Mutant Allele Fraction Research Articles

Predicting recurrent glioblastoma clinical outcome to immune checkpoint inhibition and low-dose bevacizumab with tumor in situ fluid circulating tumor DNA analysis

ObjectiveMost recurrent glioblastoma (rGBM) patients do not benefit from immune checkpoint inhibition, emphasizing the necessity for response biomarkers. This study evaluates whether tumor in situ fluid (TISF) circulating tumor DNA (ctDNA) could serve as a biomarker for response to low-dose bevacizumab (Bev) plus anti-PD-1 therapy in rGBM patients, aiming to enhance systemic responses to immunotherapy.MethodsIn this phase II trial, 32 GBM patients with first recurrence after standard therapy were enrolled and then received tislelizumab plus low-dose Bev each cycle. TISF samples were analyzed for ctDNA using a 551-gene panel before each treatment.ResultsThe median progression-free survival (mPFS) and overall survival (mOS) were 8.2 months (95% CI, 5.2–11.1) and 14.3 months (95% CI, 6.5–22.1), respectively. The 12-month OS was 43.8%, and the objective response rate was 56.3%. Patients with more than 20% reduction in the mutant allele fraction and tumor mutational burden after treatment were significantly associated with better prognosis compared to baseline TISF-ctDNA. Among detectable gene mutations, patients with MUC16 mutation, EGFR mutation & amplification, SRSF2 amplification, and H3F3B amplification were significantly associated with worse prognosis.ConclusionsLow-dose Bev plus anti-PD-1 therapy significantly improves OS in rGBM patients, offering guiding significance for future individualized treatment strategies. TISF-ctDNA can monitor rGBM patients' response to combination therapy and guide treatment.Clinical trial registrationThis trial is registered with ClinicalTrials.gov, NCT05540275.

Urine Liquid Biopsies via Highly Integrated Digital PCR System for Accurate Detection of Bladder Cancer

AbstractBladder cancer (BC) is a prevalent urological tumor with high recurrence rates, requiring long‐term monitoring. Although cystoscopy is the primary diagnostic method, its invasiveness and cost hinder routine screening and follow‐up. This study aimed to develop a novel diagnostic tool utilizing newly developed on‐chip heating dPCR platform, which features integrated and rapid temperature control capabilities, for non‐invasive BC detection. The dPCR platform is improved by integrating a multi‐color detection system, enabling precise quantification of mutant allelic fraction (MAF) of TERT promoter mutations with a limit of detection (LOD) of 0.29%. Diagnostic performance is enhanced by integrating the NRN1 methylation biomarker and employing machine learning to optimize biomarker weighting. Testing the model on urine samples from controls (n = 35) and BC patients (n = 41) yielded a sensitivity of 0.92, specificity of 0.94, and an AUC of 0.98, surpassing conventional cytology in sensitivity while maintaining comparable specificity. Furthermore, the model effectively differentiated between normal controls and different stages, achieving accuracies of 0.92, 0.71, and 0.79 for NC, stage I, and stage II+ respectively. These findings suggest the proposed dPCR assays could serve as a sensitive and non‐invasive approach for BC detection in clinical practice.

Phase II study of a PARP inhibitor, talazoparib, in HER2- metastatic breast cancer (MBC) with a somatic BRCA1/2mutation identified in a cell-free DNA or tumor tissue genotyping assay.

TPS1143 Background: PARP inhibitors are approved for germline BRCA1/2mutant MBC but the applicability of these well-tolerated oral targeted therapies is limited, as germline BRCA1/2mutations account for 5% of breast cancer. We demonstrated that pathogenic somatic BRCA1/2mutations are detectable in cell-free DNA and tumor tissue genotyping assays in a subset of HER2- MBC, in patients who are not known germline BRCA1/2carriers. In a circulating tumor cell culture model derived from a patient with a pathogenic somatic BRCA1 mutation, we demonstrated that the growth inhibitory effect of a PARP inhibitor paralleled germline BRCA1/2mutant cultures. We hypothesize that somatic BRCA1/2mutations may be amenable to PARP inhibition. In this study, we are evaluating the efficacy of a PARP inhibitor in somatic BRCA1/2mutant MBC. The results may expand the population of patients who benefit from PARP inhibitors. Methods: In this phase II investigator-initiated clinical trial, 30 patients with HER2- MBC with pathogenic somatic BRCA1/2mutations (in the absence of a known germline BRCA1/2mutation) identified in a CLIA certified cell-free DNA or tumor tissue genotyping assay are being enrolled at 7 academic centers. Mutations are vetted for pathogenicity by genetic counselors using validated genomic databases such as ClinVar. Patients with metastatic triple-negative or hormone receptor positive (HR)+/HER2- breast cancer who received ≥1 prior therapy in the metastatic setting are eligible. Any number of prior therapies are acceptable, including a prior platinum (in absence of disease progression). Patients must have adequate baseline performance status and organ function. Patients are treated with talazoparib 1 mg/day until disease progression. Disease assessments occur at baseline and every 3 months via CT chest, abdomen, and pelvis, and bone scan. The primary endpoint is progression free survival (PFS) via RECIST 1.1. This study has 81% power to demonstrate the 12-week PFS is ≥53% (4% alpha). Secondary endpoints include objective response rate and toxicity (NCI CTCAE v 5.0). Exploratory endpoints, determined using serial cell-free DNA collected at baseline and monthly, include identifying resistance mutations, and evaluating the impact of mutant allele fraction and BRCA1/2reversion mutations on response. The Cancer Risk B (CR-B) assay, a novel method to identify double-strand break repair mutations in circulating blood cells, will be correlated with response. The study (NCT03990896) is open at Massachusetts General Hospital, University of California San Francisco, MD Anderson, Emory, Northwestern, and Vanderbilt and will open at Cornell, with 17 patients enrolled as of 2/2024. Clinical trial information: NCT03990896 .

Impact of intratumor heterogeneity (ITH) on survival outcomes in patients with non-small cell lung cancer (NSCLC) with high tumor mutational burden (TMB-H).

e20519 Background: Heterogeneity among cancer cells within a tumor significantly impacts cancer development and resistance to treatment. Currently, no standard measure exists for assessing ITH in routine clinical settings. In this study, we applied a quantitative metric to assess intratumor genetic diversity in NSCLC, using a large, multicenter database. Methods: We accessed the Tempus database for next-generation sequencing results from patients with NSCLC who underwent immune checkpoint inhibitor (ICIs) monotherapy as their first-line treatment. We calculated the mutant-allele tumor heterogeneity (MATH), defined as the ratio of the distribution's range to the median of mutant-allele fractions across tumor-specific mutated loci. The primary outcomes were progression-free survival (PFS) and overall survival (OS). Results: A total of 160 pembrolizumab Significant differences in PFS between high and low MATH groups (MATH-H and MATH-L, respectively), using the 50th percentile as the cutoff, were observed among patients with stage 4 adenocarcinoma who had TMB-H above the 25th percentile (Table). In this subgroup, with 27 patients in the MATH-H/TMB-H group and 22 in the MATH-L/TMB-H group, the MATH-H/TMB-H group showed improved PFS (median PFS 5.19 vs. 1.61 months; p = 0.02). Differences in PFS were also observed among patients with TP53, KEAP1, and MET mutations (p < 0.001, p = 0.02, and p = 0.004, respectively). TP53 mutations were found in 27.6% of the MATH-H group and 27.8% of the MATH-L group, KEAP1 mutations in 8.2% and 10.1%, respectively, and MET mutations in 5.1% and 8.9%, respectively. No significant PFS differences were found in groups with low TMB, other stages, or squamous histology. Conclusions: Our findings suggest that higher genetic diversity within stage 4 TMB-H lung adenocarcinoma, as quantified by MATH, might be linked to improved clinical outcomes among those who receive pembrolizumab as first-line therapy and supports further exploration of MATH as a predictive/prognostic biomarker. [Table: see text]

Somatic KMT2D loss-of-function mutations in lung squamous cell carcinoma: a single-center cohort study.

The significant progress has been made in targeted therapy for lung adenocarcinoma (LUAD) in the past decade. Only few targeted therapeutics have yet been approved for the treatment of lung squamous cell carcinoma (LUSC). Several higher frequency of gene alterations are identified as potentially actionable in LUSC. Our work aimed to explore the complex interplay of multiple genetic alterations and pathways contributing to the pathogenesis of LUSC, with a very low frequency of a single driver molecular alterations to develop more effective therapeutic strategies in the future. We retrospectively analyzed the targeted next-generation sequencing (NGS) data (approximately 600 genes) of 335 patients initially diagnosed with non-small cell lung cancer (NSCLC) at our institution between January 2019 and March 2023 and explored the somatic genome alteration difference between LUSC and LUAD. We analyzed that the presence of loss-of-function (LoF) mutations (nonsense, frameshift, and splice-site variants) in histone-lysine N-methyltransferase 2D (KMT2D) was much more prevalent in LUSC (11/53, 20.8%) than in LUAD (6/282, 2.1%). Moreover, our data indicated TP53 co-mutated with KMT2D LoF in 90.9% (10/11) LUSC and 33.3% (2/6) LUAD. Notably, the mutation allele fraction (MAF) of KMT2D was very similar to that of TP53 in the co-mutated cases. Genomic profiling of driver gene mutations of NSCLC showed that 81.8% (9/11) of the patients with LUSC with KMT2D LoF mutations had PIK3CA amplification and/or FGFR1 amplification. Our results prompted that somatic LoF mutations of KMT2D occur frequently in LUSC, but are less frequent in LUAD and therefore may potentially contribute to the pathogenesis of LUSC. Concurrent TP53 mutations, FGFR1 amplification, and PIK3CA amplification are very common in LUSC cases with KMT2D LoF mutations. It needs more deeper investigation on the interplay of the genes and pathways and uses larger cohorts in the future.

Microfluidics-based EGFR mutation detection and its implication in the resource-limited clinical setting.

Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 μ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.

Abstract PS15-03: Vepdegestrant, a PROteolysis TArgeting Chimera (PROTAC) estrogen receptor (ER) degrader, plus palbociclib in ER–positive/human epidermal growth factor receptor 2 (HER2)–negative advanced breast cancer: phase 1b cohort

Abstract Background: Vepdegestrant (ARV-471) is an oral PROTAC ER degrader. In a phase 1/2 study (NCT04072952), vepdegestrant monotherapy had a favorable safety profile and encouraging clinical activity, and showed robust ER degradation. The phase 1b cohort of this study is evaluating vepdegestrant in combination with the cyclin-dependent kinase (CDK)4/6 inhibitor palbociclib (palbo). In xenograft models, vepdegestrant plus palbo showed substantially greater tumor growth inhibition vs fulvestrant plus palbo, supporting investigation in patients with breast cancer. Methods: Eligible patients for the phase 1b combination cohort had ER+/HER2- locally advanced/metastatic breast cancer and had received ≥1 prior endocrine therapy and ≤2 chemotherapy regimens for advanced disease; prior CDK4/6 inhibitor treatment was permitted. Vepdegestrant was given orally once daily (QD) continuously at doses of 180 mg (n=2), 200 mg (n=21), 400 mg (n=3), or 500 mg (n=20); palbo 125 mg was given orally QD for 21 days followed by 7 days off treatment in 28-day cycles. The primary endpoints were dose-limiting toxicities (DLTs) in the first cycle and safety (adverse events [AEs] and laboratory abnormalities). Enrollment in these 4 dose levels is complete; we report data as of June 6, 2023. Results: Across 46 patients in the phase 1b cohort, 45 (97.8%) patients were female with a median age of 62.0 y (range: 29–78). Patients had received a median of 4 prior therapies (range: 1–11) in any setting, including 87.0% prior CDK4/6 inhibitors (78.3% prior palbo), 80.4% prior fulvestrant, and 76.1% prior chemotherapy (45.7% in the metastatic setting). There were no DLTs. Treatment-emergent AEs (TEAEs) leading to dose reductions or discontinuation of vepdegestrant occurred in 5 and 4 patients, respectively. TEAEs leading to dose reductions or discontinuation of palbo occurred in 34 and 8 patients, respectively. Grade 3/4 treatment-related AEs (TRAEs) to either vepdegestrant or palbo in ≥10% of patients were neutropenia (89.1%), decreased white blood cell count (15.2%), and decreased platelet count (10.9%); there were no grade 5 TRAEs and no patients had febrile neutropenia. The clinical benefit rate (rate of confirmed complete response, partial response, or stable disease ≥24 wks) in patients treated with vepdegestrant plus palbo was 63.0% (95% CI: 47.5–76.8). The objective response rate in evaluable patients with measurable disease at baseline (n=31) was 41.9% (95% CI: 24.5–60.9). Pharmacokinetics (PK) showed dose-dependent exposure for vepdegestrant, consistent with data for vepdegestrant administered as monotherapy; palbo exposure was similar across dose levels of vepdegestrant and modestly higher compared with historical palbo PK data. Circulating tumor DNA levels were evaluated in patients who received vepdegestrant 200 mg QD plus palbo and demonstrated substantial and sustained decreases in ESR1 mutant allele fraction across multiple treatment cycles. Conclusions: The combination of vepdegestrant plus palbo showed promising clinical activity in heavily pretreated patients with ER+/HER2- advanced breast cancer who had received extensive prior treatment. The safety profile of vepdegestrant plus palbo was generally consistent with the known safety profiles of the 2 agents except for an increased occurrence of grade 3/4 neutropenia, which was readily managed with laboratory monitoring and dose reductions of palbo. There is an ongoing study lead-in to the global VERITAC-3 study (NCT05909397) that is evaluating 2 doses of palbo (100 mg and 75 mg) in combination with vepdegestrant 200 mg QD to determine the recommended phase 3 combination to compare with letrozole plus palbo as first-line treatment for ER+/HER2- advanced breast cancer. Citation Format: Erika Hamilton, Rinath Jeselsohn, Sara Hurvitz, Dejan Juric, Hyo Han, Melinda Telli, George Zahrah, Rita Nanda, Yuanyuan Zhang, Weiwei Tan, Elizabeth Duperret, Eric Zhi, Cecile Mather, Anne Schott. Vepdegestrant, a PROteolysis TArgeting Chimera (PROTAC) estrogen receptor (ER) degrader, plus palbociclib in ER–positive/human epidermal growth factor receptor 2 (HER2)–negative advanced breast cancer: phase 1b cohort [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 PS15-03.

Abstract PO5-14-05: Circulating tumor DNA fraction correlates with residual cancer burden post-neoadjuvant chemotherapy in triple negative breast cancer patients

Abstract Background: Pathologic response after preoperative/neoadjuvant chemotherapy is a continuum that can range from no residual cancer to extensive disease. Viable cancers can shed tumor DNA into the blood and we hypothesized that plasma ctDNA levels can reflect pathologic response as continuum. Patients and methods: Post-neoadjuvant plasma samples were obtained after completion of chemotherapy but before surgery from patients with stage I to III TNBC who received treatment with durvalumab concurrent with weekly nab-paclitaxel followed by dose dense doxorubicin/cyclophosphamide in a single arm neoadjuvant clinical trial (NCT02489448). Up to 3 mL of blood was collected into EDTA- and heparin- containing vacutainer tubes, and plasma cfDNA was extracted and twice purified in the lab at Predicine Inc. Pathologic response was quantified on a continuous scale using the Residual Cancer Burden (RCB) score, and also categorized as RCB-0 (complete pathologic response, n=21), -I (minimal residual cancer, n=7), -II (moderate residual cancer, n=13), and -III (extensive residual cancer, n=3). Median follow-up was 35 months (range 25-70 months). Tumor whole exome sequencing (WES) data was previously generated using HiSeq 4000 at the Yale Center for Genome Analysis (dbGaP: PRJNA558949). A personalized MRD panel of up to 60 personalized variants per patient was designed based on the tumor WES data, and ultra-deep next-generation sequencing (100,000X) of plasma cfDNA was performed using the PredicineBEACON assay for MRD detection by Predicine. CtDNA positivity (MRD) was defined as a weighted score equivalent to 2 or more tumor variants detected in the plasma. cfDNA tumor fraction was measured based on mutant allele fraction of observed variants. Adjusted tumor fraction could be confidently measured for 37 out of 44 patients that had enough confidently called personalized ctDNA variants. We compared tumor fraction versus RCB score as continuous variables, and also compared tumor fraction between RCB categories as ordinal variables. Invasive disease-free survival was examined by Kaplan-Meier analysis. Bioinformatic data analysis was performed using Graphpad Prism and R. Results: 44 of 48 plasma samples yielded successful cfDNA generation and sequencing. Six patients (13.6%) were MRD positive. Among these 6 cases, 3 had RCB-0 response (14.3% of RCB 0 cases), 1 had RCB II response (7.7% of RCB II cases), and 2 had RCB III response (66.7% of RCB III cases). The median cfDNA tumor fraction of patients with pathological complete response to treatment (pCR) was significantly lower than patients with residual disease (RD) (0.06% vs. 0.3%, p=0.02). There was a positive correlation between RCB scores and tumor fraction (r= 0.45, Spearman). Tumor fraction was more strongly correlated with later recurrence than a binary ctDNA positivity call. Tumor fraction was significantly higher in patients that later experienced local or metastatic recurrence (n=8) compared to those with no evidence of disease (n=29) (Wilcoxon test, p=0.02). Both ctDNA positive RCB III cases had metastatic recurrence while the remaining 4 ctDNA positive cases currently remain recurrence-free. Mutational genomic landscape of both tumor WES and plasma ctDNA samples identified hotspot mutations in 19 cancer-associated genes. Novel hot-spot mutations were observed in the plasma of two patients following treatment. TP53 mutations were highly prevalent in tissue and plasma, however no significant association of mutation of any gene with treatment response was detected. Conclusions: PredicineBeacon MRD assay was successfully performed on 3 mL plasma samples collected using from EDTA and heparin-containing tubes. Tumor fraction correlated with residual disease, RCB score and disease recurrence. Citation Format: Naing Lin Shan, Billie Gould, Kim Blenman, Julia Foldi, Pan Du, Frank Zhang, Myles Walsh, Lajos Pusztai. Circulating tumor DNA fraction correlates with residual cancer burden post-neoadjuvant chemotherapy in triple negative breast cancer patients [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 PO5-14-05.

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.

Abstract 6557: Longitudinal cell-free tumor load dynamics represent an early endpoint for immunotherapy response in non-small cell lung cancer

Abstract Background: Liquid biopsies can inform treatment strategies by rapidly and accurately assessing tumor burden; with emerging data supporting the clinical utility of circulating cell-free tumor DNA (ctDNA) as an early endpoint of immunotherapy response. Methods: We performed targeted error-correction sequencing of ctDNA (n=330) and matched white blood cell (WBC) DNA (n=109) from 109 patients with advanced non-small cell lung cancer (NSCLC) who received immune checkpoint inhibitor monotherapy (n=79) or immunotherapy-containing combination regimens (n=30). Variant cellular origin was determined using a tumor-independent WBC DNA-informed approach. The maximum mutant allele fraction (maxMAF) of tumor-derived mutations was tracked longitudinally to assess changes in total cell-free tumor load (cfTL). Results: Among 3,418 variants with resolved cellular origin, 2,452 (72%) were tumor-derived, 125 (4%) were germline, while 841 (25%) were related to clonal haematopoiesis (CH). Cell-free DNA fragments harbouring tumor-derived mutations were significantly shorter than fragments containing wild-type alleles; however, fragment length alone was insufficient to distinguish CH variants from wild-type. Overall, 97 (89%) patients with >1 detectable tumor mutation were considered for cfTL tracking and molecular response assessment. Clearance of cfTL indicated molecular response (mR) whereas persistence indicated molecular progression (mPD). Time-course analyses, aiming to establish the optimal timing for molecular response assessment, revealed that early cfTL clearance within 3-9 weeks was significantly linked to improved survival outcomes. Notably, blood tumor mutation burden above or below 16 mutations/megabase showed no association with overall survival (OS; log rank p=0.98) or progression-free survival (PFS; log rank p=0.95). Of 81 patients evaluated for early on-therapy molecular responses, 28 (35%) were classified as having an mR, while 53 (65%) patients were classified in the mPD category. The median time to mR was 5 weeks in patients receiving immune checkpoint monotherapy and 6 weeks in patients receiving chemo-immunotherapy combination regimens. Patients in the mR group achieved longer OS (log rank p=0.001) and PFS (log rank p=0.0003) compared to those with mPD. Assessment of landmark PFS at 6 months demonstrated a significant association between ctDNA molecular response and durable clinical benefit (DCB) in patients receiving immune checkpoint monotherapy (p < 0.001, Fisher’s exact test), and a numerical association with DCB in patients on chemo-immunotherapy (p=0.07, Fisher’s exact test). Conclusions: Longitudinal tracking of ctDNA provides a real-time and accurate approach for monitoring immunotherapy response. These findings underscore the clinical utility of employing ctDNA molecular response to inform clinical decision-making. Citation Format: Lavanya Sivapalan, Bahar Alipanahi, Archana Balan, Jamie E. Medina, Noushin Niknafs, Erica Peters, Jaime Wehr, Alissa Konicki, Mimi Najjar, Bryan Chesnick, Gavin Pereira, Sarah Rongione, Nisha Rao, Michael Rongione, Brittany Shavatt, Lorenzo Rinaldi, Benjamin Levy, Victor E. Velculescu, Patrick M. Forde, Valsamo Anagnostou. Longitudinal cell-free tumor load dynamics represent an early endpoint for immunotherapy response in non-small cell lung cancer [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 6557.

Abstract 988: Genome-wide repeat landscapes in cancer and cell-free DNA

Abstract Introduction: Repeat sequences comprise >50% of the human genome and structural and epigenetic changes in these regions are implicated in cancer. However, no systematic analysis of the compendium of repeat sequences has ever been performed in human cancer or cell-free DNA (cfDNA), largely due to the inability to identify and quantify repeat sequences genome-wide. We describe here the first comprehensive analysis of genome-wide repeat landscapes in cancer and demonstrate their utility in cfDNA liquid biopsies. Methods: We developed ARTEMIS (Analysis of RepeaT EleMents in dISease) an alignment-free, genome-wide approach for analyzing repeat landscapes in short read sequencing. This approach uses a de novo search of short sequences (kmers) in the telomere to telomere (chm13) reference genome to identify 1.2 billion 24-mers uniquely defining 1280 individual repeat types occurring genome-wide across 57 subfamilies and 6 families. We analyzed ARTEMIS kmers in whole genome sequences of 525 matched tumor/normal pairs from breast, colorectal, liver, lung, ovarian, cervical, prostate, thyroid, head and neck, gastric, and bladder cancers in the Pan Cancer Analysis of Whole Genomes (PCAWG), and in low coverage (1-2x) whole genome sequences of 1450 cfDNA samples from individuals with and without 8 types of cancer. Results: Analysis of ARTEMIS kmer repeat landscapes in 525 PCAWG tumors identified changes in all 1280 repeat element types, including 820 novel elements not previously known to be altered in cancer. A median of 807 repeat elements (range 246-1280) were altered in each tumor compared to its matched normal. The majority of changes were in repeat elements not previously described as altered in tumorigenesis and were most frequently found within Satellites, LINEs and SINEs, though changes were also observed in LTRs, Transposable Elements, and RNA Elements. A cross-validated cfDNA model using repeat landscapes (ARTEMIS) and fragmentation features (DELFI) detected individuals in a diagnostic cohort (n=287) across all stages of lung cancer with high performance (AUC 0.91, 95% CI 0.88-0.95) and was externally validated in a separate population (n=513). The locked model generated scores that correlated with circulating tumor mutant allele fractions for patients (n=19) undergoing targeted lung cancer therapy (r=0.80, p<2.2e-16), and stratified progression-free survival (p<0.001). ARTEMIS repeat landscape analyses of cfDNA also detected liver cancer in a high-risk cohort (n=208) of individuals with cirrhosis or viral hepatitis (AUC 0.91, 95% CI 0.87-0.95), and identified tissue of origin among seven tumor types (n=423). Conclusions: ARTEMIS reveals genome-wide repeat landscapes in human cancer, including in 820 novel elements not previously known to be altered in tumorigenesis. These repeat landscapes that can now be described are evaluable in the circulation and provide an avenue for noninvasive detection and characterization of cancer. Citation Format: Akshaya Annapragada, Noushin Niknafs, James R. White, Daniel C. Bruhm, Christopher Cherry, Jamie E. Medina, Vilmos Adleff, Carolyn Hruban, Dimitrios Mathios, Zachariah H. Foda, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. Genome-wide repeat landscapes in cancer and 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 988.

First report of ATP2A2 somatic mosaicism occurring during embryogenesis in transient acantholytic dermatosis

AbstractTransient acantholytic dermatosis (TAD) is a relatively common skin disease that typically affects older individuals, which shows clinical and histologic similarities to autosomal dominant Darier disease. TAD was recently shown to be caused by somatic ATP2A2 damaging variants. In this study, we performed Sanger sequencing and droplet digital PCR (ddPCR) in a Japanese elderly male with TAD to identify ATP2A2 somatic mosaicism occurring during embryogenesis for the first time and mutant allelic fraction (MAF). Sanger sequencing revealed a known heterozygous substitution c.1645C>T, p.Arg549* in ATP2A2 from blood and the affected skin containing the epidermis and dermis, whereas the signals of the mutated allele were weaker, suggesting that discrepancy of signal intensities demonstrates the presence of somatic mosaicism. By ddPCR, the normal and mutant allele were identified in genomic DNAs and the MAFs were 20.1% (affected skin) and 14.7% (blood), respectively. In the present case, somatic mosaicisms observed in ectodermal (epidermis) and mesodermal (dermis and blood) origin DNA can be explained by a mutational event during the early‐stage differentiation of embryonic epiblast in embryogenesis, and mutant embryonic cells somewhat preferentially differentiate to form the epidermis, rather than the dermis and blood. Disrupted intracellular Ca2+ homoeostasis through clinical deterioration together with ATP2A2 somatic mosaicism in this patient might result in transient skin eruptions.

Development and application of an ultra-sensitive ctDNA mutation profiling assay in monitoring therapy response and drug resistance.

264 Background: Next-generation sequencing (NGS) assays offer efficient high-throughput profiling methods for analyzing circulating tumor DNA (ctDNA) in blood or other liquid biopsy specimens. The ctDNA mutant allele fractions of patients after treatment is often very low and below the detection limit of regular cfDNA assays. Here we report the development and clinical application of a CLIA-certified ultra-sensitive NGS cfDNA assay in detecting the low-frequency ctDNA mutations and monitoring therapy response and drug resistance in patients with metastatic gastroesophageal adenocarcinoma under combined blockade treatments. Methods: PredicineCARE ULTRA cfDNA assay with 100,000X sequencing depth was validated under CLIA settings. Limit of blank (LoB) and limit of detection (LoD) were established by profiling 38 healthy donor plasma samples, 23 contrived human cfDNAs samples from patients with various cancers, and 30 replicates of well-established ctDNA reference standards. In silico down-sampling of FASTQ files was carried out to test the sensitivity of this assay with a lower coverage (50,000X). This assay was then adopted for longitudinal sequencing of 57 serial plasmas from 6 patients with HER2+ gastroesophageal adenocarcinomas (GEC) who underwent combined treatments (capecitabine, oxaliplatin, bevacizumab and trastuzumab) at different time points (baseline, response, and progression). Results: PredicineCARE ULTRA cfDNA assay exhibited exceptional sensitivity in calling single-nucleotide variants and indels, with a 98.0% detection rate at a LoD of 0.075% mutant allele frequency (MAF) for 60 ng cfDNA. This is significantly lower than the LoD (0.25% MAF) of a standard PredicineCARE cfDNA assay. The ultra-sensitive assay also displayed decent analytical specificity (>99.99%) and precision (intra-assay 100%; inter-assay 99.41%). In 6 GEC patients’ longitudinal samples, PredicineCARE ULTRA cfDNA assay detected 36 additional somatic mutations with the MAFs spanning in a range from 0.024% to 0.1% beyond 124 baseline mutations identified using standard PredicineCARE. In two representative cases presented here, all baseline mutations (genes TP53 and PIK3CA etc.) were consistently reported in the follow-up time points with a noticeable MAF reduction (lowest MAFs from 0.046% to 0.024%) during response. Subsequent elevations were observed in more genes, including low-frequency mutations (KRAS and ERBB2) not detected at baseline or only detected in PredicineCARE ULTRA. Conclusions: This study demonstrated the promise and clinical utility of the ultra-sensitive cfDNA molecular profiling at 100,000X sequencing depth in treatment selection, therapy monitoring, and drug resistance studies.

Rapid detection of mutations in CSF-cfTNA with the Genexus Integrated Sequencer.

Genomic alterations are fundamental for molecular-guided therapy in patients with breast and lung cancer. However, the turn-around time of standard next-generation sequencing assays is a limiting factor in the timely delivery of genomic information for clinical decision-making. In this study, we evaluated genomic alterations in 54 cerebrospinal fluid samples from 33 patients with metastatic lung cancer and metastatic breast cancer to the brain using the Oncomine Precision Assay on the Genexus sequencer. There were nine patients with samples collected at multiple time points. Cell-free total nucleic acids (cfTNA) were extracted from CSF (0.1-11.2ng/μl). Median base coverage was 31,963× with cfDNA input ranging from 2 to 20ng. Mutations were detected in 30/54 CSF samples. Nineteen (19/24) samples with no mutations detected had suboptimal DNA input (< 20ng). The EGFR exon-19 deletion and PIK3CA mutations were detected in two patients with increasing mutant allele fraction over time, highlighting the potential of CSF-cfTNA analysis for monitoring patients. Moreover, the EGFR T790M mutation was detected in one patient with prior EGFR inhibitor treatment. Additionally, ESR1 D538G and ESR1::CCDC170 alterations, associated with endocrine therapy resistance, were detected in 2 mBC patients. The average TAT from cfTNA-to-results was < 24h. In summary, our results indicate that CSF-cfTNA analysis with the Genexus-OPA can provide clinically relevant information in patients with brain metastases with short TAT.

RLP system: A single-tube two-step approach with dual amplification cascades for rapid identification of EGFR T790M

BackgroundThe detection of cancer gene mutations in biofluids plays a pivotal role in revolutionizing disease diagnosis. The presence of a large background of wild-type sequences poses a challenge to liquid biopsy of tumor mutation genes. Suppressing the detection of wild-type sequences can reduce their interference, however, due to the minimal difference between mutant and wild-type sequences (such as single nucleotide variants differing by only one nucleotide), how to suppress the detection of wild-type sequences to the greatest extent without compromising the sensitivity of mutant sequence detection remains to be explored. SignificanceThe RLP system addresses the incompatibility between RPA and RT-PCR reactions through a physical separation strategy. Besides, due to the remarkable flexibility of locked nucleic acid probes, the RLP system emerges as a potent tool for detecting mutations across diverse genes. It excels in sensitivity and speed, tolerates plasma matrix, and is cost-effective. This bodes well for advancing the field of precision medicine. ResultsThe recombinase-assisted locked nucleic acid (LNA) probe-mediated dual amplification biosensing platform (namely RLP), which combines recombinase polymerase amplification (RPA) and LNA clamp PCR method in one tube, enabling highly sensitive and selective detection of EGFR T790M mutation under the help of well-designed LNA probes. This technique can quantify DNA targets with a limit of detection (LoD) at the single copy level and identify point mutation with mutant allelic fractions as low as 0.007 % in 45 min. Moreover, RLP has the potential for the direct detection of plasma samples without the need for nucleic acid extraction and the cost of a single test is less than 1USD. Furthermore, the RLP system is a cascading dual amplification reaction conducted in a single tube, which eliminates the risk of cross-contamination associated with opening multiple tubes and ensures the reliability of the results.

Accurate Detection of Multiple Tumor Mutations in Formalin-Fixed Paraffin-Embedded Tissues by Coupling Sequence Artifacts Elimination and Mutation Enrichment With MeltArray

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary source of DNA for companion diagnostics (CDx) of cancers. Degradation of FFPE tissue DNA and inherent tumor heterogeneity constitute serious challenges in current CDx assays. To address these limitations, we introduced sequence artifact elimination and mutation enrichment to MeltArray, a highly multiplexed PCR approach, to establish an integrated protocol that provides accuracy, ease of use, and rapidness. Using PIK3CA mutations as a model, we established a MeltArray protocol that could eliminate sequence artifacts completely and enrich mutations from 23.5- to 59.4-fold via a single-reaction pretreatment step comprising uracil-DNA-glycosylase excision and PCR clamping. The entire protocol could identify 13 PIK3CA hotspot mutations of 0.05% to 0.5% mutant allele fractions within 5 hours. Evaluation of 106 breast cancer and 40 matched normal FFPE tissue samples showed that all 47 PIK3CA mutant samples were from the cancer tissue, and no false-positive results were detected in the normal samples. Further evaluation of 105 colorectal and 40 matched normal FFPE tissue samples revealed that 11 PIK3CA mutants were solely from the cancer sample. The detection results of our protocol were consistent with those of the droplet digital PCR assays that underwent sequence artifact elimination. Of the 60 colorectal samples with next-generation sequencing results, the MeltArray protocol detected 2 additional mutant samples with low mutant allele fractions. We conclude that the new protocol provides an improved alternative to current CDx assays for detecting tumor mutations in FFPE tissue DNA.

Lasofoxifene versus fulvestrant for ER+/HER2− metastatic breast cancer with an ESR1 mutation: results from the randomized, phase II ELAINE 1 trial

Acquired estrogen receptor alpha (ER/ESR1) mutations commonly cause endocrine resistance in ER+ metastatic breast cancer (mBC). Lasofoxifene, a novel selective ER modulator, stabilizes an antagonist conformation of wild-type and ESR1-mutated ER-ligand binding domains, and has antitumor activity in ESR1-mutated xenografts. In this open-label, randomized, phase II, multicenter, ELAINE 1 study (NCT03781063), we randomized women with ESR1-mutated, ER+/human epidermal growth factor receptor 2 negative (HER2-) mBC that had progressed on an aromatase inhibitor (AI) plus a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) to oral lasofoxifene 5 mg daily or IM fulvestrant 500 mg (days 1, 15, and 29, and then every 4 weeks) until disease progression/toxicity. The primary endpoint was progression-free survival (PFS); secondary endpoints were safety/tolerability. A total of 103 patients received lasofoxifene (n= 52) or fulvestrant (n= 51). The most current efficacy analysis showed that lasofoxifene did not significantly prolong median PFS compared with fulvestrant: 24.2 weeks (∼5.6 months) versus 16.2 weeks (∼3.7 months; P= 0.138); hazard ratio 0.699 (95% confidence interval 0.434-1.125). However, PFS and other clinical endpoints numerically favored lasofoxifene: clinical benefit rate (36.5% versus 21.6%; P= 0.117), objective response rate [13.2% (including a complete response in one lasofoxifene-treated patient) versus 2.9%; P= 0.124], and 6-month (53.4% versus 37.9%) and 12-month (30.7% versus 14.1%) PFS rates. Most common treatment-emergent adverse events with lasofoxifene were nausea, fatigue, arthralgia, and hot flushes. One death occurred in the fulvestrant arm. Circulating tumor DNA ESR1 mutant allele fraction (MAF) decreased from baseline to week 8 in 82.9% of evaluable lasofoxifene-treated versus 61.5% of fulvestrant-treated patients. Lasofoxifene demonstrated encouraging antitumor activity versus fulvestrant and was well tolerated in patients with ESR1-mutated, endocrine-resistant mBC following progression on AI plus CDK4/6i. Consistent with target engagement, lasofoxifene reduced ESR1 MAF, and to a greater extent than fulvestrant. Lasofoxifene may be a promising targeted treatment for patients with ESR1-mutated mBC and warrants further investigation.

Abstract B084: Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification

Abstract Background: Amplification of KRASG12C mutant allele has been reported as one of the resistance mechanisms in tumors progressed to Sotorasib, the FDA approved KRASG12C(OFF) inhibitor. An identical mechanism was observed in one of our H358 xenograft tumors relapsed to Sotorasib. The cell line generated from relapsed tumor represents an appropriate in vitro model to study therapeutic opportunities in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification. Methods: We generated five Sotorasib resistant cell lines using relapsed tumors of Sotorasib-treated H358 xenografts (MR1-MR5) and two vehicle-treated tumors (MV1 and MV2). Whole exome sequencing (WES), Taqman copy number analysis and digital PCR to study mutant allelic fractions were performed to investigate acquired mechanisms of resistance. We have also generated a Dox-inducible system for H358 cells to overexpress KRASG12C protein and mimic KRAS amplification. Results: We observed loss of sensitivity to Sotorasib in models MR1-MR5, when compared to MV1 and MV2. Western blot analysis show EMT-associated signatures in all resistant lines except MR2. The WES of resistant lines shows higher allelic frequency of KRASG12C mutation in MR2, which is homozygous (through loss of heterozygosity). The dPCR analysis further confirmed higher mutant allelic frequency in MR2. Copy-number analysis revealed the presence of more than 10 copies of KRAS in MR2. MR2 model represents the best model to investigate therapeutic options in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification. Both RAS-RAF Proximity Ligation and Raf-RBD pull down assays confirm enhanced RAS signaling in MR2 cells. The combination of KRASG12C(OFF) and SHP2 inhibitors, or single agent KRASG12C(ON) or RASMULTI(ON) inhibitors suppressed enhanced MAPK signaling in MR2 cells and drove tumor suppressions in MR2 mice xenografts. Conclusions: The stronger inhibition of KRAS pathway is necessary to suppress enhanced MAPK signaling in KRASG12C mutated lung cancer that developed secondary resistance to Sotorasib treatment through KRASG12C amplification. These preclinical data suggest that KRASG12C(OFF)/SHP2 inhibitors combo, or single agent KRASG12C(ON)I or RASMULTI(ON)I represent potential therapeutic strategies for these relapsed tumors. Citation Format: Hitendra S Solanki, Denis Imbody, Bina Desai, Ryoji Kato, Paul A Stewart, Yaakov Stern, Anurima Majumder, Liznair Bridenstine, Bhaswati Sarca, Daria Miroshnychenko, Ida Aronchik, Andriy Marusyk, Eric B Haura. Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B084.

Analytical and Clinical Validation of Duoseq, a Novel Assay for Rapid, on-Site Clinical DNA and RNA Sequencing of Hematologic Malignancies

Introduction: Next generation sequencing (NGS) has become a critical component of the workup of malignancies. NGS can provide important diagnostic information including mutations, chromosomal copy number alterations and translocations (from DNAseq) as well as gene expression and fusions (from RNAseq). Incorporation of NGS in the clinic remains difficult. DNAseq and RNAseq remain distinct assays with challenging bioinformatics and turnaround times (TAT) that can last several weeks. Duoseq is a novel assay for combined DNA- and RNAseq in a single workflow that detects single nucleotide variants (SNVs), insertions/deletions (indels), splice site variants (SSVs), structural variants (SVs) and Epstein-Barr virus (EBV) status. Initially implemented for hematologic malignancies, Duoseq assesses nearly all known gene drivers (N=475) and is compatible with both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. It is available as a reagent kit and includes cloud-based bioinformatics, enabling any clinical lab with an Illumina sequencer to perform NGS in-house with a TAT of 2-3 days. Methods/Results: Duoseq was validated for concordance with commercial DNA- and RNAseq kits (New England BioLabs (NEB)). Sequencing libraries were prepared on DNA alone, RNA alone and total nucleic acid (TNA) from cell lines in accordance with kit protocols and captured using the Duoseq panel. Results were equivalent between Duoseq and individual (NEB) library preparations with nearly complete concordance in DNA variants (99.4%, P&amp;lt;10 -6) and RNA expression (R 2=0.97, P&amp;lt;10 -6). Separately, copy number alteration calls across 42 cases had high concordance between Duoseq and Affymetrix array approaches (P&amp;lt;10 -6). Analytical validation was performed at two CLIA laboratories to establish assay precision and reproducibility, sensitivity (LoD), and specificity. Analytical validation was performed on well characterized and control samples that were selected to harbor an array of clinically important variants at known allele frequencies. Precision and reproducibility testing confirmed Duoseq's ability to produce accurate results independent of external variables. We tested inter-lab reproducibility, intra-day and inter-day repeatability and technician-to-technician reproducibility. In each case, Duoseq achieved &amp;gt;98% precision and reproducibility for each variant class (SNVs, indels/SSVs and SVs). We confirmed the LoD using TNA from the same samples diluted in normal TNA to create 3-point curves that spanned our expected LoD. We confirmed the assay to be 100% accurate down to ≥5% mutant allele fraction (MAF) for SNVs, ≥10% MAF for indels/SSVs, and ≥20% tumor purity for SVs. Specificity was additionally assessed by including normal cell lines in each run to determine the incidence of cross-contamination indicated by false positives. Analytical specificity was 100%, indicating no instances of sample cross-contamination. We assessed the clinical accuracy of Duoseq using FFPE myeloid and lymphoid specimens (N=153) collected at both laboratories and characterized via orthogonal methods including Foundation Medicine (SNVs and indels/SSVs), fluorescent in situ hybridization (translocations) and in situ hybridization (EBV status). Duoseq achieved a positive predictive agreement of 98% for SNVs (N=139), 96% for indels/SSVs (N=55), 95% for translocations (N=282), and 100% for EBV status (N=58). Cell of origin (COO) for diffuse large B cell lymphoma (n=42) using Hans criteria and had high concordance (90%) with the COO calls rendered from Duoseq expression data. Given Duoseq's array of measurements and short TAT, we plan a potential application in “match” clinical trials where patients are enrolled in treatment arms based on molecular profiling. In ongoing work to test a potential trial workflow, we enrolled four institutions that will provide cases to a central site for pathology review and molecular profiling enabling trial arm assignment. Specimen enrollment is anticipated to be completed to allow for results to be presented at the meeting. Conclusions: Duoseq is comparable to current methods and provides a cost and time effective option for library preparation.The large gene panel enables testing a broad range of clinical features in a single assay, including genes required for diagnosis and prognostication in heme malignancies. These results support clinical use of Duoseq.

Analytical Performance and Concordance with Next-Generation Sequencing of a Rapid, Multiplexed dPCR Panel for the Detection of DNA and RNA Biomarkers in Non-Small-Cell Lung Cancer

FDA approval of targeted therapies for lung cancer has significantly improved patient survival rates. Despite these improvements, barriers to timely access to biomarker information, such as nucleic acid input, still exist. Here, we report the analytical performance and concordance with next-generation sequencing (NGS) of a highly multiplexed research-use-only (RUO) panel using digital PCR (dPCR). The panel’s analytical sensitivity and reactivity were determined using contrived DNA and RNA mixes. The limit of blank was established by testing FFPE curls classified as negative by pathology. Concordance was established on 77 FFPE samples previously characterized using the Oncomine Precision Assay®, and any discordant results were resolved with Archer Fusionplex® and Variantplex® panels. The analytical sensitivity, reported as the estimated mutant allele fraction (MAF), for DNA targets ranged from 0.1 to 0.9%. For RNA targets (ALK, RET, ROS, NTRK 1/2/3 Fusions, and MET Exon 14 skipping alteration), the analytical sensitivity ranged from 23 to 101 detected counts with 5 ng of total RNA input. The population prevalence-based coverage ranged from 89.2% to 100.0% across targets and exceeded 99.0% in aggregate. The assay demonstrated &gt;97% concordance with respect to the comparator method.