ctDNA Next-generation Sequencing Research Articles

Multi-cancer early detection (MCED) in circulating tumor DNA (ctDNA) using an ultrasensitive mutation-based amplicon next-generation sequencing (NGS) assay.

e15042 Background: Cancer remains a leading cause of mortality globally. For most cancers, routine screening options are still unavailable. Blood-based MCED has emerged as a promising strategy to bridge this gap. Mutation-based NGS in ctDNA detects highly specific cancer signals, an attribute critical for MCED assays. We developed an ultrasensitive mutation-based NGS assay optimized to detect cancer signals in 50 cancers and predict tissue-of-origin (TOO) for up to 21 cancer classes. Methods: An amplicon-based NGS assay incorporating mirror barcodes for error correction was designed to detect cancer-associated alterations (CAAs), including single nucleotide variants (SNVs), insertion-deletion mutations (indels), and viral DNA (EBV and 20 genotypes of HPV), in 50 cancers. Analytical validation was performed using reference standards and clinical validation was performed in a retrospective case-control cohort of 601 subjects comprising cell-free DNA (cfDNA) from 313 self-declared healthy donors and 288 cancer patients (30.9% early-stage, 50.3% late-stage and 9.4% hematolymphoid cancers). Tissue-of-origin (TOO) was predicted for 21 cancer classes using a weighted sum model. Where relevant, matched buffy coat genomic DNA (gDNA) was analyzed to rule out clonal hematopoiesis. Results: Analytical validation demonstrated a limit of detection (LoD) of 0.05% variant allele frequency (VAF) for indels and 0.07% VAF for SNVs in cfDNA. LoD was matched in gDNA (0.04% and 0.05% VAF for indels and SNVs respectively). In clinical validation, the assay demonstrated a specificity of 99.0% (310/313) and an overall sensitivity of 80.9% (233/288), with sensitivities of 64.0%, 91.7%, and 85.2% in early-stage, late-stage, and hematolymphoid cancers respectively. The mean number of CAAs detected in early-stage, late-stage, and hematolymphoid CAA-positive cancers was 2.1, 3.1, and 4.6. Inclusion of viral targets aided the detection of nasopharyngeal (NPC), cervical, and p16+ oropharyngeal (oPSCC) cancers. EBV DNA was detected in 91.7% of NPC cases. In cervical cancer and p16+ oPSCC, HPV DNA (genotypes 16, 18, 33 and 39) was detected in 71.4% and 90.9% of cases respectively. Conversely, none of the p16- oPSCC cancers had detectable HPV DNA. Across the 21 TOO classes, a high confidence prediction could be made in 62.4% (mean 73.6%, range 26.7%-100%) of cases with an overall accuracy of 87.7% (mean 84.9%, range 33.3%-100%). Overall TOO prediction performance was modeled in a simulated cohort based on cancer incidence in Singapore and Hong Kong, giving an accuracy of 87.3% and 88.7% respectively. Conclusions: The mutation-based NGS assay sensitively detects cancer signals and accurately predicts the tumor origin for early-stage, late-stage, and hematolymphoid cancers. Inclusion of viral targets additionally enables early detection of virus-associated cancers.

Clinical utility of an ultra-sensitive ctDNA NGS assay for the detection and monitoring in HR-positive, HER2-negative breast cancer.

e13076 Background: Breast cancer is the most diagnosed cancer in women globally, mainly as hormone receptor HR-positive, HER2-negative. While endocrine therapies have markedly improved clinical outcomes, the threat of disease progression persists. This study introduces an ultra-sensitive circulating tumor DNA (ctDNA) Next-Generation Sequencing (NGS) assay that was designed for in-depth genomic profiling of breast cancer patients undergoing endocrine therapy, aimed at detecting treatment resistance and monitoring disease progression. Methods: In this ongoing investigation, 46 patients with advanced HR-positive, HER2-negative breast cancer were enrolled, and plasma samples were collected subsequent to first-line aromatase inhibitor (AI) treatment and before the initiation of second-line treatment with fulvestrant. The study employed the PredicineCARE ULTRA, an ultra-sensitive liquid biopsy assay with a proprietary NGS panel and exceeds 100,000x coverage, allowing for the ultra-sensitive identification of genomic alterations, surpassing the detection capabilities of standard ctDNA NGS assays with approximately 20,000x coverage. Results: Within the cohort of 46 patients, the ultra-sensitive assay detected 193 somatic mutations, 95 gene copy number variants (CNVs), and a gene fusion involving FGFR3. The most commonly mutated genes, with a prevalence of 20% or more, were TP53 (33%), PIK3CA (26%), ATM (24%), ESR1 (22%), and BRCA2 (20%). These findings were benchmarked against results from a down-sampling algorithm designed to emulate ctDNA assays with 20,000x coverage. The ultra-sensitive assay revealed 31.3% more mutations (193 vs. 147) and 30.1% more CNVs. It was especially proficient in detecting mutations with allele frequencies (MAF) below 0.1%, identifying 10 additional mutations not observed at the standard ctDNA assay sensitivity, with the lowest MAF detected being 0.03% compared to the 0.1% threshold of the standard assay. This heightened sensitivity was evident in genes associated with targeted therapies and drug resistance, identifying 30.7% more variations in PIK3CA and 11.1% more in ESR1, with the lowest MAFs of 0.08% and 0.06%, respectively. The FGFR3-BAIAP2L1 gene fusion was also uniquely detected by the ultra-sensitive assay, with a MAF of 0.09%. Conclusions: The ultra-sensitive ctDNA NGS assay demonstrated superior detection of low-frequency mutations in HR-positive, HER2-negative breast cancer, significantly outperforming standard liquid biopsy assays. This enhanced sensitivity may offer profound implications for identifying more diagnostically positive patients who may benefit from targeted therapy such as PIK3CA and ESR1 inhibitors. This ultra-sensitive ctDNA assay will also enable early detection of treatment resistance and refined disease monitoring, potentially guiding more effective personalized therapies.

Abstract PO3-23-09: ESR1 mutations drive resistance to CDK4/6 inhibitors in ER+ Breast Cancer

Abstract Mutations in ESR1 (the gene encoding the estrogen receptor, ERα) are major drivers of resistance to antiestrogen therapy in ER+ breast cancer. However, it remains unclear whether ESR1 mutations also drive resistance to CDK4/6 inhibitors (CDK4/6i). We analyzed de-identified next generation sequencing (NGS) data of tumor tissue DNA (n=1,820) and circulating tumor (ct) DNA (n=2,138) from patients with ER+/HER2- metastatic breast cancer. NGS of tumor tissue DNA and ctDNA previously had been performed using the Tempus xT tumor assay (595-648 genes) and Tempus xF liquid biopsy (105-523 genes), respectively. Patients were stratified into treated (xT:1,070; xF:1,885) or untreated (xT:750; xF:253) with CDK4/6i prior to biopsy, and untreated patients were restricted to those biopsied within 30 days of metastatic diagnosis. ESR1 was the only gene for which mutations were significantly enriched in patients who received CDK4/6i vs. those who did not in both xT (32% vs. 4.8%, q< 0.001) and xF (29% vs. 9.5%, q< 0.001) cohorts. To determine whether ESR1 mutations drive resistance to CDK4/6i, we used MCF-7 cells carrying ESR1 wild-type (WT), Y537S, or D538G ‘hotspot’ knock-in mutations to assess cell proliferation upon treatment with the CDK4/6i palbociclib. Cells harboring the Y537S and D538G mutations displayed a 3-fold increase in the IC50 of palbociclib single agent compared to ESR1WT cells. When treated with palbociclib + fulvestrant or palbociclib + estrogen-free medium (mimicking estrogen suppression in patients treated with aromatase inhibitors), cells harboring the Y537S and D538G mutations displayed an 11.2 to 46.9-fold increase in the IC50 of palbociclib. Treatment with palbociclib arrested growth of MCF-7 ESR1WT xenografts established in ovariectomized nude mice whereas MCF-7 ESR1Y537S and MCF-7 ESR1D538G tumors continued to grow. These results suggest that ESR1 mutations enable cell proliferation and tumor growth in the presence of CDK4/6i and the combination of CDK4/6i + antiestrogens. Next, we performed RNA-seq to investigate the mechanisms underlying ESR1 mutation-mediated resistance to CDK4/6i. Gene set enrichment analysis (GSEA) of the RNA-seq data showed significant upregulation of cell cycle-related Hallmark gene signatures such as E2F targets, G2/M checkpoints, and mitotic spindle in ESR1Y537S vs. ESR1WT and ESR1D538G vs. ESR1WT MCF-7 cells upon treatment with palbociclib. Upregulation of these gene signatures in ESR1-mutant vs. WT cells was also observed upon treatment with palbociclib + fulvestrant and palbociclib in estrogen-free media, suggesting that ESR1 mutations hamper the inhibitory effects of CDK4/6i alone or in combination with antiestrogens on cell cycle progression. Additionally, RNA-seq analysis showed that cells expressing either the Y537S or D538G mutation had elevated basal gene signatures (METABRIC_basal, TCGA_basal, and Huper_basal) compared to ESR1WT cells, consistent with previous studies suggesting that ESR1 mutations promote a luminal-to-basal subtype switch in ER+ breast cancers. We are currently investigating whether this lineage reprogramming underlies resistance to CDK4/6i in ESR1-mutant cells. In addition, we will validate our findings by analyzing the tumor tissue transcriptome from the patients included in this study. Conclusions: ESR1 mutations are enriched following treatment with CDK4/6i in a cohort of 3,958 patients with ER+/HER2- metastatic breast cancer. Mutations in ESR1 in ER+ breast cancer cells directly promote resistance to CDK4/6i alone or CDK4/6i + antiestrogens in vitro and in vivo. Citation Format: M Rosario Chica-Parrado, Chang-Ching Lin, Ellen Jaeger, Michelle Harris, Lei Guo, Emmanuel Bikorimana, Fabiana Napolitano, Calvin Chao, Ariella Hanker, Carlos Arteaga. ESR1 mutations drive resistance to CDK4/6 inhibitors in ER+ Breast Cancer [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 PO3-23-09.

Abstract 2498: Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue

Abstract Introduction Tumor profiling including RAS, BRAF, HER2, and MSI/MMR status, is required to determine the treatment for patients with metastatic colorectal cancer (mCRC) at the time of diagnosis. While comprehensive tumor profiling using tissue-based next-generation sequencing (NGS) is increasingly adopted for initial profiling in mCRC, the role of circulating tumor DNA (ctDNA) NGS as initial testing in patients with sufficient tumor tissue is not clearly understood. We assessed the clinical value of ctDNA sequencing compared to tumor NGS in patients with newly diagnosed mCRC who have sufficient tumor specimens. Methods We prospectively enrolled consecutive patients with newly diagnosed mCRC at the National Cancer Center Korea. As per the institutional protocol for mCRC, initial tumor profiling was performed on primary tumor tissue using an in-house NGS panel (NCC PCP ver.3), which included 525 genes. For ctDNA sequencing, patients were evaluated using the AlphaLiquid®100 comprehensive cancer panel (IMBdx, Inc.), which included 118 genes, before the initiation of chemotherapy. Additionally, immunohistochemical (IHC) testing for HER2 and polymerase chain reaction (PCR)/IHC testing for MSI and/or MMR were performed to assess the accuracy of HER2 and MSI/MMR status. Results A total of 188 patients were enrolled. In 139 eligible patients, 275 potentially actionable mutations were found in 12 selected CRC-related genes (APC, TP53, KRAS, NRAS, BRAF, FBXW7, PIK3CA, ERBB2, SMAD4, NF1, EGFR, MET). Of these, 32% were found both in ctDNA and tissue; 54% were found in ctDNA only; 12% in tissue only, and 2% were discordant. For RAS/BRAF mutations, which are required for anti-EGFR treatment decisions, the concordance rate between ctDNA and tissue NGS was 83.1%, and the concordance was higher in patients with higher ctDNA concentrations. For 9 patients with potentially actionable copy number variations (CNV) in EGFR, HER2, MET, and FGFR1, 3 cases were found by both assays; 4 were found by ctDNA only, and 2 were found by tissue only. ctDNA NGS correctly predicted MSI/MMR status in 2 out of 4 patients with MSI-H/dMMR; in the 2 other patients, the MSI and MMR statuses were different, suggesting potential false positivity. In addition, the fold changes in ctDNA dynamics during treatment significantly correlated with changes in tumor size and CEA levels, as well as with droplet digital PCR copy number fold changes. In a patient with MET amplification, ctDNA NGS identified MET Y1230H as a potential acquired resistance mutation after crizotinib treatment, which responded to cabozantinib but not to capmatinib. Conclusions Initial tumor profiling using ctDNA NGS yielded outcomes comparable to those of tumor tissue NGS in guiding treatment for patients with newly diagnosed mCRC, thereby suggesting its utility as an initial profiling method in mCRC. Citation Format: Yongjun Cha, Bun Kim, Dong Woon Lee, Hyun Yang Yeo, Chang Won Hong, Kyung Su Han, Byung Chang Kim, Hee Jin Chang. Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue [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 2498.

Abstract 946: Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration

Abstract Introduction: Olaparib, an oral poly polymerase (PARP) inhibitor has received approval for treating malignancies displaying homologous recombination deficiency (HRD). Current data is insufficient in establishing the role of PARP inhibitors in non-small cell lung cancer (NSCLC) in the setting of third generation EGFR tyrosine kinase inhibitor (TKI) use. Preclinical investigation suggests heightened PARP inhibitor sensitivity of TKI-resistant EGFR mutant NSCLC cells compared to TKI-sensitive cells. Methods: We utilized The Cancer Genome Atlas (TCGA) to investigate the prevalence of HRD gene mutations in patients with NSCLC harboring EGFR mutations. We also present a case demonstrating a favorable response to the dual therapy of olaparib and osimertinib in NSCLC harboring EGFR, RAD50, and ARID1A mutations that progressed on osimertinib. Results: There were 2298 patients in the NSCLC cohort (MSKCC database). Among 687 patients with EGFR mutations, 24 patients (3.5%) exhibited ARID1A mutations, 8 patients (1.2%) had BRCA1 mutations, 11 patients (1.6%) had BRCA2 mutations, and 5 (0.7%) had RAD50 mutations. A 71-year-old female presented with stage IV adenocarcinoma of the lung. She had recurrent malignant pericardial effusion requiring pericardiocentesis for previous cardiac tamponade. Computed tomography (CT) of the chest revealed a 3cm irregular left hilar mass with bilateral mediastinal and hilar lymph nodes. Tissue next generation sequencing (NGS) at baseline identified EGFR exon19 deletion along with RAD50 R726H which is a HRD pathway gene. Circulating tumor DNA (ct-DNA) NGS (Guardant360) showed EGFR E746_A750del 4.8%. The patient achieved partial response to osimertinib for 28 months. Follow-up CT showed disease progression. Olaparib 200mg twice daily was added to osimertinib based on repeat ct-DNA NGS indicating a new ARID1A alteration (splice site SNV) along with the known BRCA1 mutation. Follow up scans currently have consistently demonstrated ongoing partial response for 19 months. Combining olaparib with osimertinib can be effective in an osimertinib-resistant NSCLC patient with EGFR mutation and HRD aberration. The favorable response could be attributed not only to pre-existing RAD50 mutation but also to acquired mutations in ARID1A HRD genes. Conclusion: HRD aberrations are uncommon in EGFR mutated lung cancer patients. Further investigation on the role of PARP inhibitor in EGFR mutated lung cancer is warranted. Citation Format: Jinah Kim, Horyun Choi, Liam Il-Young Chung, Young Kwang Chae. Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration [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 946.

Abstract 977: Germline BRCA1/2 mutations detected by circulating tumor DNA testing in breast cancer patients: A retrospective mutiinstitutional analysis

Abstract Background: The use of circulating tumor (ct) DNA testing allows to detect resistant and actionable somatic alterations and it could be associated with the incidental identification of germline mutations. An univocal variant allele frequency (VAF) threshold to distinguish germinal form somatic mutations has not been assessed yet. We aimed to correlate BRCA1/2 ctdNA detected mutations with germline mutational status to determine potential VAF cutoff. Materials and methods:We retrospectively analyzed the incidence of both somatic (s) and germinal (g) BRCA1/2 alterations in a multiinstitutional retrospective breast cancer (BC) cohort to assess the VAF threshold for the likelihood of germline mutations’ detection by ctDNA next-generation sequencing (Guardant 360) testing. Clinical variables were analyzed using descriptive analyses and receiving operating characteristic (ROC) curves were generated to determine the VAF cutoff. Results: Two hundred and fourteen patients (pts) with sBRCA1/2 mutations (including variant of uncertain significant and synonymous) detected by ctDNA testing, referred to the enrolling centers between January 2015 to May 2023 were included in the analysis. The median age at diagnosis was 50 years old (Interquartile range (IQR)43,5-61.5), 43% had a family history of cancer. Hormone receptor positive/HER2 negative was the most represented subtype (68%) followed by the HER2 positive (16%) and triple negative (16%) ones. At ctDNA baseline, 95.8% of pts had a metastatic disease; the main sites of metastases were bone (67.4%) and visceral (59.3%). Ninetyfour (43.9%) and 137 (64%) pts had a sBRCA1 and sBRCA2 alterations respectively, while 17 (7.9%) had a co-mutation in BRCA1 and BRCA2. The mean VAF value for sBRCA1 alterations was 10.1% (standard deviation (SD) 19.5%, range [0.04%-84.3%]) while for sBRCA2 alterations was 10.8% (SD 18.9%, range [0.21%-80.3%]). The germinal testing, performed per standard of care, was available for 100 pts (46.7%). Among these, 42 (42%) had at least a pathogenic variant detected. A gBRCA1 mutation was present in 11 (11%) pts while 25 (25%) had a gBRCA2 mutation. Comparing the somatic and germinal testing, 9/100 pts had a concordance for BRCA1 detection, the 2 discordant cases had both a low allele frequency sBRCA2 alterations; the concordance for BRCA2 detection was instead 100%. An optimal cutoff of 38.4% (AUC 0.98) for BRCA1 and 19.5% (AUC 0.96) for BRCA2 was assessed by ROC analysis as the likelihood of a germline meaning of a somatic mutation detected by ctDNA analysis. Conclusion: The identification of BRCA1/2 alterations in ctDNA could guide the use of germline testing. The VAF cutoff identified for the likelihood of a germinal mutation is lower than expected, suggesting that a wider population should be screened for germinal mutation with relevant impact on the therapeutic choices and family screening. Citation Format: Letizia Pontolillo, Carolina Reduzzi, Andrew A. Davis, Arielle J. Medford, Annika Putur, Lorenzo Gerratana, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Surbhi Warrior, Mara S. Serafini, Eleonora Nicolò, Laura Munoz-Arcos, Jeannine Donahue, Charles S. Dai, Jennifer C. Keenan, Amir Behdad, William J. Gradishar, Diana Giannarelli, Emilio Bria, Cynthia X. Ma, Aditya Bardia, Massimo Cristofanilli. Germline BRCA1/2 mutations detected by circulating tumor DNA testing in breast cancer patients: A retrospective mutiinstitutional analysis [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 977.

Concurrent Tissue and Circulating Tumor DNA Molecular Profiling to Detect Guideline-Based Targeted Mutations in a Multicancer Cohort

Tissue-based next-generation sequencing (NGS) of solid tumors is the criterion standard for identifying somatic mutations that can be treated with National Comprehensive Cancer Network guideline-recommended targeted therapies. Sequencing of circulating tumor DNA (ctDNA) can also identify tumor-derived mutations, and there is increasing clinical evidence supporting ctDNA testing as a diagnostic tool. The clinical value of concurrent tissue and ctDNA profiling has not been formally assessed in a large, multicancer cohort from heterogeneous clinical settings. To evaluate whether patients concurrently tested with both tissue and ctDNA NGS testing have a higher rate of detection of guideline-based targeted mutations compared with tissue testing alone. This cohort study comprised 3209 patients who underwent sequencing between May 2020, and December 2022, within the deidentified, Tempus multimodal database, consisting of linked molecular and clinical data. Included patients had stage IV disease (non-small cell lung cancer, breast cancer, prostate cancer, or colorectal cancer) with sufficient tissue and blood sample quantities for analysis. Received results from tissue and plasma ctDNA genomic profiling, with biopsies and blood draws occurring within 30 days of one another. Detection rates of guideline-based variants found uniquely by ctDNA and tissue profiling. The cohort of 3209 patients (median age at diagnosis of stage IV disease, 65.3 years [2.5%-97.5% range, 43.3-83.3 years]) who underwent concurrent tissue and ctDNA testing included 1693 women (52.8%). Overall, 1448 patients (45.1%) had a guideline-based variant detected. Of these patients, 9.3% (135 of 1448) had variants uniquely detected by ctDNA profiling, and 24.2% (351 of 1448) had variants uniquely detected by solid-tissue testing. Although largely concordant with one another, differences in the identification of actionable variants by either assay varied according to cancer type, gene, variant, and ctDNA burden. Of 352 patients with breast cancer, 20.2% (71 of 352) with actionable variants had unique findings in ctDNA profiling results. Most of these unique, actionable variants (55.0% [55 of 100]) were found in ESR1, resulting in a 24.7% increase (23 of 93) in the identification of patients harboring an ESR1 mutation relative to tissue testing alone. This study suggests that unique actionable biomarkers are detected by both concurrent tissue and ctDNA testing, with higher ctDNA identification among patients with breast cancer. Integration of concurrent NGS testing into the routine management of advanced solid cancers may expand the delivery of molecularly guided therapy and improve patient outcomes.

Association of longitudinal changes of genomic alteration of circulating tumor DNA with treatment response in pancreatic cancer.

701 Background: Recently, the evaluation of circulating tumor DNA (ctDNA) in pancreatic cancer (PC) patients has gained attention for detection of mutation profile. However, the concordance between tissue next-generation sequencing (NGS) and ctDNA NGS remains unclear. Additionally, the dynamics of genomic alterations during treatment or disease progression are not well understood. Methods: In this prospective cohort study conducted at Seoul National University Hospital from Jan 2022, tissue samples from endoscopic ultrasound-guided fine needle biopsies and blood samples were collected from PC patients. Genomic DNA from the treatment naïve primary tissue and ctDNA from the blood obtained before any treatment and during follow-up with anti-cancer treatment were obtained. Genomic alternation was analyzed based on targeted NGS based panel deep sequencing including 41 genes. Results: The concordance of genomic alteration profiles between tissue NGS and ctDNA NGS was evaluated in 55 patients, and the mutation detection rate of patients who were diagnosed as stage I or II disease were 100% (14 of 14) in tissue NGS and 86% (12 of 14) in ctDNA NGS. That of patients who were diagnosed as stage III or IV disease was 92.7% in tissue NGS (38 of 41) and 90% (37 of 41) in blood ctDNA NGS. Among the study patients, the longitudinal changes of genomic mutation profile were evaluated in the patients at the first treatment response assessment, and patients with progressive disease showed significantly greater amounts of mutations including newly detected Tier1/2 variants than baseline and the previous response assessment. Conclusions: In conclusion, there is comparable mutation detection between tissue genomic DNA and blood ctDNA. The significant increase in ctDNA mutations during disease progression suggests their association with immune evasion and cancer evolution. Further studies utilizing our prospective cohort are needed to explore clinical implications of these results.

Comprehensive Genomic Analysis of Patients With Non-Small-Cell Lung Cancer Using Blood-Based Circulating Tumor DNA Assay: Findings From the BFAST Database of a Single Center in Taiwan.

The Blood First Assay Screening Trial (BFAST) is a prospective study using next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) in treatment-naïve advanced/metastatic non-small-cell lung cancer (NSCLC). We compared liquid biopsy to tissue testing and analyzed genomic alterations in Taiwanese patients with NSCLC using the BFAST database. A total of 269 patients underwent FoundationOne Liquid Companion Diagnostic (F1LCDx) assay at the National Taiwan University Hospital, of whom 264 underwent tissue-based genetic testing also. We analyzed the actionable mutations and the concordance between tissue-based genetic testing, which was limited to EGFR, ALK, ROS1, and BRAF, in a real-life clinical setting and blood-based NGS in the clinical trial. Additionally, we analyzed the co-occurring genomic alterations from the blood-based ctDNA assay. A total of 76.2% patients showed actionable mutations. Standard tissue testing did not detect known driver alterations in about 22.7% of the patients (sensitivity, 70.24%). Liquid NGS detected additional mutations (RET, KRAS, MET, and ErbB2) in 14% of the patients, which went undetected by the standard-of-care testing. The complementary use of ctDNA NGS increased the detection rate by 42%. The F1LCDx assay had a sensitivity of 83.41%. Lower tumor and metastasis stages predicted nondetected blood-based NGS ctDNA results. Common co-occurring mutations in the blood-based NGS ctDNA assay were TP53, DNMT3A, TET2, PIK3CA, CTNNB1, and RB1. Among the patients with EGFR-mutated NSCLC, TET2 co-occurring alterations correlated with shorter progression-free survival of EGFR tyrosine kinase inhibitor treatment. NGS ctDNA analysis in comprehensive genetic testing improves actionable mutation identification, vital for treating Asian NSCLC cases with high actionable mutation rates. Lower stages correlated with undetected blood-based NGS ctDNA assay results.

Characterization of the Thyroid Cancer Genomic Landscape by Plasma-Based Circulating Tumor DNA Next-Generation Sequencing.

Background: The limited availability of targeted therapies in thyroid cancer (TC) has challenged conventional treatment algorithms and has established urgency for the identification of targetable genomic abnormalities. In addition to widely adopted tissue-based next-generation sequencing (NGS), plasma-based circulating tumor DNA (ctDNA) NGS is rapidly emerging as a genomic biomarker detection method and is steadily gaining utility across solid tumors. To date, plasma-based genomic alterations in TC have not been determined. Herein, we profile potential actionable mutations detected through ctDNA in patients with TC subtypes. Methods: A retrospective data analysis of the Guardant Health, Inc. database was performed using the commercially available Guardant360® plasma-NGS test on TC samples from adult patients collected between 2016 and 2021. The landscape of genomic alterations and blood tumor mutation burden (bTMB) were analyzed in patients with different types of TC: anaplastic TC (ATC), papillary TC (PTC), follicular TC (FTC), oncolytic carcinoma of the thyroid (OCA), poorly differentiated TC (PDTC), medullary TC (MTC), and TC not otherwise specified (TC NOS). Results: Of the 1094 patients included most of the patients n = 876 had TC NOS, and 20% had a specific diagnosis (92 ATC, 62 PTC, 14 FTC, 16 OCA, 2 PDTC, and 32 MTC patients). The median age was 65 (range 10-98) and 47.3% were male. 78.3% of patients had one or more genomic alteration detected by ctDNA NGS. TP53 (46.9%) was the most common mutation detected among all TC. BRAFV600E was detected in 27.2% of ATC, 35.7% of PTC, and in none of FTC. RAS was detected in 18.5% of ATC, 11.9% of PTC, and 62.5% of FTC. RET, ALK, and NTRK fusions were seen in 1.1%, 0.5%, and 0.2% of all TC, respectively. RET mutations were detected in 66.7% of MTC. bTMB analysis was performed on 159 patients. The mean bTMB was higher in ATC compared with other types of TC (p = 0.0011, 0.0557, and <0.0001, respectively). Conclusions: Plasma-based comprehensive NGS is a promising NGS method in TC; however, future validation of the clinical utility by analysis of paired tumor and plasma samples is needed.

Interplay between ESR1/PIK3CA codon variants, oncogenic pathway alterations and clinical phenotype in patients with metastatic breast cancer (MBC): comprehensive circulating tumor DNA (ctDNA) analysis

Backgroundalthough being central for the biology and druggability of hormone-receptor positive, HER2 negative metastatic breast cancer (MBC), ESR1 and PIK3CA mutations are simplistically dichotomized as mutated or wild type in current clinical practice.MethodsThe study analyzed a multi-institutional cohort comprising 703 patients with luminal-like MBC characterized for circulating tumor DNA through next generation sequencing (NGS). Pathway classification was defined based on previous work (i.e., RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Single nucleotide variations (SNVs) were annotated for their oncogenicity through OncoKB. Only pathogenic variants were included in the models. Associations among clinical characteristics, pathway classification, and ESR1/PIK3CA codon variants were explored.ResultsThe results showed a differential pattern of associations for ESR1 and PIK3CA codon variants in terms of co-occurring pathway alterations patterns of metastatic dissemination, and prognosis. ESR1 537 was associated with SNVs in the ER and RAF pathways, CNVs in the MYC pathway and bone metastases, while ESR1 538 with SNVs in the cell cycle pathway and liver metastases. PIK3CA 1047 and 542 were associated with CNVs in the PI3K pathway and with bone metastases.ConclusionsThe study demonstrated how ESR1 and PIK3CA codon variants, together with alterations in specific oncogenic pathways, can differentially impact the biology and clinical phenotype of luminal-like MBC. As novel endocrine therapy agents such as selective estrogen receptor degraders (SERDS) and PI3K inhibitors are being developed, these results highlight the pivotal role of ctDNA NGS to describe tumor evolution and optimize clinical decision making.

Abstract PR10: A decade in the making: Screening ctDNA detection in HPV-associated HNSCC

Abstract Introduction HPV-associated head and neck squamous cell carcinomas (HPV+HNSCC) release circulating tumor DNA (ctDNA), including ctHPVDNA, into the blood, which is detectable at the time of diagnosis in nearly all patients with HPV+HNSCC using droplet digital PCR (ddPCR)-based approaches. We have previously demonstrated proof of principle that using these approaches, ctHPVDNA is detectable in a minority of patients up to 3 years prior to diagnosis, suggesting HPV+HNSCC may be reliably detected many years prior to clinical detection when using more sensitive approaches. Here, we applied a custom whole genome hybrid-capture-based next generation sequencing (NGS) assay, termed HPVseek, to plasma samples collected years prior to HPV+HNSCC diagnosis to test this hypothesis. Method Plasma samples from 28 patients collected 1-11 years prior to HPV+HNSCC diagnosis were obtained from the Mass General Brigham Biobank, along with 1:1 age and gender matched controls. Cell free DNA was extracted and run on HPVseek and our existing ddPCR assay. Pre-existing cut-points for a positive result were applied. In cases where tissue blocks were available from the time of diagnosis, DNA was extracted and underwent HPVseek for confirmatory molecular fingerprinting of the virus. Result 19 of 28 patients (68%) had detectable ctHPVDNA with lead times ranging from 1.0 to 7.5 years. Of the 11 samples obtained between 1-3 years before diagnosis, 11(100%) were ctHPVDNA positive. All controls were negative. HPV genotype distribution in positive samples was: 16 (n=16), 35 (1), 56 (1), and 59 (1) in blood with genotypes matching clinical genotype testing in 27/28 cases. 11/28 (39%) were positive on ddPCR, all of which were also positive on NGS. Molecular fingerprinting of viral genomes revealed unique viral genomes in each case and matching viral genomes within paired blood and tissue blocks, ruling out contamination and demonstrating the same inciting virus pre-diagnosis to diagnosis. Seven patients had diagnostic and post-diagnostic blood samples available as part of our ongoing prospective ctHPVDNA studies, allowing continuous tracking of cancer status along a time of up to 12 years, providing a global picture of HPV+HNSCC. Conclusion Here we demonstrate that HPV+HNSCC is detectable in the blood as ctDNA &amp;gt;seven years prior to diagnosis, and was detectable in 100% of cases up to three years prior to diagnosis, using an ultrasensitive custom ctDNA NGS assay. Findings were orthogonally validated using molecular fingerprinting in tissue and blood. This study has broad implications for the field of liquid biopsy cancer screening, demonstrating perhaps the longest lead times ever reported for ctDNA detection prior to cancer clinical diagnosis and suggesting HPV+HNSCC could be screened for, detected, and potentially treated years before clinical cancer detection. Citation Format: Shun Hirayama, Brian Y. Zhao, Saskia Naegele, Vasileios Efthymiou, Julia Mendel, Adam Fisch, Michael S. Lawrence, Viktor Adalsteinsson, Anthony J. Iafrate, Dipon Das, Daniel L. Faden. A decade in the making: Screening ctDNA detection in HPV-associated HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR10.

Clinical utility of next generation sequencing (NGS) on circulating tumor DNA (ctDNA) in patients (pts) with pancreatic cancer (PC).

4154 Background: ctDNA provides opportunities for identifying targeted therapies, a source to conduct NGS when tissue acquisition is infeasible, detection of minimal residual disease (MRD), and identification of resistance mutations. There are limited large data sets to inform clinical utility and limited correlation of NGS of ctDNA and tumor in PC. Herein, we evaluate the ctDNA detection rate in multiple cohorts of PC and report the ctDNA-tissue genotype concordance in PC at Memorial Sloan Kettering (MSK). Methods: Pts with PC at MSK who had ctDNA prospectively collected using the MSK-ACCESS 129 gene ctDNA NGS assay, were identified. ctDNA detection was defined as the identification of a mutation, copy number (no.) alteration or structural variant. Tissue-based NGS using MSK-IMPACT gene assay was performed for pts with adequate tissue, and matched with ctDNA by date. Clinical, pathologic and outcome data were abstracted. Data are summarized with descriptive statistics. Overall survival (OS) estimated by Kaplan-Meier method from date of ctDNA draw to death/last follow up. Results: From 08/2019 to 07/2022, N= 414 pts with PC and ≥ 1 ctDNA sample included. Median age 69 years (range 29-92), female N=206 (50%). Stage at ctDNA collection: Stage I-III N= 203 (49%); Stage IV N= 211 (51%). ctDNA detection rate by no. of involved organs; 0 organs (MRD), N=7/30 (23%); 1-2 organs, N=158/270 (58.5%); 3-4 organs, N=82/102 (80.4%); &gt;5 organs, 12/12 (100%). CtDNA detection rate by site of metastasis; Liver only, 75/99 (76%); peritoneum only, 15/25 (60%); lung only, 10/22 (45%). In pts with detected ctDNA median CA 19-9 was 494 U/mL (range 0, 247674) vs 100 U/mL (range 1, 22360) in pts with undetected ctDNA. In pts with detected ctDNA median CEA was 7 ng/mL (range 1, 2020) vs 4 ng/mL (range 1, 99) in pts with undetected ctDNA. In stage IV untreated pts, median OS 13 months(m) (95% CI; 7.3, 16) and 10 m (95% CI; 5.6, -) for ctDNA detected vs undetected, respectively. ctDNA detection rates by stage, and concordance between ctDNA and tissue-based NGS for common driver mutations KRAS, TP53, CDKN2A and SMAD4 in N=131 matched pairs are summarized in the table. Conclusions: ctDNA detection rates are high (89%) in pts with untreated stage IV PC, with high concordance between ctDNA and tissue-based NGS (87% - 95%). In untreated stage I-III PC, detection and concordance rates are lower (&lt;50%). Detection rates are associated with disease burden, site of metastasis, CA 19-9/CEA levels. ctDNA is a promising tool in detection of somatic alterations in PC, particularly in stage IV disease, and is a complementary adjunct to tumor-based NGS. [Table: see text]

Tumor-agnostic genomic and clinical analysis of solid tumors with BRAF fusions.

3146 Background: Approximately 15% of cancers harbor BRAF alterations, of which ~5% are BRAF fusions. BRAF fusions are class II alterations that have worse outcomes than class I V600E alterations. While prior investigational therapies for BRAF fusions had disappointing outcomes, novel therapies are in clinical trials, underscoring the need to better characterize these tumors. Methods: Data from adult and pediatric patients with BRAF fusion+ cancers identified between January 2014 and November 2022 utilizing a center-wide next generation sequencing (NGS) program of &gt;99,000 sequenced samples were analyzed. DNA-based hybrid capture tumor NGS (MSK-IMPACT), ctDNA targeted NGS (MSK-ACCESS), and/or RNA anchored multiplex PCR tumor NGS (MSK-Fusion) were used. Fusions were manually reviewed and considered to have oncogenic potential if they had an in-frame protein fusion involving a non-BRAF partner gene and an intact BRAF kinase domain (exons 11-18). Fusions not previously reported in OncoKB, COSMIC, TCGA, NIH gene, Fusion GDB2, and PubMED were classified as novel. All cases underwent clinical data curation including baseline demographic, tumor characteristics, and treatment histories. Results: 212 patients (0.2%) with BRAF-fusion positive solid tumors were identified. 194 were identified by DNA-based tumor NGS (96 had sufficient tissue for confirmatory RNA-based NGS, all of which were positive). Six were identified by ctDNA NGS and 12 by RNA-based tumor NGS only (5 had insufficient tissue for DNA-based tumor NGS). 83 unique 5’ fusion partners were found, of which 42 were novel. The most frequent tumor types were pilocytic astrocytoma (n=30, 14%), prostate CA (n=28, 13%), melanoma (n=24, 11%), lung CA (n=21, 10%), and colon CA (n=15, 7%). 44% of patients with pilocytic astrocytoma had BRAF fusions, of which 90% were BRAF-KIAA1549. Concomitant alterations (≥10% frequency) included TP53 (24%), TERT (18%), CDKN2A deletions (13%), and CDKN2B deletions (12%). 20% (n=43) were treated with MAPK-pathway directed therapies spanning multiple histologies and lines of therapies. Of the 212 patients, 17 had acquired BRAF fusions after targeted therapy for another oncogene ( EGFR mutation (n=11), BRAF V600E (n=4), FGFR fusion (n=1), NTRK fusion (n=1)). The majority of patients with acquired BRAF fusions had EGFR-mutant lung adenocarcinoma (n=11, 76%); the median time from EGFR targeted therapy initiation to BRAF fusion detection was 25 months (range 16-38 months). Conclusions: A wide variety of adult and pediatric solid tumors harbored de novo BRAF fusions. Complementary RNA sequencing optimized fusion identification in many cases. Multiple novel fusion partners were found. Acquired BRAF fusions were identified after targeted therapy for a variety of distinct oncogenes, the majority of which were EGFR mutations.

Predictive biomarkers for treatment with amivantamab plus lazertinib among EGFR-mutated NSCLC in the post-osimertinib setting: Analysis of tissue IHC and ctDNA NGS.

9013 Background: Amivantamab (ami) is a bispecific antibody targeting EGFR and MET with immune cell-directing activity, and lazertinib (laz) is a brain-penetrant, third-generation EGFR tyrosine kinase inhibitor. In a previous report, the overall response rate (ORR) of ami+laz in the post-osimertinib (osi) setting was 36%, with median duration of response of 9.6 months (Bauml JCO 2021; 39:15_suppl, 9006). Exploratory analyses suggested certain immunohistochemistry (IHC) and ctDNA-based next-generation sequencing (NGS) biomarkers may enrich for response. A prospective study was conducted to further evaluate these potential biomarkers. Methods: CHRYSALIS-2 (NCT04077463), an ongoing phase 1/1b study, examines the efficacy (ORR per RECIST v1.1) of ami+laz in patients (pts) with EGFR-mutated NSCLC. Cohort D enrolled osi-relapsed, chemotherapy-naïve pts with EGFR exon 19 deletion or L858R mutated advanced NSCLC who had tumor tissue for IHC staining and plasma for ctDNA NGS collected after their most recent therapy. Training and validation subsets identified biomarker criteria and predictive validity, respectively. A Bayesian posterior probability (biomarker positive group ORR is ≥35% and negative group ORR is ≤20%) was predefined to be ≥85% for acceptance. Results: There were 101 response-evaluable pts enrolled in Cohort D. A total of 87 pts had detectable baseline ctDNA and 77 pts had evaluable MET IHC. The training set (n = 50) identified IHC-based MET expression of 3+ staining on ≥25% of tumor cells (MET+) as a potential predictive biomarker, and the validation set (n = 27) confirmed these results. Of those with MET IHC data, 28/77 (36%) were MET+. The overall ORR for MET+ was 61% (95% CI, 41–78) and 12% (95% CI, 5–25) for MET-, with median PFS for MET+ not reached (95% CI, 4.3–NE) vs 4.1 months (95% CI, 2.8–5.7) for MET-. NGS evaluation of ctDNA did not identify predictive biomarkers meeting predetermined criteria. Of note, only 1 pt was identified by ctDNA as having MET amplification (the pt was also MET+ by IHC). Further details will be provided at the time of presentation. Conclusions: MET+ by IHC may be a predictive biomarker for response to ami+laz in the post-osi, chemotherapy-naïve setting. In contrast, molecular profiling with ctDNA failed to identify a subgroup of pts more likely to benefit. Ami+laz represents a potential chemotherapy-free option for pts with EGFR-mutated advanced NSCLC who have progressed on osi and are also MET+ by IHC. Clinical trial information: NCT04077463 .

Clinical outcomes of immune checkpoint inhibitor treatment in patients with classic cold tumors identified to have a high tumor mutational burden via ctDNA.

2561 Background: Some cancers, including breast, ovarian, pancreatic, and prostate cancers, are traditionally considered “cold” tumors, less likely to trigger an immune response and/or respond to immune checkpoint inhibitors (ICIs). However, some patients with these cancer types may benefit from ICIs. Here we investigate if a high blood tumor mutational burden (bTMB-H) may predict benefit to ICIs in such tumor types. Methods: We retrospectively queried the Guardant Health database for patients with advanced breast (n = 8531), ovarian (n = 1106), upper GI/pancreatic (n = 5110), and prostate (n = 7153) cancer who had ctDNA next-generation sequencing (Guardant360, Redwood City, CA) as part of routine clinical care from 2020-2022. Mutation profiles were evaluated for patients with bTMB-H (based on previously reported 80th percentiles for each tumor type, breast ≥15.31mut/Mb, ovarian ≥14.98mut/Mb, pancreatic ≥11.36mut/Mb, prostate ≥13.4mut/Mb) vs those with bTMB below this threshold (bTMB-L). A subset of cases were analyzed in a multi-institutional clinical cohort with a bTMB of ≥10mut/Mb (n = 35). Results: bTMB-H was observed in 9.5% of patients with breast cancer, 5.1% ovarian, 2.0% pancreatic, and 4.4% prostate. Genes more often mutated in patients with pTMB-H vs pTMB-L in each cancer type included TP53 and PIK3CA in breast cancer; TP53 and CHEK2 in ovarian; ATM and CHEK2 in pancreatic; TP53 and AR in prostate (all p values &lt; 0.01). Patients in the clinical cohort (n = 35) had a median age of 74 (range: 40-85) 29/35 White / 4/35 Black, 27 prostate, 1 breast, 3 upper GI. Patients underwent first ctDNA analysis after a median of 3 (range 0-11) lines of therapy. The median bTMB detected was 16.59 mut/Mb (range: 10.02-152.85); six patients (6/35) were MSI-H. Twenty-three patients received ICI after a median of 6 (range: 0-8) lines of therapy, either alone (9, 39%) or in combination with hormonal therapy (5, 22%), chemotherapy (3, 13%) or targeted therapy (6, 26%). The median time to progression on ICI was 5.0 (95% CI, 3.6-6.4) months and median duration of response was 19.8 months (12.1-20.2). The ORR for evaluable patients was 13% and 74% of patients had progressed on therapy at time of data cut-off. Conclusions: "Cold” tumors represent a meaningful portion of patients with advanced cancer in which ICIs are not typically leveraged, and ctDNA was a feasible tool for identifying patients with bTMB-H who achieved durable responses from ICI-based therapy. The efficacy in this clinical cohort of traditionally “cold” tumors approaches other pan-cancer cohorts that include more traditionally immunoresponsive tumor types; further research is warranted to explore predictive value of bTMB, including potential tumor-specific thresholds, for optimizing patient selection for ICIs.

Co-mutation of ATM and ASXL1 and relationship to durable response of a novel PARP1/2 inhibitor, venadaparib, in patients with pancreatic cancer.

e15019 Background: Homologous recombination repair (HRR) alterations such as BRCA mutation (BRCAm) and ATM mutation (ATMm) are established predictive biomarkers for poly (adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitor in pancreatic cancer (PC). Mutations in the ASXL1 gene (ASXL1m) have been associated with hematologic malignancies. In solid cancers, however, associations between HRR and non-HRR mutation such as ASXL1m as well as treatment response in patients with those are not well known. The aim of this analysis was to explore genetic alterations co-existing with HRR mutation to better predict efficacy from a monotherapy of venadaparib, a PARP inhibitor under development, in patients with metastatic PC. Methods: Patients with HRR mutation and no standard treatment or prior treatment failure were enrolled in a phase Ib basket trial (NCT04174716) to receive venadaparib monotherapy. Tumor response to treatment was evaluated according to RECIST 1.1. Exploratory blood samples were collected to analyze genetic alterations using ctDNA next-generation sequencing (NGS) (GuardantOMNI Gene Panel version 1.0, Redwood City, CA) on Day 1 pre-dose. Prevalence of co-occurring mutations was estimated and mutual exclusivity between HRR and non-HRR mutations were statistically tested by Fisher’s exact test using the cBioPortal for Cancer Genomics ( http://cbioportal.org ) in The Cancer Genome Atlas (TCGA) Pan-cancer and AACR Project Genie v13.0 registries. Efficacy between patients with co-mutations and single mutation only were presented. Results: In public dataset, the prevalence of ATMm and ASXL1m was 3.0% and 0.9% in TCGA pan-cancer study (n = 10,967), 3.0% and 1.7% in Genie v13.0 (n = 131,996), respectively. In both datasets, co-mutation of ATM and ASXL1 was tested significant (Log2 Odds Ratio 2.663 and 1.176 respectively; q-value 0.001 and 0.006 respectively). In the basket trial, 8 patients were enrolled, with 50% male, and a median age 66 (range 47-72). 6 patients had 2 prior palliative chemotherapy. 6 patients had ATMm and 2 had BRCA2m confirmed by enrolling institution at baseline. All patients had responded to prior platinum containing regimen. ctDNA NGS samples were available in 6 patients. Patients with ATMmASXL1m (n = 3) had a 33% overall response rate (1 partial response, 2 stable disease, range of progression-free survival (PFS), 23-113 weeks), while those with ATMmASXL1wt (n = 1) had a 0% (1 progressive disease (PD), PFS 9 weeks) and others (ATMwtASXL1wt, n = 2, 1 PD; ctDNA not available, n = 2, 2 PD) had a 0% response rate (range PFS 3-16 weeks). Two patients with BRCA2m only had PD on weeks 3 and 8. Conclusions: In this exploratory analysis, patients with co-occurring ATMm and ASXL1m showed higher tumor response. This signals potential value from ATM and ASXL1 co-mutation predicting efficacy of Venadaparib in PC and warrants further validation. Clinical trial information: NCT04174716 .

China special issue on gastrointestinal tumors-NTRK fusion in a large real-world population and clinical utility of circulating tumor DNA genotyping to guide TRK inhibitor treatment.

Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are rare oncogenic drivers and targets of TRK inhibitors in solid tumors. Little is known about NTRK fusion in Chinese patients with pan-cancer. Our study investigated the prevalence and genomic features of NTRK1/2/3 gene fusions in 67 883 Chinese patients with pan-cancer using next-generation sequencing (NGS) data and circulating tumor DNA (ctDNA) NGS to guide TRK inhibitor treatment and resistance monitoring. The prevalence of NTRK fusion (tissue NGS) in the pan-cancer population was 0.18%, with 46 unique NTRK-fusion partner pairs, of which 33 were not previously reported. NTRK2 breakpoint occurred more frequently in intron 15 than intron 12. In colorectal cancers (CRCs), compared to NTRK-negative tumors, NTRK-positive tumors displayed higher tumor mutational burden (TMB) levels (54.6 vs 17.7 mut/Mb, P < .0001). In microsatellite instability-high (MSI-H) CRC, patients with NTRK fusion had a significantly lower TMB than NTRK-negative cases (69.3 vs 79.9 mut/Mb, P = .012). The frequency of NTRK fusion in a ctDNA NGS cohort of 20 954 patients with cancer was similar to that of the tissue NGS cohort. In eight NTRK fusion ctDNA-positive patients, larotrectinib induced objective response in 75% of patients and median progression-free survival was 16.3 months. Blood samples collected from a patient with disease progression after larotrectinib treatment revealed NTRK3 G623R as the potential resistance mechanism. Our study revealed previously unreported NTRK fusion partners, associations of NTRK fusion with MSI and TMB, and the potential utility of ctDNA to screen candidates for TRK inhibitors and monitor drug resistance.

Mutational heterogeneity of imatinib resistance and efficacy of ripretinib vs sunitinib in patients with gastrointestinal stromal tumor: ctDNA analysis from INTRIGUE.

397784 Background: Ripretinib, a switch-control tyrosine kinase inhibitor (TKI), is indicated for patients (pts) with gastrointestinal stromal tumor (GIST) who received prior treatment with ≥3 TKIs, including imatinib. Sunitinib is approved for advanced GIST after imatinib failure. Circulating tumor DNA (ctDNA) analysis may provide insight into the efficacy of these agents in second-line advanced GIST. Here, we present exploratory baseline ctDNA results from INTRIGUE. Methods: INTRIGUE is an open-label, phase 3 study that enrolled adult pts with advanced GIST who progressed on or had intolerance to imatinib (NCT03673501). Randomization was 1:1 to ripretinib 150 mg once daily (QD) or sunitinib 50 mg QD (4 wks on/2 wks off). Baseline peripheral whole blood was analyzed by Guardant360, a 74-gene ctDNA next-generation sequencing (NGS)-based assay. Only KIT mutations are reported here. Results: Of 453 pts in the overall intent-to-treat (ITT) population, 362 (80%) samples were analyzed. ctDNA was detected in 280/362 (77%), with KIT mutations detected in 213/280 (76%). Common resistance mutations were in the KIT activation loop (AL; exons 17/18; 89/213, 42%) and ATP-binding pocket (ATP-BP; exons 13/14; 81/213, 38%). Efficacy in pts with detectable ctDNA in the KIT exon 11 and overall ITT populations was consistent with the primary analysis based on tumor data used for randomization. Pts with KIT exon 11 + 17/18 (−9/13/14) mutations had superior progression-free survival (PFS), objective response rate (ORR), and overall survival (OS) with ripretinib vs sunitinib, whereas pts with exon 11 + 13/14 (−9/17/18) mutations had better PFS, ORR, and OS with sunitinib vs ripretinib (Table). Subgroup safety profiles were consistent with the primary analysis. Conclusions: While KIT ATP-BP mutations predicted clinical benefit from sunitinib vs ripretinib, pts harboring resistance mutations in the KIT AL derived meaningful clinical benefit from ripretinib but not sunitinib. This study demonstrates the value of ctDNA NGS-based sequencing of the complex landscape of KIT mutations to predict the clinical benefit of ripretinib or sunitinib as second-line therapy in pts with advanced GIST. Clinical trial information: NCT03673501 . [Table: see text]

Abstract CT212: A ctDNA-directed, multi-center phase II study of molecular response adaptive immuno-chemotherapy in patients with non-small cell lung cancer: Analysis of Stage 1 of CCTG BR.36

Abstract Introduction: Analyses of circulating tumor DNA (ctDNA) have shown promise in capturing tumor burden dynamics during immune checkpoint blockade (ICB), with the potential to allow patients with primary resistance to be rapidly identified and redirected to alternative therapies. Methods: BR.36 is an international multicenter, open label, biomarker-directed, phase II trial of molecular response-adaptive immuno-chemotherapy for patients with treatment-naïve non-small cell lung cancer-NSCLC (NCT04093167). The trial consists of two stages; stage 1 was a single-arm study to validate the ctDNA approach, with stage 2 focusing on ctDNA guided treatment selection. Key eligibility criteria included EGFR and ALK mutation negative, ICB and chemotherapy-naïve metastatic NSCLC with PD-L1 expression ≥1%. Primary objectives were to ascertain ctDNA molecular response, determine its timing and concordance with radiologic RECIST/iRECIST response. Secondary objectives included the evaluation of time to ctDNA molecular response and its correlation with progression-free (PFS) and overall survival (OS). Liquid biopsy analyses utilized a validated tumor-agnostic white blood cell (WBC) DNA-informed 33-gene panel approach. Ultra-sensitive error-correction ctDNA next-generation sequencing (NGS) was performed at baseline/cycle 1 (C1D1), cycle 2 (C2D1) and cycle 3 (C3D1) of pembrolizumab (200mg IV q3 weeks). The maximum variant allele fraction (maxVAF) of tumor-derived variants was tracked from the pre-treatment to on-therapy timepoints. Results: Activated October 17, 2019, stage 1 completed accrual of 50 patients on April 5, 2022; data lock date was September 20, 2022. Most patients were ever-smokers (98%), had no prior systemic therapy (92%), stage IV NSCLC (98%), adenocarcinoma (76%) with PD-L1 tumor proportion score of ≥ 50% (96%); cohort consisted of 82% white, 52% female, 56% age&amp;gt;65 and 76% ECOG PS 1 participants. Median follow-up was 13.5 months (range 2.5-23.0). Best overall response rate (ORR) by RECIST was 32% with a median duration of 10.1 months. Matched WBC DNA analyses allowed for effective removal of germline and clonal hematopoiesis variants, followed by evaluation of ctDNA response. MaxVAF clearance signified molecular response (mR), while maxVAF persistence indicated molecular disease progression (mPD). Among 35 evaluable patients (77.8%), 15 were classified in the mR category, with an evaluable mR rate of 43% (90% CI: 0.29-0.58). The median time to molecular response was 2.1 months (90% CI: 1.5-2.6). The sensitivity of ctDNA molecular response for RECIST and iRECIST response was 82% (90% CI: 52%-97%) and 83% (90% CI: 56%-97%), with a specificity of 75% (90% CI: 56.5%-88.5%) and 78% (90% CI: 60%-91%) respectively. Patients with mR attained longer PFS (5.03 vs 2.6 months,) and OS (not reached vs 7.23 months); ctDNA response also provided prognostic information for patients with RECIST stable disease. Conclusions: ctDNA molecular responses were concordant with radiologic response assessments, but importantly better predicted PFS and OS for patients with stable disease or better. These findings are now incorporated into the interventional second stage of the trial. Citation Format: Valsamo K. Anagnostou, Cheryl Ho, Garth Nicholas, Rosalyn Anne Juergens, Adrian Sacher, Andrea Fung, Paul Wheatley-Price, Scott Laurie, Benjamin Levy, Julie Brahmer, Archana Balan, Noushin Niknafs, Egor Avrutin, Liting Zhu, Mark Sausen, Penelope Bradbury, Jill O'Donnell-Tormey, Pierre-Olivier Gaudreau, Keyue Ding, Janet Dancey. A ctDNA-directed, multi-center phase II study of molecular response adaptive immuno-chemotherapy in patients with non-small cell lung cancer: Analysis of Stage 1 of CCTG BR.36 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT212.