Actionable Genomic Alterations Research Articles

KRAS Allelic Variants in Biliary Tract Cancers

Biliary tract cancers (BTCs) contain several actionable molecular alterations, including FGFR2, IDH1, ERBB2 (formerly HER2), and KRAS. KRAS allelic variants are found in 20% to 30% of BTCs, and multiple KRAS inhibitors are currently under clinical investigation. To describe the genomic landscape, co-sequence variations, immunophenotype, genomic ancestry, and survival outcomes of KRAS-mutated BTCs and to calculate the median overall survival (mOS) for the most common allelic variants. This retrospective, multicenter, pooled cohort study obtained clinical and next-generation sequencing data from multiple databases between January 1, 2017, and December 31, 2022. These databases included Princess Margaret Cancer Centre, MD Anderson Cancer Center, Foundation Medicine, American Association for Cancer Research Project GENIE, and cBioPortal for Cancer Genomics. The cohort comprised patients with BTCs who underwent genomic testing. The main outcome was mOS, defined as date of diagnosis to date of death, which was measured in months. A total of 7457 patients (n = 3773 males [50.6%]; mean [SD] age, 63 [5] years) with BTCs and genomic testing were included. Of these patients, 5813 had clinical outcome data available, in whom 1000 KRAS-mutated BTCs were identified. KRAS allelic variants were highly prevalent in perihilar cholangiocarcinoma (28.6%) and extrahepatic cholangiocarcinoma (36.1%). Thirty-six KRAS allelic variants were identified, and the prevalence rates in descending order were G12D (41%), G12V (23%), and Q61H (8%). The variant G12D had the highest mOS of 25.1 (95% CI, 22.0-33.0) months compared with 22.8 (95% CI, 19.6-31.4) months for Q61H and 17.8 (95% CI, 16.3-23.1) months for G12V variants. The majority of KRAS-mutated BTCs (98.9%) were not microsatellite instability-high and had low tumor mutational burden (ranging from a median [IQR] of 1.2 (1.2-2.5) to a mean [SD] of 3.3 [1.3]). Immune profiling through RNA sequencing of KRAS and NRAS-mutated samples showed a pattern toward a more immune-inflamed microenvironment with higher M1 macrophage activation (0.16 vs 0.12; P = .047) and interferon-γ expression compared with wild-type tumors. The G12D variant remained the most common KRAS allelic variant in all patient ancestries. Patients with admixed American ancestry had the highest proportion of G12D variant (45.0%). This cohort study found that KRAS allelic variants were relatively common and may be potentially actionable genomic alterations in patients with BTCs, especially perihilar cholangiocarcinoma and extrahepatic cholangiocarcinoma. The findings add to the growing data on genomic and immune landscapes of KRAS allelic variants in BTCs and are potentially of value to the planning of specific therapies for this heterogeneous patient group.

Gastric cancer actionable genomic alterations across diverse populations worldwide and pharmacogenomics strategies based on precision oncology

Introduction: Gastric cancer is one of the most prevalent types of cancer worldwide. The World Health Organization (WHO), the International Agency for Research on Cancer (IARC), and the Global Cancer Statistics (GLOBOCAN) reported an age standardized global incidence rate of 9.2 per 100,000 individuals for gastric cancer in 2022, with a mortality rate of 6.1. Despite considerable progress in precision oncology through the efforts of international consortia, understanding the genomic features and their influence on the effectiveness of anti-cancer treatments across diverse ethnic groups remains essential.Methods: Our study aimed to address this need by conducting integrated in silico analyses to identify actionable genomic alterations in gastric cancer driver genes, assess their impact using deleteriousness scores, and determine allele frequencies across nine global populations: European Finnish, European non-Finnish, Latino, East Asian, South Asian, African, Middle Eastern, Ashkenazi Jewish, and Amish. Furthermore, our goal was to prioritize targeted therapeutic strategies based on pharmacogenomics clinical guidelines, in silico drug prescriptions, and clinical trial data.Results: Our comprehensive analysis examined 275,634 variants within 60 gastric cancer driver genes from 730,947 exome sequences and 76,215 whole-genome sequences from unrelated individuals, identifying 13,542 annotated and predicted oncogenic variants. We prioritized the most prevalent and deleterious oncogenic variants for subsequent pharmacogenomics testing. Additionally, we discovered actionable genomic alterations in the ARID1A, ATM, BCOR, ERBB2, ERBB3, CDKN2A, KIT, PIK3CA, PTEN, NTRK3, TP53, and CDKN2A genes that could enhance the efficacy of anti-cancer therapies, as suggested by in silico drug prescription analyses, reviews of current pharmacogenomics clinical guidelines, and evaluations of phase III and IV clinical trials targeting gastric cancer driver proteins.Discussion: These findings underline the urgency of consolidating efforts to devise effective prevention measures, invest in genomic profiling for underrepresented populations, and ensure the inclusion of ethnic minorities in future clinical trials and cancer research in developed countries.

Abstract PO4-24-08: Genomic Landscape of Malignant Phyllodes Tumors Identifies Subsets for Targeted Therapy

Abstract Introduction: Phyllodes tumors (PT) are rare fibroepithelial tumors of the breast, comprising ~1% of all breast cancers, a subset of which have considerable malignant potential. Malignant phyllodes tumors (MPTs) have aggressive biological behavior and high local and distant recurrence rates. Distant metastases of MPTs have a poor prognosis given the limited treatment options available. Surgery remains the primary treatment modality for these patients; however, initial investigations suggest a potential for targeted therapies in managing this disease. The identification of targetable opportunities and a comprehensive understanding of the mutational profiles of MPTs could greatly enhance treatment options. Therefore, we aimed to assess the molecular landscape of these tumors. Methods: Phyllodes tumor samples underwent molecular profiling at Caris Life Sciences through DNA (592-gene panel or whole exome) and RNA sequencing (whole transcriptome). The MPTs were classified into primary and metastatic based on the specimen biopsy site. PD-L1+ expression was tested by IHC (SP142; ≥2+, ≥5%). Tumor mutational burden (TMB)-High was defined as ≥10 mutation/Mb. Immune cell fractions in the tumor microenvironment (TME) were estimated using quanTIseq (Finotello, 2019). Mann-Whitney U, Chi-square, and Fisher-Exact tests were applied where appropriate. Results: We identified 57 analyzed MPTs, all from female patients, with a median age of 53 years (range: 19-88). Approximately half of samples (53%, n=30) were labeled as from primary breast sites, and the remaining sites were metastatic (47%, n= 27). Of metastatic sites, lung was the most common location (74.1%, n=20/27), followed by bone, sacrum, pancreas and small bowel. Alterations in MPT include TERT promoter (56.8%, n=21/37), MED12 (51.6%, n=16/31), TP53 (40.0%, n=22/55), and NF1 (31.8%, n=14/44) mutations, with less frequent mutations of EGFR (10.5%, n=6/57), PIK3CA (7.0%, n=4/57), and BRAF (3.5%, n=2/57). As compared to primary MPT, metastatic MPT had roughly 2-fold higher prevalence of NF1 (45.5%: n=10/22 vs 18.2% n=4/22, p=0.05), KMT2D (25.9%: n=7/27 vs 10.7%: n=3/28, p=0.18), and RB1 (25.0%: n=6/24 vs 9.5%: n=2/21, p=0.25) though not significant. Multiple gene mutations were observed exclusively in lung metastases, as compared to non-lung sites including: TERT promoter (64.3%: n=9/14 vs 0.0%, p=0.08), MED12 (60.0%: n=6/10 vs 0.0%, p =0.044), and RB1 (35.3%: n=6/17 vs 0.0%, p=0.01). Conversely, NF1 (43.8%: n=7/16 vs 50.0%: n=3/6, p=1.0) and KMT2D (20.0%: n=4/20 vs 42.9%: n=3/7, p=0.32) mutation prevalence was higher in other metastatic sites. PD-L1+ expression was observed in 13.3% (n=7) of MPT overall, with similar prevalence among local and metastatic sites. No dMMR/MSI-H was observed, while low LOH (those with genomic LOH in < 16% of segment analyzed) was seen across MPT. Compared to a large cohort of breast adenocarcinoma samples (n=9,926) representing both HER2+ and HER2- tumors, MPT had low ERBB2 expression comparable to HER2- samples. B cells, M2 macrophages and neutrophils had the highest median cell fractions in the TME of MPT. Additionally, one MPT sample harbored a pathogenic NTRK1 fusion (TPM4:NTRK1), and treatment with larotrectinib for over 16 months suggests a clinical response to therapy. Conclusions: Our study demonstrates the importance of employing next generation sequencing (NGS) in MPTs to detect actionable genomic alterations. To effectively analyze fusions, an NGS panel that include RNA sequencing is recommended, considering the occurrence of NTRK1 fusion reported herein. Although HER2 transcriptional expression was low, further investigations examining HER2 IHC in PTs are still necessary. These finding therefore highlight the importance of NGS in phyllodes tumors research, as it may uncover potential targeted treatment options for patients. Citation Format: Rani Bansal, Tolulope Adeyelu, Andrew Elliott, Antoinette Tan, Jane Meisel, Matthew Oberley, Stephanie Graff, Jorge Reis-Filho, George Sledge Jr, Sarah Sammons, Laura Rosenberger. Genomic Landscape of Malignant Phyllodes Tumors Identifies Subsets for Targeted Therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-08.

Mutational landscape of inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is the most pro-metastatic form of BC. Better understanding of its enigmatic pathophysiology is crucial. We report here the largest whole-exome sequencing (WES) study of clinical IBC samples.MethodsWe retrospectively applied WES to 54 untreated IBC primary tumor samples and matched normal DNA. The comparator samples were 102 stage-matched non-IBC samples from TCGA. We compared the somatic mutational profiles, spectra and signatures, copy number alterations (CNAs), HRD and heterogeneity scores, and frequencies of actionable genomic alterations (AGAs) between IBCs and non-IBCs. The comparisons were adjusted for the molecular subtypes.ResultsThe number of somatic mutations, TMB, and mutational spectra were not different between IBCs and non-IBCs, and no gene was differentially mutated or showed differential frequency of CNAs. Among the COSMIC signatures, only the age-related signature was more frequent in non-IBCs than in IBCs. We also identified in IBCs two new mutational signatures not associated with any environmental exposure, one of them having been previously related to HIF pathway activation. Overall, the HRD score was not different between both groups, but was higher in TN IBCs than TN non-IBCs. IBCs were less frequently classified as heterogeneous according to heterogeneity H-index than non-IBCs (21% vs 33%), and clonal mutations were more frequent and subclonal mutations less frequent in IBCs. More than 50% of patients with IBC harbored at least one high-level of evidence (LOE) AGA (OncoKB LOE 1–2, ESCAT LOE I–II), similarly to patients with non-IBC.ConclusionsWe provide the largest mutational landscape of IBC. Only a few subtle differences were identified with non-IBCs. The most clinically relevant one was the higher HRD score in TN IBCs than in TN non-IBCs, whereas the most intriguing one was the smaller intratumor heterogeneity of IBCs.

Targeting KRASG12C in Non-Small-Cell Lung Cancer: Current Standards and Developments.

Among the most common molecular alterations detected in non-small-cell lung cancer (NSCLC) are mutations in Kristen Rat Sarcoma viral oncogene homolog (KRAS). KRAS mutant NSCLC is a heterogenous group of diseases, different from other oncogene-driven tumors in terms of biology and response to therapies. Despite efforts to develop drugs aimed at inhibiting KRAS or its signaling pathways, KRAS had remained undruggable for decades. The discovery of a small pocket in the binding switch II region of KRASG12C has revolutionized the treatment of KRASG12C-mutated NSCLC patients. Sotorasib and adagrasib, direct KRASG12C inhibitors, have been approved by the US Food and Drug Administration (FDA) and other regulatory agencies for patients with previously treated KRASG12C-mutated NSCLC, and these advances have become practice changing. However, first-line treatment in KRASG12C-mutated NSCLC does not differ from NSCLC without actionable driver genomic alterations. Treatment with KRASG12C inhibitors is not curative and patients develop progressive disease, so understanding associated mechanisms of drug resistance is key. New KRASG12C inhibitors and several combination therapy strategies, including with immune checkpoint inhibitors, are being studied in clinical trials. The aim of this review is to explore the clinical impact of KRAS, and outline different treatment approaches, focusing on the novel treatment of KRASG12C-mutated NSCLC.

Clinical and Diagnostic Utility of Genomic Profiling for Digestive Cancers: Real-World Evidence from Japan.

The usefulness of comprehensive genomic profiling (CGP) in the Japanese healthcare insurance system remains underexplored. Therefore, this large-scale study aimed to determine the usefulness of CGP in diagnosing digestive cancers. Patients with various cancer types recruited between March 2020 and October 2022 underwent the FoundationOne® CDx assay at the Keio PleSSision Group (19 hospitals in Japan). A scoring system was developed to identify potentially actionable genomic alterations of biological significance and actionable genomic alterations. The detection rates for potentially actionable genomic alterations, actionable genomic alterations, and alterations equivalent to companion diagnosis (CDx), as well as the signaling pathways associated with these alterations in each digestive cancer, were analyzed. Among the 1587 patients, 547 had digestive cancer. The detection rates of potentially actionable genomic alterations, actionable genomic alterations, and alterations equivalent to CDx were 99.5%, 62.5%, and 11.5%, respectively. APC, KRAS, and CDKN2A alterations were frequently observed in colorectal, pancreatic, and biliary cancers, respectively. Most digestive cancers, except esophageal cancer, were adenocarcinomas. Thus, the classification flowchart for digestive adenocarcinomas proposed in this study may facilitate precise diagnosis. CGP has clinical and diagnostic utility in digestive cancers.

Optimizing the Use of Next-Generation Sequencing Assays in Patients With Urothelial Carcinoma: Recommendations by the 2023 San Raffaele Retreat Panel

BackgroundThe application of precision medicine in clinical practice implies a thorough evaluation of actionable genomic alterations to streamline therapeutic decision making. Comprehensive genomic profiling of tumor via next-generation sequencing (NGS) represents a great opportunity but also several challenges. During the 2023 San Raffaele Retreat, we aimed to provide expert recommendations for the optimal use of NGS in urothelial carcinoma (UC). Materials and MethodsA modified Delphi method was utilized, involving a panel of 12 experts in UC from European and United States centers, including oncologists, urologists, pathologists, and translational scientists. An initial survey, conducted before the meeting, delivered 15 statements to the panel. A consensus was defined when ≥70% agreement was reached for each statement. Statements not meeting the consensus threshold were discussed during the meeting. ResultsNine of the 15 statements covering patient selection, cancer characteristics, and type of NGS assay, achieved a consensus during the survey. The remaining six statements addressing the optimal timing of NGS use, the ideal source of tumor biospecimen for NGS testing, and the subsequent need to evaluate the germline nature of certain genomic findings were discussed during the meeting, leading to unanimous agreement at the end of the conference. ConclusionThis consensus-building effort addressed multiple unanswered questions regarding the use of NGS in UC. The opinion of experts was in favor of broader use of NGS. In a setting where recommendations/guidelines may be limited, these insights may aid clinicians to provide informed counselling and raise the bar of precision and personalized therapy.

Abstract LB099: Identifying actionable genomic alterations in non-small cell lung cancer through the analysis of extracellular vesicles in plasma

Abstract Lung cancer is the second most common cancer worldwide, accounting for 2.2 million new cases and 1.8 million deaths in 2020. Personalized targeted therapy based on actionable molecular markers has completely transformed the therapeutic landscape in NSCLC, which has improved considerably the overall survival. However, ~50% of NSCLC patients with tissue biopsy inaccessible or inadequate, limiting biomarker testing and access to targeted therapies. The availability of a wide range of highly effective targeted therapy options for NSCLC justifies the significance of developing non-invasive and cost-effective detection assays for guiding targeted therapy selection in NSCLC.Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles of sub-micrometer size that are secreted by virtually all cell types. They contain hundreds of different proteins, thousands of intact RNA species, and double-stranded DNA (dsDNA) fragments that sample the entire human genome. Recent findings further corroborate the potential role of EVs in early cancer screening, cancer diagnosis, treatment selection and monitoring. In this study, we used lipid nanoprobe (LNP) for EV isolation from plasma and droplet digital PCR assay for detecting actionable genomic alternation to aid targeted therapy selection in NSCLC. Specifically, we extracted EV associated DNA/RNA from plasma first, and then we used the individual ddPCR assays for detecting KRAS mutations, EGFR mutations, and ALK fusions from isolated EVs associated nucleic acids, respectively. We identified KRAS mutations G12C/G12S/G13C/G12A/Q61H, EGFR L858R mutation and exon 19 deletion, and ALK-EML4 gene fusion variant 3 from 12 NSCLC patients that matched the mutation status obtained from patient tissue specimens. Only in 1 out 13 patients, EV mutation status did not match the mutation status from the tissue specimen. This data indicated that clinical actionable gene mutations are present in EVs associated nucleic acids isolated from plasma samples of NSCLC patients. We anticipate that this novel non-invasive EVs-based actionable genomic alterations detection test would efficiently assist physicians/oncologists in selecting optimal targeted therapies for cancer treatment, as recommended by National Comprehensive Cancer Network (NCCN) guidelines for NSCLC. Citation Format: Hongzhang He, Jadranka Milosevic, Siyang Zheng. Identifying actionable genomic alterations in non-small cell lung cancer through the analysis of extracellular vesicles in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB099.

Identification of barriers to implementation of precision oncology in patients with rare cancers.

Established treatment options for rare cancers are limited by the small number of patients. The current comprehensive genomic profiling (CGP) testing might not fully exploit opportunities for precision oncology in patients with rare cancers. Therefore, we aimed to explore the factors associated with CGP testing utility in rare cancers and identify barriers to implementing precision oncology. Patients who underwent CGP testing at our institution between September 2019 and June 2021 were enrolled in this retrospective study. Based on their results, the patients received molecularly targeted drugs or immune checkpoint inhibitors. Univariate and multivariate analyses evaluated the association between patient characteristics and the proportion of patients receiving molecularly targeted drugs. Overall, 790 patients underwent CGP testing. Among them, 333 patients with rare cancers were identified, of whom 278 (83.5%) had actionable genomic alterations, 127 (38.1%) had druggable genomic alterations, and 25 (7.5%) received genomically matched therapy. The proportion of patients receiving molecularly targeted drugs was significantly higher among those with treatment options with evidence levels A-D (8.7%) than those without treatment options with evidence levels A-D (2.9%). A potential barrier to CGP testing utility in rare cancers is the limited number of molecularly targeted drugs with clinical evidence. We propose that CGP testing be performed in patients with rare cancerswho have treatment options with evidence levels A-D to maximize CGP testing utility in real-world practice.

Abstract 6580: A Novel EGFR x cMET bispecific ADC PRO1286 demonstrated broad antitumor activity and promising tolerability in preclinical models

Abstract EGFR and cMET are frequently co-expressed on tumor cells, often upregulated as the escape mechanism for each other, and both well-validated targets in oncology. Small molecule TKIs and amivantamab have been approved for non-small cell lung cancer with relevant actionable genomic alterations (AGA), and monoclonal antibodies offer treatment options for EGFR-expressing head and neck and colorectal cancers without AGA. Yet high unmet need in these tumors still exists. We sought to develop an EGFR and cMET dual-targeting ADC using our proprietary technology platform with carefully chosen design features for the many tumors expressing EGFR and cMET with or without AGA. PRO1286 is comprised of a monovalent bispecific human IgG1 (dubbed as “Bsab”) with fine-tuned affinity on EGFR and cMET, and the proprietary topoisomerase 1 inhibitor-based linker-drug, sesutecan. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). PRO1286 at DAR8 displays high hydrophilicity and excellent stability and developability in vitro. PRO1286 binds selectively and specifically to each target with a low nanomolar EC50 in the CHO-K1 cells overexpressing each target. PRO1286 exhibited efficient internalization (~50-80% internalized in 4 hrs) and strong cytotoxicity (sub-nanomolar IC50) against a broad panel of tumor cell lines with higher potency than the parent mAbs. PRO1286 produced marked and dose dependent anti-tumor activity in mouse xenograft models encompassing a wide range of target expression levels, tumor types (head and neck, lung, esophageal, gastric, colorectal), and genomic landscape (with or without AGA in EGFR or cMET; with diverse additional co-mutations). Sustained tumor growth inhibition or complete remission was observed in several xenograft models after treatment with a single dose (2 mg/kg) of PRO1286. PRO1286 was more efficacious than benchmarking molecules in the mouse studies. In an exploratory toxicity study in cynomolgus monkeys, PRO1286 was well tolerated at 30 mg/kg with the main toxicity being payload-driven and residing in bone marrow. PRO1286 exhibited a stable PK profile that was similar to that of the Bsab in rats and favorable PK characteristics in monkeys. In summary, PRO1286 demonstrated promising physiochemical properties, PK/PD, antitumor activity, and tolerability in preclinical studies. It may carry an expanded therapeutic index compared to other EGFR- and/or cMET-directed ADCs. It may also differentiate from existing EGFR- and/or cMET-targeting agents on broad coverage of EGFR and cMET-expressing tumors with or without the respective AGAs. Effort is under way to advance a DAR optimized molecule to the clinic for the treatment of many EGFR- or cMET-expressing solid tumors. Citation Format: Yang Xiao, Haiyu Huang, Jianhe Yin, Xuan Qiu, Lei Wang, Haidong Liu, Baiteng Zhao, Zhu Chen. A Novel EGFR x cMET bispecific ADC PRO1286 demonstrated broad antitumor activity and promising tolerability in preclinical models [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 6580.

Abstract 5710: The brain penetrant CDK4/6 inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of breast cancer, CRC and NSCLC

Abstract Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of cyclin-dependent kinase (CDK)s are often drivers of cancer pathogenesis. CDK4/6 are critical mediators of cellular transition into S phase and important for cancer initiation, growth, and survival. Previously we reported a novel brain penetrant CDK4/6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/6 and >2000-fold selectivity against other CDKs. PRT3645 inhibits cellular phosphorylation of Rb with nanomolar potency. Furthermore, PRT3645 exhibits favorable in-vivo safety pharmacology and ADME properties, including increased brain penetration, and demonstrates oral bioavailability across rodents, dog and non-human primates. Here, we explored the therapeutic potential of PRT3645 in tumor models harboring actionable genomic alterations but lacking effective targeted treatments. Specifically, we explored the combination of PRT3645 with a selective estrogen receptor degrader (SERD), an approved treatment for patients with HR+/HER2-, ESR1-mutated advanced or metastatic breast cancer. In an ESR1 mutated breast cancer PDX model, PRT3645 not only exhibited monotherapy activity but also induced significant tumor regression in combination with a SERD through inhibiting ER-mediated signaling. We also investigated PRT3645 single agent activity and combination with a clinically validated MEK inhibitor in a BRAF class III mutant colorectal CDX model. PRT3645 was well-tolerated and demonstrated significant anti-tumor efficacy, which was further enhanced by combination therapy. To further explore comprehensive inhibition of cell cycle progression and overcome potential resistance to CDK4/6 inhibition, we investigated dual inhibition of CDK4/6 and CDK2. CDK2 is a crucial player in regulating the cell cycle by controlling the late G1/S transition, promoting DNA replication, and contributing to the DNA repair processes. Additionally, CDK2 activation has been identified as a potential mechanism of resistance to CDK4/6 inhibition. We evaluated CDK2 and CDK4/6 dual inhibition in CDKN2A loss or CCNE amplified cancer cell lines and observed enhanced proliferation suppression, cell cycle arrest and senescence induction, underscoring the potential of this combination for improved therapeutic efficacy across various cancer types. In summary, PRT3645 displays an excellent balance of potency, selectivity, pharmacokinetic parameters across species as well as brain penetrance. In preclinical studies, PRT3645 was highly efficacious when combined with other targeted therapies, offering potential benefits across a wide spectrum of cancer types. In addition, the concept of dual inhibition of CDK4/6 and CDK2 presents a rational and promising approach to enhance the efficacy of cancer therapy. Citation Format: Yue Zou, Kirsten Gallagher, Srijita Dhar, Andrew Busking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Andrew Combs, Kris Vaddi, Sandy Geeganage, Peggy Scherle, Sang Hyun Lee. The brain penetrant CDK4/6 inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of breast cancer, CRC and NSCLC [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 5710.

Abstract 6457: The real-world data of comprehensive genomic profiling tests with tissue specimens from lung cancer patients in Japan

Abstract Introduction: Molecularly targeted therapy improves prognosis of lung cancer patients with specific genomic alterations, and thus it is important to identify actionable genomic alterations leading to appropriate therapy. In Japan, comprehensive genomic profiling (CGP) tests have been covered by insurance since June 2019. 99.7% of the CGP data under insurance coverage have been registered with the nationwide database: Center for Cancer Genomics and Advanced Therapeutics (C-CAT). C-CAT data include CGP test results and clinical information such as companion diagnostic results for EGFR, ALK, ROS1, and BRAF as well as therapeutic history. The aim of this study is to evaluate the usefulness of CGP tests for identifying actionable genomic alterations in real-world lung cancer patients. Methods: Among the 3,240 lung cancer patients registered in the C-CAT database by September 2023, 2,076 non-small cell lung cancer cases were tested using tissue specimens and analyzed retrospectively. The analysis was based on the levels of evidence defined by C-CAT. Level A indicated approved drugs for the particular cancer type, while level C indicated approved drugs or presence of positive clinical trials regardless of cancer type. Level F indicated that the genomic alteration was an oncogenic marker. Results: The cases were divided into two groups: 335 cases with known genomic alterations with positive results from companion diagnostic tests or use of specific inhibitors and 1,741 cases without. Genomic alterations with level A, A to C, and A to F were identified in 430 cases (24.7%), 1174 cases (67.4%), and 1718 cases (98.7%) with unknown genomic alterations, respectively. Genomic alterations with level A included rearrangement of RET (43 of 1741 cases, 2.5%), ALK (14 cases, 0.8%), ROS1 (8 cases, 0.5%), and NTRK1 (2 cases, 0.1%), and mutations of EGFR (124 cases, 7.1%), MET (38 cases, 2.2%), ERBB2 (122 cases, 7.0%), KRAS G12C (66 cases, 3.8%), and BRAF V600E (15 cases, 0.9%). Genomic alterations with level C included mutations in KRAS (185 cases, 10.6%), PIK3CA (99 cases, 5.7%), BRAF (55 cases, 3.2%), and PTEN (32 cases, 1.8%) and amplifications in ERBB2 (147 cases, 8.4%) and MET (68 cases, 3.9%). Among cases with known genomic alterations, ALK and ROS1 fusion genes were not detected in 11 of 49 (22.4%) and 17 of 46 (37.0%), while EGFR and BRAF mutation was not detected in 11 of 218 (5.0%) and 3 of 4 cases (75.0%), respectively. The detection rate of fusion genes was lower than that of mutated genes (70.5% vs 93.7%, p<0.001). Conclusion: In non-small cell lung cancer patients with unknown driver genes, driver oncogenes with corresponding approved therapy were found in a quarter of the patients. CGP tests offer the possibility of increasing the opportunities of receiving new therapies. CGP tests may have a lower detection rate for fusion genes compared with other mutations. Citation Format: Koki Fujii, Momoko Morishita, Michiko Ueki, Hiroaki Ikushima, Hideaki Isago, Kousuke Watanabe, Katsutoshi Oda, Hidenori Kage. The real-world data of comprehensive genomic profiling tests with tissue specimens from lung cancer patients in Japan [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 6457.

Clinical Management of Patients with Non-Small Cell Lung Cancer, Brain Metastases, and Actionable Genomic Alterations: A Systematic Literature Review.

Nearly 60% of patients with non-small cell lung cancer (NSCLC) present with metastatic disease, and approximately 20% have brain metastases (BrMs) at diagnosis. During the disease course, 25-50% of patients will develop BrMs. Despite available treatments, survival rates for patients with NSCLC and BrMs remain low, and their overall prognosis is poor. Even with newer agents for NSCLC, options for treating BrMs can be limited by their ineffective transport across the blood-brain barrier (BBB) and the unique brain tumor microenvironment. The presence of actionable genomic alterations (AGAs) is a key determinant of optimal treatment selection, which aims to maximize responses and minimize toxicities. The objective of this systematic literature review (SLR) was to understand the current landscape of the clinical management of patients with NSCLC and BrMs, particularly those with AGAs. A Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA)-compliant SLR was conducted to identify studies in patients with BrMs in NSCLC. Searches used the EMBASE and MEDLINE® databases, and articles published between January 1, 2017 and September 26, 2022 were reviewed. Overall, 179 studies were included in the SLR. This subset review focused on 80 studies that included patients with NSCLC, BrMs, and AGAs (19 randomized controlled trials [RCTs], two single-arm studies, and 59 observational studies). Sixty-four of the 80 studies reported on epidermal growth factor receptor (EGFR) mutations, 14 on anaplastic lymphoma kinase (ALK) alterations, and two on both alterations. Ninety-five percent of studies evaluated targeted therapy. All RCTs allowed patients with previously treated, asymptomatic, or neurologically stable BrMs; the percentage of asymptomatic BrMs varied across observational studies. Although targeted therapies demonstrate systemic benefits for patients with NSCLC, BrMs, and AGAs, there remains a continued need for effective therapies to treat and prevent BrMs in this population. Increased BBB permeability of emerging therapies may improve outcomes for this population.

The evolution of metastatic upper tract urothelial carcinoma through genomic-transcriptomic and single-cell protein markers analysis

The molecular characteristics of metastatic upper tract urothelial carcinoma (UTUC) are not well understood, and there is a lack of knowledge regarding the genomic and transcriptomic differences between primary and metastatic UTUC. To address these gaps, we integrate whole-exome sequencing, RNA sequencing, and Imaging Mass Cytometry using lanthanide metal-conjugated antibodies of 44 tumor samples from 28 patients with high-grade primary and metastatic UTUC. We perform a spatially-resolved single-cell analysis of cancer, immune, and stromal cells to understand the evolution of primary to metastatic UTUC. We discover that actionable genomic alterations are frequently discordant between primary and metastatic UTUC tumors in the same patient. In contrast, molecular subtype membership and immune depletion signature are stable across primary and matched metastatic UTUC. Molecular and immune subtypes are consistent between bulk RNA-sequencing and mass cytometry of protein markers from 340,798 single cells. Molecular subtypes at the single-cell level are highly conserved between primary and metastatic UTUC tumors within the same patient.

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.

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 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]

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.

Genomic Landscape of NSCLC in the Republic of Ireland

IntroductionThe identification of genomic “targets” through next-generation sequencing (NGS) of patient’s NSCLC tumors has resulted in a rapid expansion of targeted treatment options for selected patients. This retrospective study aims to identify the proportion of patients with advanced NSCLC in the Republic of Ireland whose tumors harbor actionable genomic alterations through broad NGS panel testing. MethodsInstitutional review board approval was obtained before study initiation. Patients with NSCLC whose tumors underwent genomic testing through the largest available NGS panel at a nationally funded Cancer Molecular Diagnostics laboratory (St. James’s Hospital) between June 2017 and June 2022 were identified. Patient demographics and tumor-related data were collected by retrospective review from all cancer centers in Ireland, referring to the Cancer Molecular Diagnostics laboratory. A total of 203 (9%) tumor samples were excluded due to insufficient neoplastic cell content. Genomic data were collected through retrospective search of Ion Reporter software. The spectrum and proportion of patients with oncogenic driver mutations were evaluated using descriptive statistics (SPSS version 29.0). ResultsIn total, 2052 patients were identified. Patients were referred from 23 different hospital sites and all four geographic regions (Leinster = 1091, 53%; Munster = 763, 37.2%; Connacht = 191, 9.3%; Ulster = 7, 0.3%). Median age was 69 (range: 26–94) years; 53% were male. The most common tumor histologic subtype was adenocarcinoma (77%, n = 1577). An actionable genomic alteration was identified in 1099 cases (53%), the most common of which was KRAS (n = 657, 32%). Less frequently, NSCLC tumors harbored the following: MET exon 14 skipping (n = 53, 2.6%), MET amplification (n = 26, 1.3%), EGFR (n = 181, 8.8%), HER2 (n = 35, 1.7%), and BRAF (n = 72, 3.5%) mutations. Fusions were detected in 76 patients (3.7%) including ALK (n = 44, 58%), RET (n = 11, 14.5%), ROS1 (n = 16, 21%), and FGFR3 (n = 5, 6.6%), whereas no NTRK fusion was identified. Co-alterations were detected in 114 patients (5.6%), the most common of which was KRAS/PIK3CA (n = 19, 17%), EGFR/PIK3CA (n = 10, 8.5%), and KRAS/IDH1 (n = 9, 8%). Other co-alterations of interest identified included KRAS G12A/ROS1 fusion (n = 1) and KRAS G12C/BRAF G469A (n = 2). ConclusionsThis is the first retrospective study to comprehensively characterize the genomic landscape of NSCLC in Ireland, using the broadest available NGS panel. Actionable alterations were identified in 53.4% of the patients, and KRAS was the most common oncogenic driver alteration. Our study revealed a lower prevalence of patients whose tumor harbors ALK, ROS1, and RET fusions, compared with similar data sets.

The landscape of actionable genomic alterations in lung adenocarcinomas in India.

Lung adenocarcinoma (LUAD), the most prevalent form of non-small cell lung cancer (NSCLC), remains a leading cause of cancer-related death globally, including in India, with a 5-year survival rate below 10%. Despite these grim statistics, recent advances in the use of next-generation sequencing (NGS) for identifying genetic alterations and the emergence of targeted therapies have opened new possibilities for personalized treatment based on distinct molecular signatures. To understand the molecular pattern of NSCLC, a retrospective study was conducted with 53 Indian LUAD patient samples, using a targeted NGS panel of 46 cancer-relevant oncogenes to identify clinically relevant variants. Pathogenic or likely pathogenic variants were detected in 94% of the 53 cases. Non-synonymous mutations, rearrangements, copy number alterations, insertions, and deletions of functional relevance were observed in 31 out of 46 genes. The most frequently mutated genes included TP53 (52.8%) and EGFR (50.9%), followed by RET, PIK3CA and ERBB2; some patients had multiple alterations in the same gene. Gender-based enrichment analysis indicated that ALK and IDH2 alterations were more prevalent in females, while TP53 and PTEN were more common in males. No significant correlation was found between mutations and other clinicopathological attributes, such as age, stage, and subtype. A higher prevalence of EGFR, RET, PIK3CA, ERBB2 and ALK mutations were observed compared to previous LUAD genetic studies coupled with a lower frequency of KRAS mutations. Clinically actionable variants were annotated using OncoKB and categorized into the four therapeutic levels based on their clinical evidence. Seventy-nine percent of cases had at least one clinically actionable alteration. Most patients (39.6%) had the highest level of actionability (Level 1) wherein an FDA-approved drug is available specifically for the observed mutation in lung cancer patients. EGFR Exon19 in-frame deletions and EGFR L858R were the most frequent among targetable variants (20.7%). These findings emphasize the importance of a selective NGS panel in enabling personalized medicine approaches by identifying actionable molecular alterations and informing the choice of targeted therapy for more effective treatment options in Indian NSCLC patients.