Actionable Alterations Research Articles

Precision needle-punch tumor enrichment from paraffin blocks improves the detection of clinically actionable genomic alterations and biomarkers.

While many molecular assays can detect mutations at low tumor purity and variant allele frequencies, complex biomarkers such as tumor mutational burden (TMB), microsatellite instability (MSI), and genomic loss of heterozygosity (gLOH) require higher tumor purity for accurate measurement. Scalable, quality-controlled, tissue-conserving methods to increase tumor nuclei percentage (TN%) from tumor specimens are needed for complex biomarkers and hence necessary to maximize patient matching to approved therapies or clinical trial enrollment. We evaluated the clinical utility and performance of precision needle-punch enrichment (NPE) compared with traditional razor blade macroenrichment of tumor specimens on molecular testing success. Pathologist-directed NPE was performed manually on formalin-fixed, paraffin embedded (FFPE) blocks. Quality control of target capture region and quantity of residual tumor in each tissue block was determined via a post-enrichment histologic slide recut. Resultant tumor purity and biomarker status were determined by the computational analysis pipeline component of the FDA-approved next-generation sequencing (NGS) assay, FoundationOne®CDx. Following NPE implementation for real-world clinical samples, assay performance and biomarker (MSI, TMB, gLOH) detection were analyzed. In real-world clinical samples, enrichment rate via NPE was increased to ~50% over a 2.5-year period, exceeding the prior use of razor blade macro-enrichment (<30% of cases) prior to NPE implementation due to proven efficacy in generating high quality molecular results from marginal samples and the ease of use for both pathologist and histotechnologists. NPE was associated with lower test failures, higher computational tumor purity, and higher rates of successful TMB, MSI and gLOH determination when stratified by pre-enriched (incipient) tumor nuclei percentage. In addition, challenging cases in which tumor content was initially insufficient for testing were salvaged for analysis of biomarker status, gene amplification/deletion, and confident mutant or wild-type gene status determination. Pathologist-directed precision enrichment from tissue blocks (aka NPE) increases tumor purity, and consequently, yields a greater number of successful tests and complex biomarker determinations. Moreover, this process is rapid, safe, inexpensive, scalable, and conserves patient surgical pathology material. NPE may constitute best practice with respect to enriching tumor cells from low-purity specimens for biomarker detection in molecular laboratories.

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

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

Comprehensive analysis of targetable alterations in urachal cancer by NGS.

663 Background: Urachal cancer (UrC) is a rare genitourinary cancer with molecular drivers commonly found in colorectal cancer and harbors fewer actionable alterations. Although prior studies have identified genomic alterations associated with UrC, these lack comprehensive genomic profiling, often using targeted panels, and lack RNA interrogation. This study presents detailed characterization of molecular profiles and the tumor microenvironment of real-world UrC patient samples. Methods: UrC samples (n = 42) were profiled using next-generation sequencing (NGS) of DNA and RNA. Prevalence was calculated for pathogenic SNVs/Indels and copy number amplifications, dMMR/MSI status assessed by IHC and NGS, PD-L1 expression measured by IHC, and TMB-H defined as ≥ 10 mut/Mb. Immune cell fractions of TME were estimated using RNA deconvolution. Mann-Whitney U, chi-square, and Fisher exact tests were applied where appropriate, with p-values adjusted for multiple comparisons. Histological features were reviewed by a genitourinary pathologist. Results: UrC samples were collected from 19 males and 23 females with a median age at sample collection was 58.5 years (range: 24-86 years). Pathology review revealed the majority were adenocarcinomas (n=41/42, 97.6%), including mucinous (n=17/41, 41.5%) and enteric adenocarcinomas (n=10/41, 24.4%). Pathogenic variants were most commonly observed in TP53 (n = 35, 83.3%), KRAS (n=15, 36%), GNAS (n=5, 12%), SMAD4 (n=4, 10%), PIK3CA (n=4, 10%), STK11 (n=3, 7%), BRCA2 (n=3, 7%), ARID1A (n=2, 5%), PTEN (n=2, 5%), ERBB2 (n=2, 5%), BRAF (n=2, 5%), and APC (n=2, 5%). MAPK pathway alterations were present in 52.3% (n=22) overall, with increased MAPK pathway activation in MAPK-altered UrC (median MPAS: 0.77 vs -0.61, p = 0.066). Loss of heterozygosity was prevalent (n = 9, 29%). While no UrC tumors had dMMR/MSI, 5% (n=2) were TMB-H and 9% (n=3) were PD-L1+. The mean fraction of T cells – CD8 (0.4%) and monocytes (0.0%) represented a smaller fraction UrC TME composition compared to macrophages M1 (5.19%) and neutrophil (6.4%). The median NK cell fraction was significantly higher in MAPK-altered UrC patients (3.5 vs 2.7%, p = 0.022), yet a transcriptional signature of response to immunotherapy (IFN-g) was similar between MAPK-altered and MAPK-WT UrC patients (median IFN: -0.366 vs -0.384, p = 0.052). Conclusions: This study provides a comprehensive molecular characterization of UrC and the associated immune landscape. While MAPK and DNA repair gene alterations were common, UrC tumors rarely harbored predictive markers of response to immunotherapy, suggesting limited efficacy in this patient population. However, the recurrence of MAPK alterations and associated pathway activation in UrC warrants further investigation of MAPK-targeted therapies in prospective clinical trials.

Plasma-based analysis of ERBB2 mutational status by multiplex digital PCR in a large series of patients with metastatic breast cancer.

Erb-b2 receptor tyrosine kinase 2 (ERBB2)-activating mutations are therapeutically actionable alterations found in various cancers, including metastatic breast cancer (MBC). We developed multiplex digital PCR assays to detect and quantify ERBB2 mutations in circulating tumor DNA from liquid biopsies. We studied the plasma from 272 patients with hormone-receptor-positive, human epidermal growth factor receptor 2-negative (HR+/HER2-) MBC to detect 17 ERBB2 mutations using a screening assay. The assay was developed on the three-color Crystal dPCR™ naica® platform with a two-step strategy for precise mutation identification. We found that nine patients (3.3%) harbored at least one ERBB2 mutation. The mutation rate was higher in patients with lobular histology (5.9%) compared to invasive breast carcinoma of no special type (2.6%). A total of 12 mutations were found with the following frequencies: L755S (25.00%), V777L (25.00%), S310Y (16.67%), L869R (16.67%), S310F (8.33%), and D769H (8.33%). Matched tumor samples from six patients identified the same mutations with an 83% concordance rate. In summary, our highly sensitive multiplex digital PCR assays are well suited for plasma-based monitoring of ERBB2 mutational status in patients with MBC.

Integrated Proteogenomic Analysis Reveals Distinct Potentially Actionable Therapeutic Vulnerabilities in Triple-Negative Breast Cancer Subtypes

Triple-negative breast cancer (TNBC) is characterized by an aggressive clinical presentation and a paucity of clinically actionable genomic alterations. Here, we utilized the Cancer Genome Atlas (TCGA) to explore the proteogenomic landscape of TNBC subtypes to see whether genomic alterations can be inferred from proteomic data. We found only 4% of the protein level changes are explained by mutations, while 21% of the protein and 35% of the transcriptomics changes were determined by copy number alterations (CNAs). We found tighter coupling between proteome and genome in some genes that are predicted to be the targets of drug inhibitors, including CDKs, PI3K, tyrosine kinase (TKI), and mTOR. The validation of our proteogenomic workflow using mass spectrometry Clinical Proteomic Tumor Analysis Consortium (MS-CPTAC) data also demonstrated the highest correlation between protein–RNA–CNA. The integrated proteogenomic approach helps to prioritize potentially actionable targets and may enable the acceleration of personalized cancer treatment.

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.

Actionable molecular alterations in veterans with advanced cholangiocarcinoma.

548 Background: Cholangiocarcinoma (CCA) requires integrated efforts to understand the heterogeneity in available molecular targets. Chemotherapy remains a non-selective standard of care, however defining distinct populations with molecular actionability remains a critical need. The Veteran Health Administration’s (VHA) National Precision Oncology Program (NPOP) was established to characterize comprehensive genomic profiles across the integrated VHA network. Methods: Veterans with CCA underwent next-generation sequencing (NGS) with FoundationOne CDx, which enhanced sensitivity in the reporting of gene fusions. Analysis included dedicated chart review for date of diagnosis and survival utilizing the VHA’s Corporate Data Warehouse. Molecular alterations included mutations(mut), amplifications (amp) and fusion/rearrangements (fus). Log-rank Kaplan-Meier survival analysis was stratified by primary tumor location, and actionable driver alterations defined by VA Clinical Pathways. Results: Veterans diagnosed with CCA (07/2008-04/2023) underwent tissue-based NGS (n=483) including 96 unique VHA sites with male predominant population (96.9%). There was a predominance of localized disease at clinical presentation (55.3%). A summary of common pathogenic variants is provided (see table). Actionable alterations defined as IDH1mut, FGFR2fus, ERBB2amp, BRAFmut (V600E), and MSI-H collectively represented 21.1% of all Veteran CCA. The largest population was intrahepatic CCA (n=327) with actionable alterations including IDH1mut (13.5%), ERBB2amp (4.3%), and FGFR2fus (4.3%). Data suggested improved overall survival (OS) in IDH1mut Intrahepatic CCA including median OS 19.8 months (mo) v. 10.7 mo (p&lt;0.03). ERBB2amp (n=32) was enriched in extrahepatic (8.1%) and gallbladder cancer (17.2%), however did not improve OS including 7.1 mo in ERBB2amp v. 13.2 mo in wildtype (p=0.15). PIK3CAmut CCA (n=35) was found to confer inferior OS across the population with median OS of 7.5 mo v. 13.2 mo in wildtype (p&lt;0.02). Additional pathologic mutations in genes of homologous recombination deficiency (31.7%), PIK3CA (7.2%), and amplifications in MDM2 (5.0%). Conclusions: Across an integrated healthcare system of Veterans, actionable molecular alterations from VA Pathways were seen in 21.1% of the population. Further data is needed to clarify the prognostic versus predictive value of integrated precision-based strategies in clinical practice. The Veteran population includes important molecular subtypes to consider in future prospective trial design. [Table: see text]

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 (&lt;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.

Evaluation of a real-world clinico-genomics database of patients with upper gastrointestinal (GI) malignancies in Japan.

400 Background: The genomic profile of gastrointestinal (GI) cancers provides essential information for precision medicine, however, there is limited availability of real-world data in Asian populations as they are not well-represented in most publicly accessible clinico-genomic databases. In this retrospective study, we evaluated the clinical and genomic characteristics of upper GI cancer patients in the database from an academia-industry collaborative nationwide cancer genome screening project in Japan. Methods: The Screening Projectfor Individualized Medicine in Japan (J-SCRUM) GI-SCREEN enrolled advanced GI cancer patients from 26 sites before planned or during systemic therapy from 2015 to 2019. Archived formalin-fixed paraffin-embedded tumor tissue was analyzed by next-generation sequencing (NGS) using Oncomine Cancer Research Panel and Oncomine Comprehensive Assay. Patients with gastric (GC), esophageal (EC) and pancreatic (PC) cancers were identified in the database. The frequency of key actionable genomic alterations in each cancer cohort was explored and compared to similar upper GI cohorts in The Cancer Genome Atlas (TCGA), a publicly available genomics database initiated in 2005 and concluded sample collection in 2013 with most samples from White cancer patients. Results: A total of 872 GC (mean age at registration [SD]: 64y [12]; percent males: 65%), 265 EC (64y [9]; 78%) and 495 PC (64y [10]; 60%) patients were identified. The majority (&gt;70%) of samples were obtained from primary sites. Adenocarcinoma was the most common histological type for GC (85%) ad PC (76%), while squamous cell (76%) was the most common among EC patients. In the GC cohort, the prevalence of key actionable genomic alterations in J-SCRUM GI-SCREEN vs TCGA (N = 380) was, TP53 mutation (49% vs 49%), ERBB2 amplification (14% vs 13%), PIK3CA mutation (11% vs 14%), MYC amplification (9% vs 12%) and MET amplification (2% vs 3%). In the EC cohort, the prevalence in J-SCRUM vs TCGA (N = 182) was TP53 mutation (77% vs 87%), NFE2L2 mutation (21% vs 10%), PIK3CA mutation (9% vs 9%) and CDKN2A mutation (8% vs 8%). In the PC cohort, the prevalence in J-SCRUM vs TCGA (N = 150) was KRAS hotspot mutation (90% vs 93%), KRAS G12D (43% vs 41%), KRAS G12V (32% vs27%), TP53 mutation (58% vs 64%), BRCA1/2 deleterious mutation (5% vs 5%), ATM deleterious mutation (6% vs 5%) and MYC amplification (5% vs 5%). Conclusions: As part of a nationwide cancer genome screening project, J-SCRUM GI SCREEN database provides comprehensive genomic information for Asian population (Japan). The prevalence of key actionable gene alterations in each tumor type was highly comparable between J-SCRUM GI-SCREEN and TCGA data.

Real-world testing, treatment patterns, and outcomes following liquid biopsy in advanced cholangiocarcinoma.

455 Background: Cholangiocarcinoma (CCA) is an aggressive cancer with a poor prognosis. Given failure rates of up to 27% of CCA tissue biopsies, liquid biopsy has become an important tool to identify actionable molecular alterations. It is estimated tissue-based testing identifies IDH1 mutations and FGFR2 fusions in 12% and 9% of patients with advanced CCA (aCCA), respectively. Here we examined the rate of molecular alteration detection using circulating tumor DNA (ctDNA) testing, treatment patterns in aCCA, and outcomes for patients receiving ivosidenib following ctDNA-detected IDH1 mutations. Methods: Real-world data was sourced from GuardantINFORM, which comprises aggregated commercial payer health claims and de-identified records from &gt;330,000 patients with clinical ctDNA testing via Guardant360 (G360), from 2014 to 2023. Patients with aCCA and &gt;1 treatment claim after G360 results were included (N=1726). Patient outcomes were assessed via real-world time to treatment discontinuation (rwTTD), real-world time to next treatment (rwTTNT), and real-world overall survival (rwOS), all assessed in months. Log rank test was used to compare Kaplan-Meier survival curves. Results: Of 1495 patients with &gt;1 ctDNA alteration detected, a guideline-recommended biomarker was identified in 18% of patients. In patients with intrahepatic CCA (n=403), 11% had an IDH1 mutation, 9% an FGFR2 fusion, 1% MSI-H detected, 1% BRAF V600E mutation, &lt;1% ERBB2 amplification and &lt;1% RET fusion. Testing was performed prior to first-line therapy, after first-line therapy and after second-line therapy in 34%, 46% and 21% of patients respectively. Gemcitabine and cisplatin represented the most common first-line therapy (48%). FOLFOX (21%), gemcitabine and cisplatin (13%), and capecitabine (11%) were the most common second-line therapies. Among ivosidenib-naïve patients with an IDH1 mutation, 58 (20%) started therapy within 90 days of ctDNA test: 36% received ivosidenib, 57% received chemotherapy, 7% received other therapy. Patients with IDH1 mutations treated with ivosidenib (n=21) had numerically improved rwTTD and rwTTNT compared to those receiving chemotherapy (n=33) [rwTTD: 4.6 (95% CI:2.6-8.6) vs 2.8 (95% CI:2.0-6.3), p=0.1011; rwTTNT 11.0 (95% CI:5.7-NE) vs 5.2 (95% CI:3.3-NE); p=0.2559]. There was no difference in rwOS between patients receiving ivosidenib vs not. However, sample size was small. Conclusions: The rate of actionable molecular alterations detected via ctDNA is 18%, comparable to reports from tissue-based testing. Patients with aCCA are often tested before starting first or second LOT. Patients with ctDNA-detected IDH1 mutations treated with ivosidenib and had a trend toward improved rwTTD and rwTTNT, although it did not reach statistical significance. This data supports the clinical utility of liquid biopsy to identify aCCA patients who may benefit from targeted therapy.

Clinical utility of upfront circulating tumor DNA (ctDNA) genotyping to guide first-line therapy in patients (pts) with metastatic colorectal cancer (mCRC): A GOZILA substudy.

157 Background: The clinical utility of upfront ctDNA genomic profiling is not established in treatment (tx)-naïve pts with mCRC. Methods: GOZILA is a nationwide plasma genomic profiling study using Guardant360 CDx for advanced solid tumors. We assessed ctDNA molecular profile and its association with treatment efficacy in pts with mCRC who were enrolled in GOZILA from January 2018 to July 2022 and underwent ctDNA genotyping before initiation of first-line systemic tx. ctDNA fraction was defined as the maximum variant allelic frequency of somatic alteration and pts were classified into three groups based on ctDNA fraction: low (&lt;1%), middle (≥1% and &lt;10%) and high (≥10%) ctDNA fraction groups. Results: This study included 418 pts with mCRC receiving upfront ctDNA genotyping. Median turnaround time of ctDNA test was 7 days, comparable with standard-of-care (SOC) tissue RAS/BRAF testing (7 days) and microsatellite instability (MSI) testing (8 days). Pathogenic alterations were detected in 384 pts (92%), and concordance rates for RAS and BRAF mutation (mut) compared to SOC tissue-based testing were 91% and 97%, respectively. Based on biomarkers identified by ctDNA genotyping, 142 (40%) pts were treated with matched targeted tx: anti-EGFR antibody for RAS/BRAF wild-type (n=137), BRAF inhibitor for BRAF V600E mutation (n=4), and anti-PD-1 antibody for MSI high (n=1). Low, middle, and high ctDNA fraction groups included 87, 97 and 234 pts, respectively, and had significantly different median progression-free survival (PFS) (p&lt;0.01, table) and overall survival (OS) (p&lt;0.01, table), respectively. In 13 pts who repeated ctDNA test before second-line tx, those with elevated ctDNA fraction compared to upfront ctDNA had significantly shorter PFS (median, 3.8 vs. 8.1 months; HR, 5.7, 95% CI, 0.9 to 35.1) and OS (median, 8.2 vs. 17.4 months; HR, 11.3, 95% CI, 1.2 to 105.9) of second-line tx that those with not elevated ctDNA fraction. Conclusions: Upfront ctDNA genotyping successfully identified actionable genomic alterations in pts with mCRC. ctDNA fraction in upfront ctDNA was significantly associated with efficacy of first-line tx and ctDNA fraction monitoring may be useful in determination of subsequent tx. [Table: see text]

A review of comprehensive genomic profiling testing in a Japanese single institution.

740 Background: In Japan, comprehensive genomic profiling (CGP) testing, including OncoGuide and FoundationOne CDx (F-1), were approved in June 2019 for patients with advanced solid tumors. Here, we report the landscape of CGP testing in our hospital. Methods: This study was conducted with a retrospective design to evaluate the clinico-genomic characteristics of patients who underwent CGP testing. OncoGuide is a panel using matched tumor tissue and blood sample, containing 124 genes, developed by the National Cancer Center. F1 is a tumor-only panel, containing 324 genes, developed by Foundation Medicine Industry. Results: Of the 460 patients enrolled in this study, 376 and 84 underwent F-1 and OncoGuide, respectively. The most common primary tumor was colorectal cancer (93 pts), followed by malignant melanoma (33 pts) and pancreatic cancer (33 pts). Actionable gene alteration was detected in 304 patients (66.1%). The detected actionable genes were TP53 (55.9%), APC (27.8%), KRAS (22.6%), and BRAF (8.5%) in colorectal cancer, BRAF (10.1%), CDKN2A (30.2%), CDKN2B (9.4%), and TERT (33.3%) in malignant melanoma, and KRAS (18.8%), TP53 (54.3%), and CDKN2A (21.8%) in pancreatic cancer. The mean time from sample shipment to result receipt was 42.5 days. As a result of CGP testing, candidate anticancer drugs were found in 304 patients: however, 108 patients were not treated due to poor general condition, complications, or age, which limited access to clinical trials. In germline findings, a pathogenic germline variant of BRCA2 was detected in one patient in OncoGuide, and presumed germline pathogenic variants of MLH1, BRCA2, and PTEN were detected in 1, 13, 26 patients in F-1. Conclusions: In Japan, CGP testing found candidate anticancer drugs, however it requires a significant amount of time to receive treatment because of limiting access to ongoing clinical trials and being typically performed after standard chemotherapy. Therefore, improving access to clinical trials, including determining the timing of testing and selecting appropriate cases, was critical.

Identification of MAPK and mTOR pathway alterations in HER2-amplified colorectal cancer.

185 Background: Human epidermal growth factor receptor 2 (HER2) amplification is now a highly actionable alteration for patients with colorectal cancer (CRC) with distinct combination therapies. Recent studies have shown that oncogenic KRAS, BRAF, and PIK3CA mutations, may serve as biomarkers of response to HER-directed treatments such as Trastuzumab and Tucatinib/Pertuzumab. In this study, we investigated the incidence of MAP kinase and mTOR pathway alternations in patients with HER2-amplified CRC. Methods: The cBioPortal for Cancer Genomics was utilized to obtain data and graphics on HER2 amplification in CRC. cBioPortal was queried to select 21 published studies that contain CRC specimens (assessed April 2023). Clinical, specimen, copy number alteration (CNA), and somatic mutation data of each study were downloaded and aggregated across all studies, followed by filtering, standardization, and harmonization to generate ~30 analysis-ready fields encompassing demographic variables, HER2 amplification, and KRAS/NRAS/PIK3CA/BRAF mutations. Results: Among a pool of 4822 CRC patients with known gender, 44.4% (2139) were female and 55.6% (2683) were male. The incidence of HER2 amplification in the overall cohort was 2.6% (123/4822) and was noted to be higher in males (3.1%, 82/2683) than in females (1.9%, 41/2139). The incidence of HER2 amplification was highest in Asian CRC patients (3.9%, 9/228), followed by Black CRC patients (3.0%, 9/298), and then Caucasian CRC patients (2.7%, 57/2118). Rates of HER2 amplification were similar in the colon and the rectum at 2.6% (61/2360 and 26/1015, respectively). After analyzing rates of concurrent MAPK and mTOR pathway mutations in patients with HER2-amplified CRC, KRAS mutations were noted to be the most common mutation in HER2-amplified colon cancer (27.9%, 17/61), but were less common in HER2-amplified rectal cancer (7.7%, 2/26). PIK3CA mutations were also common, with an incidence of 13.1% (8/61) in HER2-amplified colon cancer and 7.7% (2/26) in HER2-amplified rectal cancer. NRAS mutations occurred at comparable, low rates in HER2-amplified colon and rectal cancer, at 3.3% (2/61) and 3.8% (1/26) respectively. No BRAF mutations were reported in either disease group. Conclusions: In this study, we identified that MAPK and mTOR pathway alterations are relatively common among patients with HER2-amplified CRC. These somatic events appear to be more common in HER2-amplified colon cancer than rectal cancer. Next generation based molecular profiling is recommended to identify potential resistance mechanisms before initiation of HER2-directed therapy.

Abstract A101: Codon-specific KRAS mutations predict survival in advanced pancreatic cancer

Abstract Background: How distinct KRAS alterations in pancreatic adenocarcinoma (PDAC) influence tumor initiation and outcomes remains unclear. Moreover, KRAS is now partly targetable with novel specific inhibitors targeting KRASG12 (G12C or G12D) but specific outcomes of these subgroups of patients is poorly described. We analyzed clinical/genomic characteristics, and outcomes of PDAC depending on codon-specific KRAS (G12 vs others), as well as gene expression profiles and in vitro drug sensibility using organoids (PDO). Methods: All metastatic patients with PDAC and available molecular profile from 2015-2022 were eligible and patients with KRAS mutation were included (NCT0493252) in a monocentric retrospective cohort. A cohort of PDO was used to assess the efficacy of 25 antitumor therapies (chemogram) and transcriptomic data from 69 KRAS-mutated tumors was also analyzed. Results: Overall, 263 patients were included, 238 KRASG12 (91%) and 24 (9%) KRASother. There was no difference between KRASG12 and KRASother regarding clinicopathological characteristics and potentially actionable alterations, except for BRAF that was found in 13% of KRASother (p=0.01). GNAS was found more altered in KRASother tumors (p=0.002) suggesting potential TIPMP precursors. Median overall survival from metastatic diagnosis was 16.7 months (95%CI:14.3-18.3) in KRASG12 and 24.9 months (95% CI:17.4-43.4) in KRASother (HR (ref:KRASG12)=0.56 (0.34-0.94), p=0.03 adjusted). First-line treatment-response was not different between groups (ORR and PFS), as confirmed with a similar PDO in vitro sensibility. Transcriptomic analyses showed a significant and exclusive upregulation of immune pathways in KRASG12 tumors. Conclusion: Codon-specific KRAS mutations are not equal and we report for the first time that KRASG12 patients have a worst prognosis than KRASother patients in PDAC. KRASG12 appears as a prognostic factor, as confirmed by transcriptomic analyses. These results warrant to be confirmed in larger scale studies. Citation Format: Alice Boilève, Adrien Rousseau, Marc Hilmi, Anthony Tarabay, Jacques R.R. Mathieu, Negaar Goudarzi, Jérôme Cartry, Sabrina Bedja, Claudio Nicotra, Maud NgoCamus, Valérie Boige, Marine Valéry, Mohamed Amine Bani, Lambros Tselikas, Antoine Italiano, Fanny Jaulin, Cristina Smolenschi, Antoine Hollebecque, Michel Ducreux. Codon-specific KRAS mutations predict survival in advanced pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A101.

Clinical practice recommendations for the use of next-generation sequencing in patients with solid cancer: a joint report from KSMO and KSP.

In recent years, next-generation sequencing (NGS)-based genetic testing has become crucial in cancer care. While its primary objective is to identify actionable genetic alterations to guide treatment decisions, its scope has broadened to encompass aiding in pathological diagnosis and exploring resistance mechanisms. With the ongoing expansion in NGS application and reliance, a compelling necessity arises for expert consensus on its application in solid cancers. To address this demand, the forthcoming recommendations not only provide pragmatic guidance for the clinical use of NGS but also systematically classify actionable genes based on specific cancer types. Additionally, these recommendations will incorporate expert perspectives on crucial biomarkers, ensuring informed decisions regarding circulating tumor DNA panel testing.

STAR-121: A Phase III Randomized Study of Domvanalimab and Zimberelimab in Combination With Chemotherapy Versus Pembrolizumab With Chemotherapy in Untreated Metastatic Non–Small Cell Lung Cancer With No Actionable Gene Alterations

IntroductionDual inhibition with a T-cell immunoreceptor with immunoglobulin and ITIM domains plus programmed death (ligand)-1 (PD[L]-1) inhibitors, with or without chemotherapy, is an emerging therapeutic strategy in metastatic non–small cell lung cancer (mNSCLC). The STAR-121 (NCT05502237) phase III, global, randomized, open-label study will investigate first-line domvanalimab (anti-TIGIT) and zimberelimab (anti–PD-1) plus chemotherapy versus pembrolizumab plus chemotherapy in mNSCLC with no actionable gene alterations. Participants and MethodsApproximately 720 participants (≥18 years old) with untreated mNSCLC and no EGFR and ALK mutations will be randomized into 3 groups (A, B, or C) in a 4:4:1 ratio and stratified by baseline PD-L1 expression (tumor cells <50% vs. ≥50%), histology (squamous vs. nonsquamous), and geographic region (East Asia vs. non-East Asia). Group A will receive domvanalimab 1200 mg plus zimberelimab 360 mg plus platinum-doublet chemotherapy (PT), group B will receive pembrolizumab 200 mg plus PT, and group C will receive zimberelimab 360 mg plus PT, every 3 weeks. Treatment will be administered until disease progression or intolerable toxicity. Dual primary endpoints are progression-free survival (by blinded independent central review [BICR]) and overall survival for group A versus B. Key secondary endpoints comprise overall response rate (by BICR), safety, and quality of life. Exploratory endpoints include efficacy and safety between groups A and C, pharmacokinetics, patient-reported outcomes, and biomarkers. ConclusionEnrollment in the STAR-121 study commenced on October 12, 2022, and is currently ongoing with completion planned by September 2024. The study completion is expected by December 2027.

Chemotherapy-free treatment targeting fusions and driver mutations in KRAS wild-type pancreatic ductal adenocarcinoma, a case series.

KRAS wild-type (WT) pancreatic ductal adenocarcinoma (PDAC) represents a distinct entity with unique biology. The therapeutic impact of matched targeted therapy in these patients in a real-world setting, to date, is less established. The aim of our study was to review our institutional database to identify the prevalence of actionable genomic alterations in patients with KRAS-WT tumors and to evaluate the therapeutic impact of matched targeted therapy in these patients. We reviewed electronic medical records of patients with KRAS-WT PDAC and advanced disease (n = 14) who underwent clinical-grade tissue ± liquid next-generation sequencing (315-648 genes for tissue) between years 2015 and 2021. Demographic and disease characteristics were summarized using descriptive parameters. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Of 236 PDAC patients, 14 had advanced/metastatic disease with KRAS-WT tumors. Median age at diagnosis was 66 years. There was a high frequency of potentially actionable genomic alterations, including three (21%) with BRAF alterations, two (14%) with fusions [RET-PCM1 and FGFR2-POC1B (N = 1 each)]; and one with a druggable EGFR (EGFR E746_A755delISERD) variant; two other patients had an STK11 and a MUTYH alteration. Five patients were treated with matched targeted therapy, with three having durable benefit: (i) erlotinib for EGFR-altered tumor, followed by osimertinib/capmatinib when MET amplification emerged (first-line therapy); (ii) pralsetinib for RET fusion (fifth line); and (iii) dabrafenib/trametinib for BRAF N486_P490del (third line). Duration of time on chemotherapy-free matched targeted therapy for these patients was 17+, 11, and 18+ months, respectively. Sustained therapeutic benefit can be achieved in a real-world setting in a subset of patients with advanced/metastatic KRAS-WT PDAC treated with chemotherapy-free matched targeted agents. Prospective studies are warranted.

Unique genomic alterations in the circulating tumor DNA of patients with solid tumors brain metastases.

Although serum circulating tumor DNA (ctDNA) is routine, data from patients with brain metastases (BrMs) is limited. We assessed genomic alterations in ctDNA from patients with solid tumor BrMs in 3 groups: Isolated BrMs with stable extracranial disease (iCNS), concurrent brain and extracranial progression (cCNS), and extracranial progression with no active BrMs (eCNS). We also compared ctDNA alterations between patients with and without BrMs. Patients with a Guardant360 ctDNA profile with (n = 253) and without BrMs (n = 449) from the Duke Molecular Registry between January 2014 and December 2020 were identified. Actionable alterations were defined as FDA-recognized or standard-of-care biomarkers. Disease status was determined via investigator assessment within 30 days of ctDNA collection. Among the 253 patients with BrMs: 29 (12%) had iCNS, 160 (63%) cCNS, and 64 (25%) eCNS. Breast (BC; 12.0%) and non-small cell lung cancer (NSCLC; 76.4%) were the most common tumor types. ESR1 (60% vs 25%, P < .001) and BRCA2 (17% vs 5%, P = .022) were more frequent in BC BrMs. In NSCLC BrMs, EGFR alterations were most frequent in the iCNS group (iCNS: 67%, cCNS: 40%, eCNS:37%, P = .08) and in patients with BrMs (36% vs 17%, P < .001). Sequencing from both brain tissue and ctDNA were available for 8 patients; 7 (87.5%) had identical alterations. This study illustrates the feasibility of detecting alterations from ctDNA among patients with BrMs. A higher frequency of actionable mutations was observed in ctDNA in patients with BrMs. Additional studies comparing ctDNA and alterations in BrMs tissue are needed to determine if ctDNA can be considered a surrogate to support treatment decisions.

Clinical utility of comprehensive genomic profiling for skin cancer: A cohort study of 522 cases in a Japanese real-world setting

BackgroundAdvanced cases of skin cancer sometimes represent a clinical challenge to treat due to limited approved treatments. Comprehensive genomic profiling (CGP) tests are indicated for metastatic or unresectable cases; however, their results and clinical utility in skin cancer remain unclear. MethodsThis is a retrospective cohort study analyzing the CGP test results of 522 cases of skin cancer submitted between June 2019 and November 2022 using a nationwide database recording clinical data and CGP test results in Japan. Results522 cases were registered. In cancer types, the most prevalent was cutaneous melanoma (CM, n=235), followed by cutaneous squamous cell carcinoma (cSCC, n=91), Extramammary Paget’s disease (EMPD, n=75), adnexal carcinoma (AC, n=58), cutaneous angiosarcoma (cAS, n=33), basal cell carcinoma (BCC, n=15), Merkel cell carcinoma (n=10) and dermatofibrosarcoma protuberans (n=5). The median TMB was highest in EMPD and cAS (5.0 mutations/Mb), whereas high TMB cases (≥10.0 mutations/Mb) were most prevalent in BCC (33 %), followed by cSCC (26 %). No cases were classified as MSI-high. NTRK1/2/3 fusions were identified in two cases: one CM and one AC. BRAF V600E mutations in non-melanoma skin cancer were observed in one case of AC. Actionable genetic alterations eligible for ongoing clinical trials utilizing unapproved drugs were found most frequently in EMPD (51 %), followed by BRAF V600E/K-negative CM (44 %). ConclusionsThis study provides an overview of CGP test results and identifies potential therapeutic targets for each type of skin cancer. These findings improve our understanding of CGP test outcomes and advance tailored therapies for skin cancer.

Front-line liquid biopsy for early molecular assessment and treatment of hospitalized lung cancer patients

BackgroundMolecular characterization is pivotal for managing non-small cell lung cancer (NSCLC), although this process is often time-consuming and patients’ conditions might worsen while molecular analyses are processed.Our primary aim was to evaluate the performance of “up-front” next-generation sequencing (NGS) through liquid biopsy (LB) of hospitalized patients with newly detected lung neoplasm in parallel with conventional diagnosis. The secondary aim included longitudinal monitoring through LB of patients with oncogenic alterations at baseline. MethodsWe enrolled 47 consecutive patients immediately after hospitalization and radiological detection of symptomatic lung neoplasm. LB from peripheral blood was performed at baseline, in parallel with conventional biopsy (CB), when feasible. Additionally, LBs were repeated during treatment in patients with actionable gene alterations at baseline. Oncomine™ Lung cfTNA Research Assay panel was employed for processing plasma samples in NGS. Results47 hospitalized patients were enrolled. LB identified 28 patients with gene alterations, including mutations of EGFR (n = 7), KRAS (n = 12), ERBB2 (n = 1), TP53 (n = 2), BRAF (n = 1), one ALK rearrangement, and 4 patients with combined mutations involving EGFR, KRAS and PIK3CA.LB and CB were consistent, except for two patients. Three patients with positive LB for oncogenic drivers did not undergo CB due to contraindications.Median time to molecular results after LB was significantly lower compared to time to molecular report after CB (11 versus 22 days, p < 0.001). ConclusionsDespite limited numbers, our study supports the role of front-line LB for improving management of symptomatic patients with lung cancer, potentially leading to early targeted therapy initiation.