Tumor Tissue Next-generation Sequencing Research Articles

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.

Clinical utility of plasma cell-free DNA in pancreatic neuroendocrine neoplasms.

In advanced pancreatic neuroendocrine neoplasms (PanNEN), there are little data detailing the frequency of genetic alterations identified in cell free DNA (cfDNA), plasma-tissue concordance of detected alterations, and clinical utility of cfDNA. Patients with metastatic PanNENs underwent cfDNA collection in routine practice. Next-generation sequencing (NGS) of cfDNA and matched tissue when available was performed. Clinical actionability of variants was annotated by OncoKB. Thirty-two cfDNA samples were analyzed from 25 patients, the majority who had well-differentiated intermediate grade disease (13/25; 52%). Genomic alterations were detected in 68% of patients and in 66% of all cfDNA samples. The most frequently altered genes were DAXX (28%), TSC2 (24%), MEN1 (24%), ARID1B (20%), ARID1A (12%), and ATRX (12%). Twenty-three out of 25 (92%) patients underwent tumor tissue NGS. Tissue-plasma concordance for select genes was as follows:DAXX (95.7%), ARID1A (91.1%), ATRX (87%), TSC2 (82.6%), MEN1 (69.6%). Potentially actionable alterations were identified in cfDNA of 8 patients, including TSC2 (4; level 3b), ATM (1; level 3b), ARID1A (2; level 4), and KRAS (1; level 4). An ETV6:NTRK fusion detected in tumor tissue was treated with larotrectinib; at progression, sequencing of cfDNA identified an NTRK3 G623R alteration as the acquired mechanism of resistance; the patient enrolled in a clinical trial of a second-generation TRK inhibitor with clinical benefit. In metastatic PanNENs, cfDNA-based NGS identified tumor-associated mutations in 66% of plasma samples with a high level of plasma-tissue agreement in PanNEN-associated genes. Clonal evolution, actionable alterations, and resistance mechanisms were detected through circulating cfDNA genotyping.

Real-world analysis of patients with advanced gastrointestinal (GI) cancers harboring inactivating TSC1 and TSC2 alterations using the Foundation Medicine genomic database.

558 Background: Genomic profiling of GI tumors can identify potentially actionable genetic alterations, ultimately leading to better outcomes in patients who currently have few treatment options. In exploratory biomarker analyses of patients with perivascular epithelioid cell tumors treated with the mTOR inhibitor nab-sirolimus (AMPECT, NCT02494570), those with inactivating alterations in the tumor suppressor genes TSC1 or TSC2 (critical negative regulators of mTOR activity) had confirmed responses (8/9 patients with inactivating TSC2 alterations and 1/5 patients with inactivating TSC1 alterations). PRECISION 1, a currently enrolling tumor-agnostic study, will evaluate nab-sirolimus in patients with TSC1 or TSC2 alterations. To enumerate the frequency of these alterations in GI cancers, we analyzed data from a real-world genomic database. Methods: Next-generation sequencing (NGS) data from Foundation Medicine’s genomic database of patients with advanced cancer were analyzed using the FoundationInsights web-based platform to identify patients with GI cancers who had inactivating TSC1 or TSC2 alterations (short variants [base substitutions, insertions and deletions], rearrangements, and copy number deletions). Tumor mutational burden (TMB), microsatellite instability status and other mutations were also characterized in this group of patients. Results: As of 29 March 2022, tumor tissue NGS data were available for 5816 patients with GI cancers bearing alterations in TSC1 or TSC2. Of these, 1642 patients had at least one inactivating alteration. The GI tumor types that most frequently expressed inactivating alterations in TSC1 or TSC2 were hepatocellular carcinoma (alterations in 6.5%); colorectal adenocarcinoma (1.7%); gastrointestinal stromal tumors (1.7%); and cholangiocarcinoma (1.6%). Of all 1642 patients, 688 were female, and most (74%) were 51–80 years of age when the sample was obtained. Of patients with inactivating TSC1 and TSC2 alterations, 37.6% were in TSC1 and 62.9% were in TSC2 and included short variants (31.3% and 48.4%), rearrangements (2.7% and 6.6%), and copy number deletions (3.5% and 7.4%), respectively. TMB was low (<6 mutations/megabase) in 58% of patients, and most patients had microsatellite stable (MSS; 71.3%) tumors. Other commonly mutated genes in this cohort were TP53 (62.7%), APC (38.5%) and KRAS (30.4%). Conclusions: TSC1 and/or TSC2 alterations were commonly observed in GI cancers. These cancers were frequently microsatellite stable and of low TMB suggesting that these are actionable alterations that may be candidates for targeted therapy. This hypothesis is being tested in the PRECISION 1 (NCT05103358) trial which is open for enrollment or just-in-time clinical trial sites and available to patients with GI cancers harboring TSC1 or TSC2 alterations.

Abstract C019: Inactivating TSC1 and TSC2 alterations, co-mutations, and genomic instability in advanced cancers: Analysis of a real-world (RW) patient (pt) population using the Foundation Medicine genomic database

Abstract Background Tumor suppressor genes tuberous sclerosis complex subunit 1 and 2 (TSC1, TSC2) are critical negative regulators of mTOR activity. In the exploratory biomarker analysis (AMPECT, NCT02494570) and an Expanded Access Program (NCT03817515), pts with inactivating alterations in TSC1 or TSC2 demonstrated responses with the mTOR inhibitor, nab-sirolimus. While these alterations are observed in most cancer histologies, characterization and distribution of TSC1 and TSC2 alterations across tumor types are not well understood. PRECISION I (NCT05103358), a currently enrolling tumor-agnostic study, will assess the clinical benefit of nab-sirolimus in pts with cancer who harbor inactivating TSC1 and TSC2 alterations. To appreciate the potential for TSC1 and TSC2 as therapeutic biomarkers, we analyzed TSC1 and TSC2 mutational data from a RW genomic database. Methods Next-generation sequencing (NGS) data from Foundation Medicine’s genomic database of pts primarily with advanced cancer were analyzed using the FoundationInsights™ web-based platform. Frequency and actionability of TSC1 and TSC2 alterations were assessed. Known or likely pathogenic inactivating TSC1 and TSC2 alterations (ie, indel, rearrangement, frameshift, biallelic loss, nonsense, and some missense mutations) were further characterized for co-mutations, tumor mutational burden (TMB), and microsatellite instability status. Results As of 29 Mar 2022, 438,974 pts with advanced cancer in the Foundation Medicine genomic database had tumor tissue NGS data available. Known/likely inactivating alterations in TSC1 and TSC2 were identified in 8464 (1.9%) pts; 4.3% had variants of unknown significance, primarily missense (~89%), that were not known to be inactivating. Of the inactivating alterations, 52.1% were TSC1 and 48.6% TSC2, including short variants, defined as base substitutions and short indels (85.0%), rearrangements (10.8%), and copy number deletions (17.9%). Alterations were most frequently identified in urinary bladder (8.6%), kidney and renal pelvis (5.8%), corpus and uterus (3.4%), soft tissue (including heart; 2.4%), and liver and intrahepatic bile duct (2.3%) cancers. The most frequently co-occurring mutations included TP53 (62.2%), CDKN2A (26.2%), and TERT (22.2%). TMB was low (<6 mutations per megabase) in 51.6% of pts with inactivating alterations in TSC1 and TSC2. Samples were mostly microsatellite stable (MSS; 82.2%) across tumor types. Conclusion In a large RW database of pts with advanced cancer, inactivating TSC1 and TSC2 variants occurred in 1.9% of pts. These occurred across common tumor types at rates as high as 8.6% (urinary bladder tumors). A majority of pts had low TMB and/or MSS status, suggesting that these are actionable alterations and not passenger events (Bennett et al, Mod Pathol 2022). Common co-mutations also have limited clinical actionability. These observations suggest cancers with TSC1 and TSC2 alterations may be candidates for targeted therapy; this hypothesis is being tested in the currently enrolling PRECISION I (NCT05103358) study. Citation Format: David J. Kwiatkowski, Norma A. Palma, Willis H. Navarro, Gopa Iyer. Inactivating TSC1 and TSC2 alterations, co-mutations, and genomic instability in advanced cancers: Analysis of a real-world (RW) patient (pt) population using the Foundation Medicine genomic database [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 C019.

Abstract B007: Concordance between tumor tissue and plasma genotyping in NCI-MATCH Trial (EAY131)

Abstract Background: Utilization of liquid biopsy has become increasingly prevalent in the field of cancer therapeutics over the past decade. However, there remain unanswered questions that need to be addressed including better understanding of concordance between tumor tissue and plasma-derived circulating tumor DNA (ctDNA) from the same patient. In this study, we present the results of plasma ctDNA analysis by next-generation sequencing (NGS) in patients participating in selected arms of the NCI-MATCH Trial, with a focus on concordance between tumor tissue and plasma findings. Methods: Our study included patients treated on the NCI-MATCH Trial (NCT02465060) who had their plasma samples collected within 90 days before commencing treatment. 16 out of 39 treatment subprotocols of NCI-MATCH Trial were included in this plasma analysis protocol. Plasma samples were tested using the Guardant360 NGS assay (Guardant Health) and tumor tissue NGS results were obtained through central testing by the Oncomine assay (ThermoFisher). Concordance was defined as the detection of the same gene alteration leading to patient enrollment in NCI-MATCH trial, in both tissue and plasma. Results: Our study included 334 patients with a variety of tumor types who had baseline plasma samples. Plasma sequencing data were available in 328 patients; of whom 326 (99.4%) had a total of 2,779 alterations in plasma (2,055 somatic [74%] and 724 presumed germline [26%]). The tissue alteration of interest was detected in plasma of 196 (76.9%) of 255 patients enrolled based on central lab testing who had contemporaneous plasma and tissue samples. The concordance rate was higher in patients with tissue biopsies obtained from metastatic sites compared to patients with tissue biopsies obtained from primary tumor (79.9% [n=183] vs 45.5% [n=10], p<0.001). Concordance was highest for copy number variations (82.5%, n=52), single nucleotide variants (79.5%, n=124) and insertions/deletions (75%, n=12), while lower concordance rates were found for gene fusions (40%, n=8). A lower concordance rate was observed in 73 patients enrolled based on outside designated lab tissue testing (43.8% [n=32]). Notably, these patients had a median interval between plasma and tissue samples of 239 days (range; 38-1917). However, concordance was also higher in this group with samples collected from metastatic sites compared to primary tumors (62.5% vs 26.5%, p=0.003). Conclusions: In the NCI-MATCH Trial, the tumor tissue alteration of interest was detected in plasma of 76.9% of patients who had contemporaneous tissue and plasma samples, suggesting a potential role for liquid biopsy in patients’ enrollment. Concordance rates varied according to type of alteration of interest, and site of tissue sample (primary versus metastatic tumor), as well as the interval between sampling of tissue and plasma. Citation Format: Mohamed A Gouda, Filip Janku, Ying Yuan, Leylah Drusbosky, Alice P Chen, Keyur Patel, Stanley R Hamilton, Mark Routbort, P. Mickey Williams, John Iafrate, Jeffrey Sklar, Brent Coffey, Richard F Little, Carlos L Arteaga, Jeffrey S Abrams, Peter J O’Dwyer, Keith T Flaherty, Lyndsay N Harris, Funda Meric-Bernstam. Concordance between tumor tissue and plasma genotyping in NCI-MATCH Trial (EAY131) [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 B007.

Genomic landscape of cancer of unknown primary site (CUP): Mayo Clinic experience.

e18807 Background: CUP represents both a diagnostic and a management challenge. Despite improvements through sophisticated imaging modalities, specific and sensitive testing using immunohistochemistry, a concrete determination of the site of origin of the primary tumor can still be a diagnostic challenge Hereby, we described the genomic landscape of patients with CUP treated at our institution. Methods: Retrospective study of patients with CUP treated at Mayo Clinic Rochester MN from 2010 to 2021. Next generation sequencing (NGS) information was abstracted, and alterations were ranked into different levels of actionability following the Onco KB framework. All data were analyzed by the SPSS version 22.0 software (SPSS, Chicago, IL, United States). Results: From a total of 214 patients, we identified 67 (32%) patients in whom NGS was performed. Among patients with available NGS the median age was 64 years (range: 35-86) and 51% were female. the most prevalent histology included adenocarcinoma (39%), carcinoma NOS (31%), and neuroendocrine tumors (12%). NGS on tumor (t-NGS) was available in 56 (84 %) patients, circulating tumor NGS (b-NGS) was available in 12 (18%) patients, and only 1 patient had both testing available. Among the 56 patients with available t-NGS, MSI-high was detected in 9 patients, whereas among the 12 patients with available b-NGS, MSI-high was detected in 8 patients; yielding an MSI-high detection rate of 16% and 67% for t-NGS and b-NGS. In this cohort, there were a total of 474 genomic alterations detected from which 103 (22 %) were able to be ranked into one therapeutic level following the OncoKB framework. Genomic alterations were most observed among patients with carcinoma (n = 50, 48.53%) adenocarcinoma (n = 33, 32.03 %), squamous cell carcinoma (n = 3, 2.91 %) and neuroendocrine tumor (n = 2, 1.94%). Following the OncoKB framework, 39-level 1 genomic alterations were detected in a total of 14 genes. The most common level 1 genomic alterations comprised the BRCA2 (20.51%), NF1 ATM (17.95%) and HER2/NEU (ERBB2) (15.38%) genes. Therapeutic level 2 included 51 genomic alterations in 13 genes of which KRAS (17.65%), BRAF (15.69%), and BRCA2 (15.69%) were the most prevalent mutated genes. Additionally, level R comprised 13 genomic alterations in the following genes: KRAS (69.23) NRAS (23.08%) and PDGFRA (7.69%). Conclusions: CUP are malignancies rich in potentially actionable alterations, and NGS of tissue and blood samples can inform systemic therapy decisions. Alteration-specific targeted therapy may be an option for a substantial proportion of patients. NGS of tumor tissue or blood should be considered for all patients with CUP.

Circulating tumor DNA (ctDNA) dynamics and survival outcomes in patients (pts) with advanced non-small cell lung cancer (aNSCLC) and high (>50%) programmed cell death-ligand 1 (PD-L1) expression, randomized to cemiplimab (cemi) vs chemotherapy (chemo).

9022 Background: While ctDNA has emerged as a promising tool for monitoring response on therapy, there is a paucity of data from prospective, randomised Phase 3 studies to establish clear criteria for the clinical application of ctDNA. In the EMPOWER-Lung 1 study, first line (1L) cemi monotherapy improved overall survival (OS) vs platinum chemo in pts with aNSCLC, PD-L1 ≥50%, and no EGFR, ALK, or ROS1 aberrations and an acceptable risk/benefit profile. We performed personalized tumor-specific analysis of ctDNA from pts treated in the EMPOWER-Lung 1 study to determine the optimal time point and magnitude of ctDNA variation that are associated with OS and progression-free survival (PFS). Methods: Tumor tissue next-generation sequencing was performed to identify tumor-specific DNA variants. ctDNA levels were monitored using personalized pt specific probe sets (Natera, Foundation Medicine) in the plasma at baseline, end of week 3 (W3) and W9. Endpoints included overall response rate (ORR) complete or partial response (CR/PR), stable disease (SD) and progressive disease (PD) per RECIST 1.1, OS and PFS. Association between ctDNA decrease and clinical endpoints was tested in 3 groups of pts defined by sensitivity analysis: no-decrease, any decrease (molecular response, MR), and complete clearance (complete MR, cMR) of ctDNA. Results: ctDNA analysis was performed on samples from 175 pts (chemo n = 89; cemi n = 86). Baseline characteristics, ORR, OS, and PFS were comparable to those of the intention to treat population. At W9, cMR/MR was associated with clinical response to cemi (CR/PR: 97%, SD:58%, PD:17%), but not with clinical response to chemo, as it was detected in the majority of chemo pts across all RECIST categories (CR/PR: 100%, SD:90%, PD:80%). In the cemi arm, ctDNA cMR was associated with the longest survival, with median OS (mOS) not-reached compared to pts with MR (mOS of 29 months) and pts with no-decrease (mOS of 8 months), as well as statistically significant hazard ratios (cMR vs MR: W9 HR = 8, 95% CI 1.8-35, p = 0.0056; cMR vs no-decrease: W9 HR = 25, 5.7–110, p = 0.000021 and W3 HR = 5.9, 1.4-25, p = 0.017). Delayed MR/cMR (i.e., MR/cMR at W9 but not at W3) was observed in 8/66 pts (12%), and transient MR (i.e., MR at W3 but not W9) in 9/66 pts (14%), with available ctDNA data at both timepoints. Conclusions: This is the largest data set to date correlating ctDNA levels with efficacy outcomes from a randomized clinical trial comparing chemo with immunotherapy in 1L aNSCLC. The results indicate that lack of treatment-induced decrease in ctDNA may identify pts with inferior OS benefit from cemi monotherapy as early as 3 weeks following initiation of therapy and can inform future use of early ctDNA response assessment in prospective interventional studies. Clinical trial information: NCT03088540 .

Plasma Sequencing for Patients with GIST-Limitations and Opportunities in an Academic Setting.

Circulating tumor DNA (ctDNA) from circulating free DNA (cfDNA) in GIST is of interest for the detection of heterogeneous resistance mutations and treatment monitoring. However, methodologies for use in a local setting are not standardized and are error-prone and difficult to interpret. We established a workflow to evaluate routine tumor tissue NGS (Illumina-based next generation sequencing) panels and pipelines for ctDNA sequencing in an academic setting. Regular blood collection (Sarstedt) EDTA tubes were sufficient for direct processing whereas specialized tubes (STRECK) were better for transportation. Mutation detection rate was higher in automatically extracted (AE) than manually extracted (ME) samples. Sensitivity and specificity for specific mutation detection was higher using digital droplet (dd)PCR compared to NGS. In a retrospective analysis of NGS and clinical data (133 samples from 38 patients), cfDNA concentration correlated with tumor load and mutation detection. A clinical routine pipeline and a novel research pipeline yielded different results, but known and resistance-mediating mutations were detected by both and correlated with the resistance spectrum of TKIs used. In conclusion, NGS routine panel analysis was not sensitive and specific enough to replace solid biopsies in GIST. However, more precise methods (hybridization capture NGS, ddPCR) may comprise important research tools to investigate resistance. Future clinical trials need to compare methodology and protocols.

Biomarkers for Systemic Therapy in Metastatic Breast Cancer: ASCO Guideline Update

To update the ASCO Biomarkers to Guide Systemic Therapy for Metastatic Breast Cancer (MBC) guideline. An Expert Panel conducted a systematic review to identify randomized clinical trials and prospective-retrospective studies from January 2015 to January 2022. The search identified 19 studies informing the evidence base. Candidates for a regimen with a phosphatidylinositol 3-kinase inhibitor and hormonal therapy should undergo testing for PIK3CA mutations using next-generation sequencing of tumor tissue or circulating tumor DNA (ctDNA) in plasma to determine eligibility for alpelisib plus fulvestrant. If no mutation is found in ctDNA, testing in tumor tissue, if available, should be used. Patients who are candidates for poly (ADP-ribose) polymerase (PARP) inhibitor therapy should undergo testing for germline BRCA1 and BRCA2 pathogenic or likely pathogenic mutations to determine eligibility for a PARP inhibitor. There is insufficient evidence for or against testing for a germline PALB2 pathogenic variant to determine eligibility for PARP inhibitor therapy in the metastatic setting. Candidates for immune checkpoint inhibitor therapy should undergo testing for expression of programmed cell death ligand-1 in the tumor and immune cells to determine eligibility for treatment with pembrolizumab plus chemotherapy. Candidates for an immune checkpoint inhibitor should also undergo testing for deficient mismatch repair/microsatellite instability-high to determine eligibility for dostarlimab-gxly or pembrolizumab, as well as testing for tumor mutational burden. Clinicians may test for NTRK fusions to determine eligibility for TRK inhibitors. There are insufficient data to recommend routine testing of tumors for ESR1 mutations, for homologous recombination deficiency, or for TROP2 expression to guide MBC therapy selection. There are insufficient data to recommend routine use of ctDNA or circulating tumor cells to monitor response to therapy among patients with MBC.Additional information can be found at www.asco.org/breast-cancer-guidelines.

Abstract 3411: Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients

Abstract Background: The analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision making and disease monitoring. A suitable diagnostic-grade platform is required for detection of tumor-specific mutations with high sensitivity in circulating cell-free DNA (ccfDNA) of cancer patients. Methods: In an international multicenter study conducted by three laboratories participating in the CANCER-ID consortium, a cohort containing 177 cell-free plasma samples at baseline, follow-up or disease progression derived from 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC), relevant mutations were tested using the UltraSEEK® Lung Panel on the MassARRAY® System covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2 and PIK3CA, and compared against mutation-specific droplet digital PCR (ddPCR) and tumor tissue next-generation sequencing (NGS) performed in routine diagnostics. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy were analyzed. Results: The UltraSEEK® Lung Panel revealed an overall detection rate of 87.2% irrespective of ccfDNA input, and a specificity of >99.5% using the reference material. When comparing the 131 variants identified in the patient-derived cell-free plasma using UltraSEEK® with mutation-specific ddPCR analyses, a concordance of 90% was found. The 77 diagnostically or clinically relevant variants identified in 66 pretreatment tumor tissue showed an overall concordance with UltraSEEK® of 73% at baseline with a false-negative rate of 3%. When restricting to therapeutically targetable mutations, the concordance elevated to 85%. A decrease in ctDNA levels at 4-6 weeks after start of treatment detected with UltraSEEK® correlated with a prolonged median PFS (46 vs 11 weeks; P<0.05) and OS (145 vs 39 weeks; P<0.001). In four out of thirteen NSCLC patients treated with targeted therapy, resistance mutations were identified using UltraSEEK® at disease progression, implicating the requirement of a therapy switch. Conclusion: Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect both mutations at diagnosis and disease progression, and to monitor treatment response. Citation Format: Paul van der Leest, Melanie Janning, Naomi Rifaela, Maria L. Aguirre Azpurua, Jolanthe Kropidlowski, Sonja Loges, Nicholas Lozano, Alexander Sartori, Darryl Irwin, Pierre-Jean Lamy, T. Jeroen N. Hiltermann, Harry J. Groen, Klaus Pantel, Léon C. van Kempen, Harriet Wikman, Ed Schuuring. Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3411.

Evaluation of tumor gene expression profile (GEP) in minority patients with stage IV non-small cell lung cancer (NSCLC).

e21043 Background: Differences in tumor gene expression profile (GEP) may contribute to disparities in lung cancer incidence and survival between racial and ethnic groups. For patients with stage IV NSCLC, tumor biomarker analysis is important for identification of potentially effective targeted therapies. However, despite advancements in biomarker testing and targeted therapy, minority patient enrollment in clinical trials remains low and it has been shown that minority patients are less likely to undergo next-generation sequencing (NGS) compared to Non-Hispanic Whites (NHW). As such, frequencies of tumor biomarkers in minority populations in the United States are not well-defined. This study aimed to determine the extent of tumor GEP diversity in minority patients vs NHW. Methods: This retrospective study included 196 adult patients with stage IV NSCLC that had a baseline NGS of tumor tissue or blood (liquid biopsy) performed between July 1, 2015 and June 30, 2021. Minority populations included Hispanic, African American, and Asian patients. Data collected utilizing the electronic medical record included patient demographics, detection of tumor biomarkers and treatment initiated. Descriptive statistics were calculated for all demographic and clinical variables. Comparisons of actionable epidermal growth factor receptor (EGFR) mutation frequency between NHW and minority patients were done using Chi-squared and Fisher’s exact tests. Results: Of the 196 patients evaluated, baseline characteristics included 46% male, 67% former smokers, 54% NHW, 31% Hispanic, 9% African American, 5% Asian, and 1% other. The median age at diagnosis was 66 ± 12 years. Actionable mutations (EGFR, KRAS, BRAF, ROS-1, ALK, MET, NTRK 1-3) were found in 50% of patients (see Table). The frequency of actionable EGFR mutations was higher in minority patients overall compared to NHW (39.3% vs 21.5%; p < 0.01). The frequency of actionable EGFR mutations was similar between NHW and Hispanic patients (21.5% vs 27.9%; p = 0.35), however differences were observed comparing NHW to African American (21.5% vs 50%; p = 0.02) and Asian (21.5% vs 90%; p < 0.01) patients, respectively. Conclusions: Among patients with stage IV NSCLC, the GEP of minority patients varied from that of NHW. Asian patients had the highest frequency of EGFR mutations, followed by African American and Hispanic patients, respectively. Larger studies are needed to validate these findings.[Table: see text]

Molecularly Targeted Therapy towards Genetic Alterations in Advanced Bladder Cancer.

Simple SummaryIn recent years, there have been several advances in the care of advanced bladder cancer, highlighted by the addition of immune checkpoint inhibitors to the care of advanced disease. Despite these advances, there is a need for further improvement; the morbidity and mortality associated with advanced bladder cancer remain high. With the recent incorporation of advanced molecular techniques, there is more clarity regarding key genetic alterations of the disease. Therapies directed at specific genetic aberrations in bladder cancer provide both proven and potential paths forward. This review discusses the key targetable genetic aberrations and summarizes the current status of targeted therapies in muscle-invasive bladder cancer.Despite the introduction of immune checkpoint inhibitors and antibody–drug conjugates to the management of advanced urothelial carcinoma, the disease is generally incurable. The increasing incorporation of next-generation sequencing of tumor tissue into the characterization of bladder cancer has led to a better understanding of the somatic genetic aberrations potentially involved in its pathogenesis. Genetic alterations have been observed in kinases, such as FGFRs, ErbBs, PI3K/Akt/mTOR, and Ras-MAPK, and genetic alterations in critical cellular processes, such as chromatin remodeling, cell cycle regulation, and DNA damage repair. However, activating mutations or fusions of FGFR2 and FGFR3 remains the only validated therapeutically actionable alteration, with erdafitinib as the only targeted agent currently approved for this group. Bladder cancer is characterized by genomic heterogeneity and a high tumor mutation burden. This review highlights the potential relevance of aberrations and discusses the current status of targeted therapies directed at them.

Nivolumab/ipilimumab with or without CBM588 in metastatic renal cell carcinoma: A randomized phase Ib study and the evolution of the functionality of microbial communities with treatment.

371 Background: The role of gut microbial composition as a determinant of clinical outcomes has been well established in several cancers, including metastatic renal cell carcinoma (mRCC) (Routy et al Science 2018). A growing body of evidence suggests that examining the metabolic function of microbial communities may provide a more insightful understanding of these associations (Helmink et al Nature Medicine 2019). Herein, we aimed to examine the effect of nivolumab/ipilimumab with or without CBM588 on clinical outcomes and gut microbiome functionality. Methods: Treatment naïve mRCC pts with clear cell and/or sarcomatoid histology and IMDC intermediate/high risk disease were enrolled and randomized into receiving nivolumab/ipilimumab or nivolumab/ipilimumab with CBM588 in 1:2 fashion. Whole metagenome sequencing was performed on stool samples collected at baseline and week 12. Generated MetaPhlan 3.0 data was run through HUMAnN 3.0 to identify differentially expressed metabolic pathways between two timepoints in each arm and with respect to treatment response. Results: A total of 30 pts were enrolled and randomized, and 29 pts were eligible for analysis as one patient was excluded as tumor tissue next-generation sequencing revealed genomic alterations pathognomonic for sarcoma after initiation of protocol-based therapy. Median age of the participants was 66 years, 21 pts (72%) were male, 10 pts (34%) had sarcomatoid features, and 29 pts (45%) had prior nephrectomy. Objective response was achieved in 58% and 20% of the pts in nivolumab/ipilimumab/CBM-588 and nivolumab/ipilimumab arm, respectively. Significant changes in 40 metabolic pathways (37 with upregulation and 3 with downregulation) in nivolumab/ipilimumab arm and 52 metabolic pathways (49 with downregulation and 3 with upregulation) in nivolumab/ipilimumab with CBM588 arm were identified. In detail, dTDP-β-L-rhamnose biosynthesis, L-lysine biosynthesis II and superpathway of pyrimidine ribonucleosides degradation pathways were found upregulated while O-antigen building blocks biosynthesis (E. coli) pathway was found downregulated after treatment with nivolumab/ipilimumab and CBM588 (p = 0.001, p = 0.007, p = 0.037, p = 0.005 respectively). Heatmaps detailing the dynamics of metabolic pathway expressions in regard to response in each arm will be presented. Conclusions: We observed an increase in the activity of the pathways associated with butyrate consumption and a resultant decrease in glycolytic dependence. Further, suppression of the pathogenic E. coli function was observed, suggesting a role for CBM588 in protection from pathogenic species. Our findings provide mechanistic evidence for the effect of the addition of CBM588 to nivolumab/ipilimumab on gut microbiome function and resultant improvement in clinical outcomes in mRCC. Clinical trial information: NCT03829111.

Genomic landscape of treatment refractory metastatic colorectal cancer

Background Metastatic colorectal cancer (mCRC) is a complex and heterogeneous disease with few standard and targeted treatment options. Next-generation sequencing of tumor tissue was performed to identify cancer driver mutations to discover possible personalized treatment options, as targeted treatment possibilities are limited for this patient population. Results of genomic sequencing in patients with treatment-refractory mCRC are described in this retrospective analysis. Material and methods Clinico-pathological characteristics and genomic sequence results of consecutive patients with refractory mCRC, referred to the Experimental Cancer Therapy Unit (ECTU) at Department of Oncology, Herlev & Gentofte Hospital in the period from 1 October 2015 to 14 December 2018 were reviewed in this retrospective analysis. Tumor tissue from the patients was analyzed by next-generation sequencing using the Oncomine Comprehensive primer panel to detect actionable variants of cancer driver mutations and microsatellite instability status. From August 2018 tumor mutational burden was also analyzed. Results A total of 80 patients with treatment-refractory mCRC and in a fairly good performance were referred to the ECTU during this period. Genomic sequencing of tumor tissue was performed for all 80 patients and a cancer driver mutation was identified in 90% (n = 72) of the patients. A total of 31.3% (n = 25) of the patients received therapy either as targetable therapy outside an available trial (n = 2), FDA approved therapy (n = 2), or treatment in phase 1 or 2 trials, independent of the genomic signature 26.3% (n = 21). Conclusion Most mCRC patients refractory to standard anti-neoplastic therapies, presented with a cancer driver mutation, however, only a few of these mutations gave rise to matched therapies as only 2.5% of the patients from this period received targeted therapy.

Exploration of Next-Generation Sequencing of Tumor Tissue and Blood in Head and Neck Squamous Cell Carcinoma

Next-generation sequencing of circulating tumor DNA (ctDNA) is a promising new tool in the analysis of tumor genomics that has traditionally been performed through sequencing of tumor tissue (tDNA). Analysis and correlation of the two platforms in each tumor type can bring valuable diagnostic, therapeutic and prognostic information. The aim of this study is to explore the genomic signature of Head and Neck Squamous Cell Carcinoma (HNSCC) in circulation and tumor tissue and understand the implications of ctDNA sequencing for prognosis and precision oncology treatments.

Role of Targeted Therapies in Management of Metastatic Urothelial Cancer in the Era of Immunotherapy.

Despite significant advances and the approval of immune checkpoint inhibitors, metastatic urothelial carcinoma (mUC) is still very hard to treat and has poor outcomes. Deeper understanding of the molecular underpinnings of mUC has identified potential biomarkers, biologic drivers, and relevant therapy targets. However, targeted therapies in mUC have had significant challenges due to molecular heterogeneity, clonal evolution, and genomic instability, and have not improved outcomes so far. Despite that, recent technological developments, clinical utilization of molecular biology, and discovery of new agents with preclinical and early clinical activity have signaled a new age of experimental therapeutics in mUC. The more frequent use of next-generation sequencing of tumor tissue and cell-free circulating tumor DNA, combined with novel agents tested in clinical trials, provides promise. There is a plethora of agents being tested in mUC, including inhibitors of receptors and signaling pathways (e.g., fibroblast growth factor receptor, human epidermal growth factor receptor, phosphatidylinositol 3-kinase/AKT/mTOR pathway), angiogenesis (e.g., vascular endothelial growth factor and its receptors), poly (ADP-ribose) polymerase (PARP) inhibitors, immuno-oncology agents, cytotoxic agents (e.g., chemotherapy, antibody drug conjugates), and epigenetic modulators, among others. Two agents, enfortumab-vedotin (antibody drug conjugate) and erdafitinib (fibroblast growth factor receptor inhibitor), have breakthrough designation by the FDA, but are not approved as of March 2019. Novel combinations with various modalities and optimal sequencing of active therapies are being investigated in clinical trials. More sophisticated patient selection, discovery and prospective validation of predictive (and prognostic) biomarkers with clinical utility, and relevant clinical trial designs are important parameters in that context. Based on the above, there is high potential for targeted therapies to be added in the growing armamentarium of mUC, and possibly complement chemotherapy and immuno-oncology agents.

Predictors of treatment response in colorectal high-grade neuroendocrine carcinomas (HGNEC): A single institution experience.

690 Background: Colorectal high-grade neuroendocrine carcinomas (HGNEC) are aggressive tumors; treatment options consist of cis/carboplatin and 5FU-based chemotherapy. To date, choice of systemic therapy and sequencing of these drugs remains poorly understood. We examined clinical and molecular characteristics of colorectal HGNEC to better define predictors of response to cis/carboplatin and 5FU-based treatment. Methods: Patients (pts) with colorectal HGNEC treated at MSKCC from 1990-2018 were identified. MANEC (mixed adeno-neuroendocrine carcinoma) were excluded. Demographics, response to first- and second-line chemotherapy (by radiology report), outcomes, and molecular data (next-generation sequencing of tumor tissue), were collected. Results: 65 pts (mean age 58, 52% male) were identified, 13 (20%) with small cell carcinomas. 52 (79%) metastatic, 13 (20%) locally advanced. 27 (42%) received surgery and 11 (17%) received radiation. 56 pts received cis/carboplatin-based therapy, partial response (PR) in 18 (32%), stable disease (SD) in 4 (7%), and progressive disease (PD) in 31 (55%); 3 (5%) did not tolerate therapy. 28 pts received 5FU-based therapy, 13 PR in (46%), SD in 6 (21%), and PD in 7 (25%); 2 (7%) did not tolerate therapy. Median overall survival was 11.4 months. 21/65 (32%) pts underwent molecular sequencing of tumor; the most common alterations were KRAS 11 (52%), TP53 13 (62%), BRAF 7 (33%), APC 8 (38%), RB1 7 (33%). Most tumors (13/21, 62%) harbored alterations in genes traditionally altered in colorectal adenocarcinoma (KRAS/BRAF/APC) and in HGNEC (TP53/RB1). There was no significant difference in response to cis/carboplatin or 5FU-based chemotherapy based on location of the primary tumor (right vs. left) (p = 0.69/0.85), histologic features of the disease (p = 0.71/0.87), and for response to cis/carboplatin by molecular alterations in KRAS (p = 0.94), BRAF (p = 0.24), APC (p = 0.28), TP53 (p = 0.58), or RB1 (p = 0.28). Conclusions: Colorectal HGNEC are highly aggressive and more effective therapies are desperately needed. In this series, OS was poor. Clinical and molecular characteristics failed to predict response to cis/carboplatin and 5FU-based chemotherapy.

High concordance in advanced cancer patient paired testing by commercially available tumor tissue NGS assays and a liquid biopsy NGS assay.

e24169Background: Next-generation sequencing (NGS) assays are increasingly being used in oncology clinical practice. With the recent FDA approval of a commercially available tumor tissue NGS assay ...

The Role of Circulating Tumor DNA in Renal Cell Carcinoma.

Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) is a novel technology that can complement tumor tissue NGS and has the potential to influence diagnosis and treatment of both localized and metastatic renal cell carcinoma (mRCC). ctDNA NGS is an attractive alternative to tumor tissue NGS because it circumvents the need for repeated, invasive tissue biopsies while providing a contemporary mutational profile of a patient's tumors. While the role of ctDNA NGS in non-small cell lung cancer and colorectal cancer is well established, studies of ctDNA NGS in mRCC are only hypothesis-generating to date. In the localized RCC setting, ctDNA has demonstrated potential as a surveillance biomarker for disease recurrence. Earlier detection of mRCC, prior to the onset of symptoms, may lead to improved clinical outcomes. NGS of ctDNA in mRCC is even more promising in patients with metastatic disease. The majority of patients with mRCC have detectable ctDNA. Thus, ctDNA could be used to select patients for biomarker-guided clinical trials, such as savolitinib in MET-positive papillary RCC. Furthermore, studies have shown that the mutational profile of mRCC in ctDNA evolves after treatment progression. The most exciting potential role for ctDNA in mRCC is as a predictive biomarker for response to immunotherapy. Studies have shown that tumor mutational burden (TMB) is predictive of response to immune checkpoint inhibitors, and hypermutated ctDNA can act as a surrogate biomarker for TMB and response to immunotherapy. While studies of ctDNA in RCC are still in their infancy, there are many promising roles for ctDNA in localized and metastatic RCC.

1265P - Correlation of somatic genomic alterations between tissue genomics and circulating tumor DNA (ctDNA) employing next generation sequencing (NGS) analysis in lung and gastrointestinal cancers

Background: Peripheral blood circulating tumor DNA (ctDNA) and tumor tissue next-generation sequencing (NGS) is routinely performed to guide therapy in cancer patients. However, little is known about the concordance or discordance between commercially available tissue genomics testing panels and ctDNA. The aim of our study was to assess concordance between matched cancer tissue genomics and blood based ctDNA in lung and gastrointestinal (GI) cancers. Methods: Tissue genomic analysis was performed with Paradigm® (n = 17)/Caris® (n = 11) and ctDNA was analyzed with Guardant360® (n = 28). Samples included, non-small cell lung cancer (n = 10), small cell lung cancer (n = 4), colorectal cancer (n = 5), hepatocellular carcinoma (n = 2), intrahepatic cholangiocarcinoma (n = 1), pancreatobiliary adenocarcinoma (n = 3), esophageal adenocarcinoma (n = 2) and gastric adenocarcinoma (n = 1). Results: We identified 6 (21%) patients with at least one gene mutation that was detected by both tissue genomic and ctDNA analysis. Total number of gene mutations identified in 28 patients were 106, but only 8 (7.5%) were detected by both tissue and ctDNA panels. When this testing was done within 90 days the concordance increased to 10.20%. Table.Table1265PPatients with at least one mutation detected by both platforms21% (n = 6/28)Mutations detectable by both platforms7.54% (n = 8/106)Mutations detectable by both platforms if interval between tissue and blood collection <90 days.10.20% (n = 5/49) Open table in a new tab Conclusions: We identified significant discordance between tissue and ctDNA mutational profiles in lung and GI cancers. Therefore, the results from NGS platforms should be interpreted with extreme caution. Our analysis reveals that these platforms should not be used interchangeably. The discordance rate may be due to tissue heterogeneity and/or spatial and temporal clonal evolution. Standardization of the sequencing techniques and education of practicing oncologists about the limitations of liquid biopsies needs to be highlighted. Legal entity responsible for the study: Saint Luke’s Health System Funding: None Disclosure: J. Subramanian: Paradigm Advisory board. All other authors have declared no conflicts of interest.