCLC recurrence. To identify ALK-TKI-resistant NSCLC at the early recurrent phase, highly sensitive and accurate methods for the detection of mutations are essential. ObjectiveThe aim of this study was to establish highly sensitive, accurate, cost-effective, and clinically practical methods for the detection of two frequent ALK-TKI resistance mutations, ALK G1202R and L1196M, by liquid biopsy. MethodsThe efficacy of oligoribonucleotide interference-PCR (ORNi-PCR) was examined by first optimizing experimental conditions to specifically amplify the ALK-TKI resistance mutant DNA corresponding to ALK G1202R and L1196M mutations. ORNi-PCR was then combined with droplet digital PCR (ddPCR) or real-time PCR to detect these mutations in cell-free DNA (cfDNA) extracted from NSCLC patients. ResultsORNi-PCR followed by ddPCR/real-time PCR detected 1–10 copy(s) of G1202R and L1196M DNA in model cfDNA. These mutations in patients’ cfDNA were identified using ORNi-PCR-based methods, whereas conventional ddPCR failed to detect them. ConclusionORNi-PCR followed by ddPCR/real-time PCR enables highly sensitive and accurate detection of ALK mutations by liquid biopsy. Although the clinical data are limited, our results show that these methods are potentially useful for identifying ALK-TKI-resistant NSCLC at the early recurrent phase.

Clinical utility of plasma cell-free DNA (cfDNA) in diffuse gliomas for the detection of IDH1 R132H mutation

Liquid biopsy for CNS tumors is in its nascent phase, hindered by the low levels of circulating tumor DNA (ctDNA). Overcoming this challenge requires highly sensitive molecular techniques. DD-PCR emerges as a standout technique due to its ability to identify rare mutations, copy number variations, and circulating nucleic acids, making it one of the best methods for identifying somatic mutations in cell-free DNA (cfDNA). Despite promising results from various studies demonstrating the feasibility of obtaining informative ctDNA profiles from liquid biopsy samples, challenges persist, including the need to standardize sample collection, storage, and processing methods, define clear assay positivity thresholds, and address the overall low assay sensitivity. Our two-phase study began by assessing DD-PCR efficacy in FFPE tissues, revealing robust concordance with immunohistochemistry. In Phase 1 (85 cases), DD-PCR on FFPE tissues demonstrated 100 % sensitivity and specificity for IDH1 R132H mutations. In Phase 2 (100 cases), analysis extended to cfDNA, maintaining high specificity (100 %) with moderate sensitivity (44.2 %). Overall concordance with immunohistochemistry was 61 %, highlighting liquid biopsy's potential in glioma management. The findings emphasized DD-PCR's clinical utility in both tissue and liquid biopsy, underscoring its role in early detection, diagnosis, and therapeutic monitoring of diffuse gliomas.

KRAS and GNAS mutations in cell-free DNA and in circulating epithelial cells in patients with intraductal papillary mucinous neoplasms-an observational pilot study.

Intraductal papillary mucinous neoplasms (IPMNs) are potential precursor lesions of pancreatic cancer. We assessed the efficacy of screening for KRAS proto-oncogene, GTPase (KRAS), and GNAS complex locus (GNAS) mutations in cell-free DNA (cfDNA)-using digital droplet polymerase chain reaction (ddPCR) and circulating epithelial cell (CEC) detection-as biomarkers for risk stratification in IPMN patients. We prospectively collected plasma samples from 25 resected patients at risk of malignant progression, and 23 under clinical surveillance. Our findings revealed KRAS mutations in 10.4% and GNAS mutations in 18.8% of the overall cohort. Among resected IPMN patients, KRAS and GNAS mutation detection rates were 16.0% and 32.0%, respectively, whereas both rates were 4.0% in conservatively managed IPMN. GNAS mutations in cfDNA were significantly more prevalent in resected IPMN (P = 0.024) compared with IPMN under surveillance. No CECs were detected. The absence of KRAS and GNAS mutations could be a reliable marker for branch duct IPMN without worrisome features. The emergence of GNAS mutations could prompt enhanced imaging surveillance. Neither the presence of established worrisome features nor GNAS or KRAS mutations appear effective in identifying high-grade dysplasia among IPMN patients.

Novel diagnostic biomarkers for pancreatic cancer: assessing methylation status with epigenetic-specific peptide nucleic acid and KRAS mutation in cell-free DNA.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor with a poor prognosis that poses challenges for diagnosis using traditional tissue-based techniques. DNA methylation alterations have emerged as potential and promising biomarkers for PDAC. In this study, we aimed to assess the diagnostic potential of a novel DNA methylation assay based on epigenetic-specific peptide nucleic acid (Epi-sPNA) in both tissue and plasma samples for detecting PDAC. The study involved 46 patients with PDAC who underwent surgical resection. Epi-TOP pancreatic assay was used to detect PDAC-specific epigenetic biomarkers. The Epi-sPNA allowed accurate and rapid methylation analysis without bisulfite sample processing. Genomic DNA extracted from paired normal pancreatic and PDAC tissues was used to assess the diagnostic efficacy of epigenetic biomarkers for PDAC. Subsequent validation was conducted on cell-free DNA (cfDNA) extracted from plasma samples, with 10 individuals represented in each group: PDAC, benign pancreatic cystic neoplasm, and healthy control. The combination of seven epigenetic biomarkers (HOXA9, TWIST, WT1, RPRM, BMP3, NPTX2, and BNC1) achieved 93.5% sensitivity and 96.7% specificity in discerning normal pancreatic from PDAC tissues. Plasma cfDNA, analyzed using these markers and KRAS mutations, exhibited a substantial 90.0% sensitivity, 95.0% specificity, and an overall 93.3% accuracy for discriminating PDAC. Notably, cancer antigen 19-9 and carcinoembryonic antigen both had an accuracy of 90.0%. Our study suggests that analyzing seven differentially methylated genes with KRAS mutations in cfDNA using the novel Epi-TOP pancreatic assay is a potential blood-based biomarker for the diagnosis of PDAC.

Serial analysis of ESR1 and PIK3CA mutations in cell-free DNA from metastatic breast cancer during 1st line endocrine therapy.

e13060 Background: Activating mutations ofESR1 and PIK3CA genes are known mechanisms of endocrine resistance. Recent analysis supported benefit of early intervention at presence of ESR1 mutation in blood (bESR1) before clinical progression. We aimed to investigate ESR1 and PIK3CA mutations detected in cell-free DNA (cfDNA) from patients with hormone receptor-positive, HER2-negative metastatic breast cancer (MBC) and its impact on progression-free survival (PFS). Methods: Thirty patients undergoing 1st line endocrine therapy (ETx) were identified from a prospective cohort. Seven ESR1 hotspot mutations in the ligand binding domain were tested in cfDNA with droplet digital polymerase chain reaction (ddPCR) assay (Genopeaks Co., Ltd). Eleven PIK3CA hotspot mutations were tested using amplicon-targeted sequencing method (Genopeaks Co., Ltd) in both tumor and cfDNA. PFS analyses were performed using Kaplan-Meier methods and compared with the log-rank test. Results: We examined serial cfDNA samples from 26 patients collected every 3-6 months. ESR1 mutations were found in 61.5% (16/26) patients at any timepoint during 1st line endocrine therapy, with D538G being the most common followed by Y537C and 68.8% (11/16) being polyclonal. Among the 50.0% (8/16) bESR1 positive patients with clinical progression, 4 had bESR1 detected before clinical progression while 4 were diagnosed clinical progression concurrently, suggesting the necessity of closer intervals of cfDNA analyses. While presence of bESR1 did not affect PFS overall, 18.8% of patients with bESR1 detected within 6 months after 1st line ETx, displayed worse outcome (median PFS 5 vs 52 months). Also, patients with clearance of bESR1 mutation in subsequent cfDNA (81.3%, 13/16) were shown to have a better outcome (median PFS 52 vs 41 months). Among 14 patients with clinical progression, 8 had bESR1 mutation. Median PFS between patients with bESR1 positive vs negative was 14 vs 9 months. PIK3CA mutations were detected in 69.5% (16/23) of cfDNA at diagnosis of distant metastasis, among them 53.3% (8/15) had co-occurrence with bESR1 mutations. While presence of PIK3CA mutation in primary tumor tissue did not affect disease-free survival nor PFS, patients with blood PIK3CA mutation at diagnosis of metastasis displayed significantly worse outcome (p = 0.041). Conclusions: Substantial number of hormone receptor-positive, HER2-negative MBC patients were detected with ESR1 and PIK3CA mutation in plasma. Though it was a small-sized analysis to accurately assess statistical significance, we observed worse outcome in patients with 1) early detection (<6 months) of bESR1 and with 2) sustained bESR1 during palliative ETx. Patients with clinical progression without bESR1 mutation displayed shorter PFS than patients with bESR1. PIK3CA mutations in cfDNA were prognostic supporting the benefit of combined targeted therapy.

Development of a nine-variant reference material panel to standardize cell-free DNA detection.

Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.

Liquid Biopsy Profiling with Multiple Tests in Patients with Metastatic Breast Cancer

The chief goal of the Blood Profiling Atlas in Cancer (BloodPAC) consortium is to promote collaborative efforts that support the development and implementation of liquid biopsy tests. Here, we report the results of a pilot study conducted by three BloodPAC members that aimed to demonstrate a multisite liquid biopsy testing framework using longitudinal blood specimens from 38 patients with metastatic breast cancer. Three laboratories receiving identical samples from two clinical sites each applied a different targeted sequencing platform to analyze mutations in cell-free DNA (cfDNA). The resulting mutational profiles reflected common breast cancer alterations, including clinically actionable mutations for 40% of hormone- receptor-positive patients. In 12 genes with shared target regions across sequencing panels, perfect inter-assay concordance was also observed for mutations detected above the lowest common assay limit of detection. Whole-genome copy number profiling of cfDNA and circulating tumor cells (CTCs) further revealed marked heterogeneity in copy number alterations and cfDNA tumor fractions across patients. Additionally, comparison of tumor fraction and CTC abundance demonstrated the complementary nature of cfDNA and CTC analyses. Overall, the framework described in this study may serve as a resource for future trials aiming to identify multimodal liquid biopsy biomarkers to guide clinical care.

Abstract PO4-19-06: Phase II study of a PARP inhibitor, talazoparib, in HER2- metastatic breast cancer with a somatic BRCA1/2 mutation present in cell-free DNA or tumor tissue genotyping

Abstract Background: PARP inhibitors improve both progression free survival (PFS) and patient quality of life in germline BRCA1/2 mutant metastatic breast cancer (MBC), which accounts for 5-10% of breast cancer, leading to their approval in this setting. We have previously shown that a subset of patients with MBC who are not germline BRCA1/2 carriers may harbor pathogenic somatic BRCA1/2 mutations that are identified by cell-free DNA and/or tumor tissue genotyping. In our prior work, we developed a circulating tumor cell culture from a patient with MBC harboring a pathogenic somatic BRCA1 mutation, and demonstrated that a PARP inhibitor induced cell growth inhibition similar to germline BRCA1/2 mutant cultures. Thus, we hypothesize that a PARP inhibitor may be effective in treating somatic BRCA1/2 mutant MBC. In this clinical trial, we are studying the efficacy of a PARP inhibitor in somatic BRCA1/2 mutant MBC. Our work may help expand the clinical application of PARP inhibitors in MBC. Trial Design: This phase II investigator-initiated clinical trial is enrolling 30 patients with MBC who have a pathogenic somatic BRCA1/2 mutation identified by a CLIA certified cell-free DNA and/or tumor tissue genotyping assay. Patients are treated with the PARP inhibitor talazoparib 1 mg/day until progression of disease. Imaging (CT chest, abdomen and pelvis, and bone scan) occurs for disease assessment at baseline and every 3 months. Cell-free DNA is collected at baseline and then monthly to evaluate changes in the genomic environment. Patients also undergo the Cancer Risk B (CR-B) assay, a novel flow variant assay to identify double-strand break repair mutations in circulating blood cells, at baseline. Eligibility Criteria: Patients must have MBC with a pathogenic somatic BRCA1/2 mutation identified in a cell-free DNA and/or tumor tissue genotyping assay, with pathogenicity confirmed by a genetics counselor using validated genomic databases such as ClinVar. Patients may have triple-negative (receipt of at least 1 prior chemotherapy) or hormone receptor positive/HER2- MBC (receipt of at least 1 prior hormone therapy). Patients must not be known germline BRCA1/2 carriers. Any number of prior therapies including a prior platinum (in absence of progressive disease on a platinum) are allowed, but patients should not have received a prior PARP inhibitor. Patients must have adequate organ function and performance status. Specific Aims: The primary aim is to determine PFS (RECIST 1.1). Secondary aims include determining the objective response rate and toxicity (NCI CTCAE v 5.0). Exploratory aims include evaluating serial changes in BRCA1/2 mutant allelic frequency in cell-free DNA, understanding the impact of BRCA1/2 reversion mutations in cell-free DNA, comparing pre- and post-treatment cell-free DNA results to understand changes in the genomic environment, studying the CR-B assay positivity rate, and correlating these biomarker analyses with treatment response. Statistical Methods: This study has 81% power to demonstrate that the 12-week PFS is 53% or higher. In contrast, there is a 4% (alpha) probability of concluding that the 12-week PFS is ≥ 53% if the true 12-week PFS is 30% or lower. If 14 or more of 30 total patients achieve PFS > 12 weeks, the null hypothesis (12-week PFS ≤ 30%) will be rejected. Present accrual and target accrual: This study (NCT03990896) is open at Massachusetts General Hospital, MD Anderson, University of California San Francisco, Emory, Northwestern, and Vanderbilt. As of 7/2023, 14/30 patients are enrolled. Funding: This study is funded by a Pfizer ASPIRE award and Conquer Cancer Foundation of ASCO–Breast Cancer Research Foundation- Career Development Award. Contact information: Neelima Vidula, MD, Massachusetts General Hospital, nvidula@mgh.harvard.edu. Citation Format: Neelima Vidula, Senthil Damodaran, Manali Bhave, Hope Rugo, Ami N. Shah, Erica Blouch, Nathan Royce Ruffle-Deignan, Ogadinma Ogbenna, Lisa E. Flaum, Massimo Cristofanilli, Joseph Sparano, Harry Ostrer, Vandana Abramson, Nora Horick, Aditya Bardia. Phase II study of a PARP inhibitor, talazoparib, in HER2- metastatic breast cancer with a somatic BRCA1/2 mutation present in cell-free DNA or tumor tissue genotyping [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-06.

Detection of circulating tumor DNA in plasma of patients with primary CNS lymphoma by digital droplet PCR

BackgroundPrimary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL.MethodsWe analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively.ResultsMYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196.ConclusionThe low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.

Abstract 6101: PLASMUT: An R Package for estimating the probability of tumor-specific mutations in cell-free DNA

Abstract Noninvasive approaches for detection of tumor-specific mutations in cell-free DNA (cfDNA) have the potential to track a patient’s response to treatment, enabling effective and timely decisions on therapy. However, mutations in cfDNA arising from clonal hematopoeisis (CH) are common and tumor biopsies for definitive identification of the origin of these mutations are not always available. Sequencing of matched cells from buffy coat and the absence of mutations in these cells has been used to rule-out white blood cell (WBC) mutations, but uneven sequencing depths between matched cfDNA and buffy coat and the fraction of mutant alleles are generally ignored by rule-based tests. A probabilistic approach that quantifies the evidence of tumor-derived mutations in cfDNA is needed. We developed a Bayesian framework to estimate the probability that a mutation identified in cfDNA is tumor specific. Our approach requires the number of reads with a mutant allele in plasma (yp) and WBCs (yw) and the corresponding number of total distinct reads at these locations. The posterior odds that a mutation is tumor-derived (S) versus CH or germline (H) is given by the ratio of the probabilities of observing the distinct reads given each model times the prior odds. Estimation of the Bayes factor is obtained by integrating over the unobserved mutant allele fractions in plasma and WBCs using Monte Carlo importance sampling. We applied this approach to 52 patients with initially unresectable colorectal cancer (CRC) liver metastases in the CAIRO5 clinical trial (NCT02162563), using ultra-deep targeted sequencing of cfDNA from plasma and matched WBCs. Among the CAIRO5 patients analyzed, we identified 95 mutations with moderate evidence of tumor-derived cfDNA mutations (Bayes factor > 10) and 19 mutations that were CH-derived (Bayes factor < 0.1). For a subset of 47 cfDNA mutations with no corresponding mutation identified by WBC sequencing, the evidence of tumor origin was highly variable (Bayes factor range: 0.03 to 5.6). While the standard rule-based approach identifies all of these mutations as tumor-derived, none of these mutations reach a moderate evidence cutpoint (all Bayes factors < 6). As a false positive would lead to identification of cfDNA mutations that do not track tumor burden, requiring even higher levels of evidence (Bayes factor > 99) for the selection of cfDNA mutations could be warranted, and still identifies one or more cfDNA mutations in 43 of the patients. This approach is implemented in the R package PLASMUT available from Bioconductor (doi:10.18129/B9.bioc.plasmut). We developed an approach that quantifies the evidence between two competing models for the origin of mutations in cfDNA. A cutpoint for determination of the probability of tumor-derived cfDNA mutations can be tailored to the disease application, balancing the potential benefits of noninvasive testing with the harms of false positives and negatives. Citation Format: Adith S. Arun, Jamie E. Medina, Stephen Cristiano, Daniel C. Bruhm, Remond J. Fijneman, Gerrit A. Meijer, Alessandro Leal, Victor E. Velculescu, Robert B. Scharpf. PLASMUT: An R Package for estimating the probability of tumor-specific mutations in cell-free DNA [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 6101.

High-efficiency EGFR genotyping using cell-free DNA in bronchial washing fluid.

EGFR mutation testing is required for treatment of lung adenocarcinoma using epidermal growth factor receptor-tyrosine kinase inhibitor. However, the amounts of tumor tissue or tumor cells obtained by bronchoscopy are often insufficient. Bronchial washing fluid, obtained by lavage with saline after tumor biopsy or brushing, and the supernatant of bronchial washing fluid are thought to contain cell-free DNA that would be potentially applicable for EGFR testing. From among patients with suspected adenocarcinoma or non-small cell lung carcinoma diagnosed from biopsy or surgical specimens at the University of Tsukuba Hospital between 2015 and 2019, cell-free DNAs from 80 specimens of supernatant of bronchial washing fluid (50 with EGFR mutation and 30 with wild type EGFR) and 8 blood serum samples were examined for EGFR mutation using droplet digital PCR. Among the 50 patients harboring EGFR mutation, the rate of positivity for cell-free DNA extracted from supernatant of bronchial washing fluid was 80% (40/50). In nine of the EGFR mutation-positive cases, tumor cells were not detected by either biopsy or cytology, but the mutation was detected in four cases (4/9, 44%). Comparison of the cell-free DNA mutation detection rate between supernatant of bronchial washing fluid and blood serum in six cases showed that mutations were detected from the former in all cases (6/6, 100%), but from the latter in only one case (1/6, 17%). Using supernatant of bronchial washing fluid samples, the detection rate of EGFR mutation was high, and EGFR mutations were detectable even when no tumor cells had been detectable by biopsy or cytology. Supernatant of bronchial washing fluid might be an effective sample source for EGFR mutation testing.

Retracted: Detection and Significance of Cell-Free DNA Mutation in Pleural Effusion in Patients with Advanced NSCLC.

[This retracts the article DOI: 10.1155/2022/3112281.].

CAR19 Therapy Drives Expansion of Clonal Hematopoiesis and Associated Cytopenias

Background: For patients with relapsed/refractory large B-cell lymphomas (rrLBCL), CD19-directed chimeric antigen receptor T cells (CAR19) improve survival compared to autologous hematopoietic cell transplantation (HCT). However, major toxicities of CAR19 therapy include prolonged cytopenias and associated infections. To better understand the impact of CAR19 on such toxicities, we studied a cohort of LBCL patients achieving durable remissions to assess immune recovery after CAR19 treatment. We (1) compared immune reconstitution in HCT vs CAR19 treated patients, (2) performed molecular analysis of post-CAR19 cytopenic marrow specimens, and (3) profiled emergent mutations in cell free DNA (cfDNA) before and after CAR19. Methods & Results: We first propensity matched rrLBCL patients treated at Stanford with either CAR19 (100% axi-cel) or HCT who achieved durable remissions. Patients were matched based on age, sex, prior systemic lines of therapy, and time from infusion. From 471 eligible cases, propensity score matching was successful in 138 (n=69 per arm). When compared to HCT-treated counterparts, CAR19-treated patients had reduced recovery of absolute lymphocyte counts (ALC) and absolute neutrophil counts (ANC; longitudinal regression day 28-800 ANOVA p<0.005), but similar recovery of hemoglobin and platelets. CAR19-treated patients experienced significantly more Grade ≥3 infections in competing risk analyses (HR 2.61, CI 1.26-5.42), and higher non-relapse mortality (log-rank p=0.027). Next, to characterize mechanisms underlying cytopenias after CAR19, we profiled marrow aspirates from cases requiring biopsy due to prolonged cytopenia or clinical concern for myeloid neoplasms. No patient had evidence of lymphoma relapse at the time of biopsy, and all were >60d from CAR19 infusion (median 377d, range 86-1561d). Among 21 patients profiled by DNA sequencing, 18 (86%) had evidence of ≥1 somatic mutation in genes canonically associated with clonal hematopoiesis (CH) in the marrow ( Fig. 1A), with the most recurrently mutated genes being DNMT3A (38%), TP53 (33%), PPM1D (24%), ASXL1 (14%), and TET2 (14%). Among 6 patients diagnosed with and requiring therapy for a myeloid malignancy, all (100%) had a canonical myeloid karyotypic aberration detected by FISH, as compared with 0 in the 14 other evaluable cases ( Fig. 1A). CAR19 cells were proportionately enriched in the marrow relative to paired peripheral blood by flow cytometry (n = 12, median 2.5-fold enrichment, Wilcoxon p<0.001). Using 10x scRNA-Seq, viable CAR19 was detectable in 7 of 8 marrows (88%) and were predominantly CD4+. We hypothesized that CH clones detectable after CAR19 would also be present in pre-CAR19 blood samples and detectable in cfDNA. To test this, we profiled 786 longitudinal samples from 141 patients using CAPP-seq and serially monitored CH clones in cfDNA. Among 141 eligible cases, 40 (28%) were evaluable for CH, in having both pre- and post-CAR19 infusion specimens, while in durable remission (median follow up 967d). Of 51 CH clones detected in post-infusion cfDNA, 46 (90.1%) were also detectable prior to CAR19, typically expanding post-infusion (median = 1.8-fold, Fig. 1B). We next tested whether expanding CH clones showed evidence for selection after CAR19. Strikingly, among 48 recurrently mutated genes tested, DNMT3A and PPM1D showed significant evidence for clonal selection and expansion after CAR19 (Q-value < 0.05). When compared to patients without emerging CH clones, patients with ≥5-fold clonal expansions of canonical CH genes (23% of cases) had reduced ANC, but not ALC, recovery (median ANC=1.43 vs 2.43 at 6-mo, Wilcoxon p<0.001). This association remained significant in multivariate longitudinal regression that included age, sex, prior lines of therapy, and pre-infusion ANC (ANOVA p = 0.007). Conclusion: CAR19 treated patients experience reduced immune reconstitution and higher infection rates than HCT treated counterparts. Bone marrow findings reveal prolonged CAR19 cytopenia to commonly be associated with clonal cytopenia of undetermined significance (CCUS). Despite durable lymphoma remissions, canonical myeloid clonal expansions are common post-CAR19, and associated with myelosuppression. Non-invasive genotyping could aid in early identification of patients at risk for prolonged cytopenias after CAR19 and inform interventions to decrease associated complications.

Clonal Hematopoiesis in Whole-Blood and Cell-Free DNA of Ovarian Cancer Patients Undergoing PARP-Inhibitor Treatment: An Exploratory Analysis of the ENGOT-ov48/Eudario Trial

Clonal hematopoiesis (CH), characterized by the expansion of somatically mutated hematopoietic stem cells, is an age-related phenomenon associated with the development of hematologic malignancies, increased cardiovascular risk, and other age-related proinflammatory conditions. In patients with solid tumors, cytotoxic therapies differentially select for clones with mutations in the DNA damage response (DDR) pathway, i.e. TP53 and PPM1D, leading to an elevated risk for therapy-related myeloid malignancies (t-MNs). Poly(ADP-ribose) polymerase inhibitors (PARPi) are frequently used to treat ovarian cancer with particularly high efficacy reported in tumors exhibiting homologous recombination (HR) deficiency caused by mutations in BRCA1/2 or other HR pathway genes. However, recent data indicate that PARPi treatment is also associated with an elevated risk for t-MNs. Here, we performed an in-depth analysis of CH in whole-blood (WB) and plasma of patients with relapsed high-grade ovarian cancer (HGOC) receiving platinum-based chemotherapy and PARPi treatment, elucidating the mutational landscape of CH, gene-specific dynamics, as well as the potential interference of CH with putative tumor-derived mutations in cell-free DNA (cfDNA). Serial specimen of 103 patients with relapsed, platinum-sensitive HGOC participating in the European Trial on Enhanced DNA Repair Inhibition in Ovarian Cancer (EUDARIO) study (ENGOT-ov48/NCT03783949) were analyzed using error-corrected targeted sequencing with a custom panel covering 45 myeloid and 27 HR-related genes. Study treatment consisted of six cycles of carboplatin-based chemotherapy followed by maintenance therapy with Niraparib ± the HSP90 inhibitor Ganetespib. A total of 128 somatic mutations with variant allele frequencies (VAFs) ≥ 1% were identified in 58 patients (56%) with a high fraction of DDR mutations (51 PPM1D mutations in 27 patients, 7 TP53 mutations in 7 patients). Age (median 62 years) and the number of previous therapy lines (median 1, range 1-5 lines) were significantly associated with the presence of CH. Specifically, prior exposure to PARPi treatment was strongly associated with both the presence of CH and the number of CH mutations. CH-positive patients had significantly shorter progression-free survival (median 7.9 months vs 10.6 months, p = 0.021 in log-rank test), and a trend for shorter overall survival (median 21.1 months vs 27.7 months, p = 0.16) in univariate survival analysis. With respect to adverse events, CH-positive patients more frequently had infections during study treatment (49% vs 26%, p = 0.025). All but 5 CH mutations with VAF ≥ 1% were also detectable in cfDNA with significant correlation of VAFs (R = 0.84, p < 0.0001). Mutations occurring solely in cfDNA (putatively tumor-derived) had a high fraction of TP53 and HR-related mutations, while DNMT3A and PPM1D, were rarely mutated (Figure 1). However, 23/87 (26%) of TP53 mutations and 31/98 (32%) of mutations in HR-related genes detected in cfDNA were of hematopoietic origin, underlining the importance of parallel sequencing of WB in liquid biopsies to avoid false positive results. The dynamics of CH clones under carboplatin and PARPi treatment were assessed in paired WB samples at initiation/end of treatment, which were available for 62 patients. 96 clones that initially had a VAF < 1% emerged during the treatment, including 56 PPM1D- and 10 TP53-mutated clones. Gene-wise comparison revealed a significantly higher median clonal fitness in PPM1D- and TP53-mutated clones compared to TET2- or DNMT3A-mutated clones (Figure 2). PPM1D-mutated clones exhibited lower fitness during PARPi treatment compared to carboplatin treatment (p = 0.0015 in Wilcoxon rank sum test), which was not the case for TP53-mutated clones. Finally, 8 samples with multiple mutations were subjected to single-cell genotyping on the Mission Bio Tapestri platform to investigate their clonal architecture. Preliminary data indicate clonal exclusiveness of co-occurring DDR mutations. In summary, our data reveals a high prevalence of CH in patients with relapsed HGOC and provides novel insights into the clonal architecture and dynamics of CH under carboplatin and PARPi treatment with a differential selection of DDR-driven clones. Moreover, we report a relevant interference of CH-derived mutations with tumor-derived mutations in cfDNA.

From Pediatric to Adult Brain Cancer: Exploring Histone H3 Mutations in Australian Brain Cancer Patients.

Genetic histone variants have been implicated in cancer development and progression. Mutations affecting the histone 3 (H3) family, H3.1 (encoded by HIST1H3B and HIST1H3C) and H3.3 (encoded by H3F3A), are mainly associated with pediatric brain cancers. While considered poor prognostic brain cancer biomarkers in children, more recent studies have reported H3 alterations in adult brain cancer as well. Here, we established reliable droplet digital PCR based assays to detect three histone mutations (H3.3-K27M, H3.3-G34R, and H3.1-K27M) primarily linked to childhood brain cancer. We demonstrate the utility of our assays for sensitively detecting these mutations in cell-free DNA released from cultured diffuse intrinsic pontine glioma (DIPG) cells and in the cerebral spinal fluid of a pediatric patient with DIPG. We further screened tumor tissue DNA from 89 adult patients with glioma and 1 with diffuse hemispheric glioma from Southwestern Sydney, Australia, an ethnically diverse region, for these three mutations. No histone mutations were detected in adult glioma tissue, while H3.3-G34R presence was confirmed in the diffuse hemispheric glioma patient.

Mismatch-Guided Deoxyribonucleic Acid Assembly Enables Ultrasensitive and Multiplex Detection of Low-Allele-Fraction Variants in Clinical Samples.

Somatic mutations are important signatures in clinical cancer treatment. However, accurate detection of rare somatic mutations with low variant-allele frequencies (VAFs) in clinical samples is challenging because of the interference caused by high concentrations of wild-type (WT) sequences. Here, we report a post amplification SNV-specific DNA assembly (PANDA) technology that eliminates the high concentration pressure caused by WT through a mismatch-guided DNA assembly and enables the ultrasensitive detection of cancer mutations with VAFs as low as 0.1%. Because it generates an assembly product that only exposes a single-stranded domain with the minimal length for signal readout and thus eliminates possible interferences from secondary structures and cross-interactions among sequences, PANDA is highly versatile and expandable for multiplex testing. With ultrahigh sensitivity, PANDA enabled the quantitative analysis of EGFR mutations in cell-free DNA of 68 clinical plasma samples and four pleuroperitoneal fluid samples, with test results highly consistent with NGS deep sequencing. Compared to digital PCR, PANDA returned fewer false negatives and ambiguous cases of clinical tests. Meanwhile, it also offers much lower upfront instrumental and operational costs. The multiplexity was demonstrated by developing a 3-plex PANDA for the simultaneous analysis of three EGFR mutations in 54 pairs of tumor and the adjacent noncancerous tissue samples collected from lung cancer patients. Because of the ultrahigh sensitivity, multiplexity, and simplicity, we anticipate that PANDA will find wide applications for analyzing clinically important rare mutations in diverse devastating diseases.

Enhanced detection of molecular residual disease (MRD) by tumor-informed and tumor-agnostic variants in circulating tumor DNA (ctDNA).

e15586 Background: Liquid biopsies have been mainly used for cancer detection, non-invasive tumor genotyping and disease surveillance. Highly sensitive liquid biopsy assays have been developed that can now be applied to detect and characterize MRD. Several techniques have been developed for the detection of MRD, mainly by tracking ultra-low-frequency somatic tumor mutations in cell-free DNA. Methods: NGS using a 1123-gene-panel were performed on the tumor tissue and white blood cells which was designed for pan-cancer covering 1.8Mbp human genome. Moreover, we used a self-designed pan-cancer panel covering 208 kb and increasing 20 or less mutations individually known to be present in the surgical tissue based on the fixed panel for postoperative plasma or follow-up plasma. In Our study, plasma were collected from 137 healthy donors and UMI sequencing with 208 kb panel for built an database of cfDNA background noise. Furthermore, we constructed two different databases of CH(clonal hematopoietic) variants and negative-background noise with 2300+ cancer patients and 1000+ tumor-plasma paired data respectively. In this way, this approach not only could track selected tumor-specific mutations, but also provided with new variants using tumor-agnostic. MRD-positive status was defined as at least one variants in the plasma. Results: To test sensitivity and specificity of the minimal residual disease detection (MRD) method, we used 208kb panel data for plasma samples from 137 healthy samples and 150 samples with CRC untreatment. The result showed an area under the curve (AUC) of 0.95, with sensitivity of 92% and specificity of 98%. To test the limit of detection of 208 kb panel, we sequenced six different dilution levels (5%, 1%, 0.5%, 0.1%, 0.05% and 0.01%) with a positive control sample containing 98 known mutations. The result showed the detection limitation of 208 kb panel achieved 0.02%. We retrospectively analyzed 3000+ patients with colorectal cancer, 35 of whom met the eligibility criteria were enrolled in this study: 1) With stage II-III CRC undergoing radical surgery; 2)Blood specimens were collected on postoperative 7 days to 3 months; 3)Follow-up duration was 2 years or relapsed; 4)Enough samples for targeting sequencing. Twelve (34%) patients with MRD+,which all patients recurred while 23(66%) patients were MRD-, which 22/23 patient did not experience local recurrence or metastasis. The Kaplan-Meier estimates showed that the positivity of ctDNA was of high predictive value for relapse (HR 21, 95%CI 18.3-28.9;P < 0.001). Conclusions: In this study, combination of tumor-informed and tumor-agnostic strategies can effectively distinguish healthy and cancer patients. Meanwhile,our study highlights that postoperative ctDNA status can effectively detect MRD and identify patients with a high risk of relapse.

Accuracy of genome-wide mutational analysis for tumor-informed ctDNA-guided MRD monitoring in bladder cancer.

4589 Background: Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard management of localized muscle-invasive bladder cancer (MIBC). However, about 45% of patients develop metastases within 2 years after RC. Biomarkers for treatment efficacy evaluation and early detection of minimal residual disease (MRD) are needed for earlier treatment initiation and monitoring of treatment response. Tumor-informed detection of mutations in cell-free DNA (cfDNA) has shown promising results to monitor MRD. However, the low tumor fraction after surgery and limited input material obtained from a typical plasma sample limits the probability of detecting low metastatic burden scenarios. Here we implemented and applied a whole-genome sequencing (WGS) approach to monitor circulating tumor DNA (ctDNA) for sensitive ctDNA detection. Methods: 110 MIBC patients undergoing NAC and RC were enrolled. cfDNA was extracted from ~1mL plasma (n=978) and procured from longitudinal plasma sampling during NAC and pre-RC and post-RC. WGS of tumor/germline pairs (30x/20x) and plasma cfDNA (>20x) was performed, facilitating detection of genome-wide genomic alterations and quantification of ctDNA using the C2inform method. Results: For each patient we developed a tumor-informed WGS model by integrating genome-wide mutation and copy number variation data coupled with advanced signal processing and AI-based error suppression. Patient-specific somatic variant patterns were used for detection and measuring ctDNA levels in low-input blood samples by WGS. Post-RC ctDNA analysis identified patients with recurrence with 78% sensitivity and 95% specificity and with a median lead time over radiographic imaging of 118 days (full follow-up included). When restricting follow-up time to 12 months following the latest ctDNA test, a sensitivity of 86% and specificity of 94% was achieved. ctDNA status was associated with recurrence-free (p<0.0001) and overall survival (p<0.0001). Furthermore, ctDNA clearance during NAC was also associated with recurrence-free survival (p=0.0051). The APOBEC associated signatures SBS2 and SBS13 were identified as the primary contributors to the mutational landscape of the primary tumors and TP53, KMT2D, PIK3CA, RB1 and KDM6A were the main affected driver genes. Half of the tumors displayed whole genome doubling. Moreover, our analysis of plasma samples after treatment showed the presence of chemotherapy-induced mutational signatures not present in the primary tumor, along with increases in copy numbers on chromosomes 19q and 20, and a focal amplification of the FGFR3 gene on chromosome 4. Conclusions: Our results highlight the clinical potential of personalized genome-wide mutation integration as an ultra-sensitive, non-invasive method for MRD detection and treatment response monitoring.

KRAS Mutation Analysis Using Cell-free DNA of Pancreatic Cancer.

Detection of pancreatic cancer using small samples of the pancreas obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) remains a challenge. The purpose of this study was to investigate whether the detection of KRAS mutations in cell-free DNA (cfDNA) extracted from supernatants of liquid-based fixed cytology (LBC) specimens obtained using EUS-FNA in solid pancreatic cancer can be an auxiliary test for differential diagnosis. The purpose of this study was to investigate whether the detection of KRAS mutations in cell-free DNA (cfDNA) extracted from supernatants of liquid-based fixed cytology (LBC) specimens obtained using EUS-FNA in solid pancreatic cancer can be an auxiliary test for differential diagnosis. This was a single-institution cohort study that included 50 patients with pancreatic lesions. cfDNA was isolated from the supernatant of fixed LBC samples, and KRAS mutation status was compared between cfDNA samples and FFPE small fragment tissues. Of the 50 cfDNA samples, 84% (42/50) were valid. KRAS mutations were detected in 57.1% (24/42) of the 42 valid samples. The sensitivity, specificity, and accuracy of KRAS mutation detection using cfDNA samples in the pancreatic lesions were 63.2% (24/38), 100.0% (4/4), and 66.7% (28/42), respectively. KRAS mutation status between FFPE small tissues and cfDNA samples were comparable. Gene mutation analysis using cfDNA from the supernatant of fixed LBC samples is an effective ancillary diagnostic tool for pancreatic cancer.