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Background/objectives: Patients with recurrent or metastatic endometrial cancer (EC) have poor prognoses with limited therapeutic options following immunotherapy or immunochemotherapy treatments. Inhibitors of KRAS mutations (KRAS-mut) have shown efficacy in early solid tumor studies, but data in EC are lacking. This study describes the frequency of KRAS-mut relative to other oncogenic alterations in EC to identify genomic characteristics of KRAS-mut tumors that could lead to novel therapeutic options., Methods: A molecular database of 7870 ECs was queried for presence of oncogenic mutations and immunotherapy biomarkers. Comparisons were performed using Fisher-Exact/ChiSquare (p-values) and adjusted for multiple tests by Benjamini-Hochberg (q) and pairwise nonparametric analysis using Wilcoxon Method., Results: KRAS-mut is a relatively frequent genotype in EC, detected in 16% of cases. Codon 12 was most frequently mutated, with G12D (31%) and G12V (27%) the most common subtypes. Biomarkers of immunotherapy response co-occur with KRAS-mut. Microsatellite instability-high and tumor mutational burden-high status were observed in 34.1% and 36.5% in KRAS-mut compared to 19.8% and 16.9% in KRAS-WT, respectively (p < 0.05). PD-L1 >1% was detected in 8.4% vs 6.4% of KRAS-mut vs KRAS-WT (p < 0.05). BRCA1/2 mutations were detected with similar low frequency (5.9% vs 4.9%) among KRAS-mut and KRAS-WT ECs (p > 0.05). KRAS-mut was inversely associated with Her-2 overexpression (1.8% KRAS-mut vs 13% KRAS-WT. (p < 0.001)., Conclusions: KRAS-mut represents a genotypically distinct group of ECs. Overlap exists with genomic predictors (TMB-high, MSI-high) of immunotherapy response, suggesting a possible biomarker-driven combination option with immunotherapy. Clinical trials to evaluate these strategies should be developed., Competing Interests: Declaration of competing interest RWH has Speaking Honoraria with AstraZeneca, Eisai, Merck, Natera, and Tesaro/GSK; and Consulting or Advisory Roles with Caris, Genelux, Natera, ImmunoGen, and GSK. PHT has done advisory boards or consulting work with Iovance, Astra Zeneca, Glaxo Smith Kline, Clovis Oncology, Zentalis, Novocure, Seagen, Abbvie, R Pharm, Aadi Pharmaceuticals, Caris, Merck, Imunon, Verastem; and has institutional grant funding from Merck and Glaxo Smith Kline. TJH has done advisory boards or consulting work with Aadi, Astra Zeneca, AbbVie (Immunogen), Caris, Clovis, Corcept, Eisai, Genmab, GSK, GOG Foundation, Merck, Pfizer (Seagen), Roche (Genentech). There are no conflicts to report for other co-authors., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Early-onset biliary tract cancer (eoBTC) is among the fast-growing subset of early-onset cancers, yet little is known about its biology. We sought to identify novel molecular characteristics of eoBTC in relation to average-onset BTC (aoBTC) using a real-world multiomics data set., Methods: The study comprised patients with BTC whose tumors underwent molecular analyses at Caris Life Sciences and were categorized by age (<50 years for eoBTC, ≥50 years for aoBTC). P values were adjusted for multiple testing and considered significant at Q < 0.05 (molecular comparisons) or Q < 0.25 (Gene Set Enrichment Analysis [GSEA]). Insurance claims data were used for survival analysis., Results: The study included 5,587 patients with BTC (453 eoBTC, median age = 44 years and 5,134 aoBTC, median age = 68 years). FGFR2 fusion (15.7% in eoBTC v 5.9% in aoBTC) and NIPBL fusion (1.1% v 0%) were significantly more prevalent in eoBTC (both Q < 0.001). The interferon gamma-IFG score (fold change [FC], 1.1; Q = 0.01) and T-cell inflammation score (FC, 17.3; Q = 0.03) were significantly higher in aoBTC. On GSEA, angiogenesis was enriched in eoBTC (normalized enrichment score [NES] = 1.51; Q = 0.16), whereas IFG (NES = -1.58; Q = 0.06) and inflammatory response (NES = -1.46; Q = 0.18) were enriched in aoBTC. The median overall survival (OS) was 16.5 (eoBTC) versus 13.3 months (aoBTC), hazard ratio = 0.86, P = .004. The median OS by FGFR2 fusion (with fusion v without) was 21.7 versus 15.0 months ( P = .47) for eoBTC and 18.6 versus 12.2 months ( P < .001) for aoBTC., Conclusion: We identified crucial differences including higher prevalence of FGFR2 fusions in eoBTC and variations in immunotherapy-related markers. Better outcomes in eoBTC were affected by the FGFR2 fusion status. Our findings underscore the need for ensuring access to next-generation sequencing testing, including prompt identification of actionable targets.

Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in cancer. HR-deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of HR deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Using whole exome and genome sequencing data, we found that a significant subset of GEA, but very few colorectal adenocarcinomas, show evidence of HR deficiency by mutational signature analysis (HRD score). High HRD gastric cancer cell lines demonstrated functional HR deficiency by RAD51 foci assay and increased sensitivity to platinum chemotherapy and PARP inhibitors. Of clinical relevance, analysis of three different GEA patient cohorts demonstrated that platinum treated HR deficient cancers had better outcomes. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by two photoproduct repair assays. Single-cell RNA-sequencing revealed that, in addition to inducing apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, validated by intracellular GSH assay. Overall, our study provides preclinical evidence that a subset of GEAs harbor genomic features of HR and NER deficiency and may therefore benefit from platinum chemotherapy and PARP inhibitors., (© 2024. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) of the head (H) and body/tail (B/T) differ in embryonic origin, cell composition, blood supply, lymphatic and venous drainage, and innervation. We aimed to compare the molecular and tumor immune microenvironment (TIME) profiles of PDAC of the H vs. B/T. A total of 3499 PDAC samples were analyzed via next-generation sequencing (NGS) of RNA (whole transcriptome, NovaSeq), DNA (NextSeq, 592 genes or NovaSeq, whole exome sequencing), and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Significance was determined as p values adjusted for multiple corrections (q) of <0.05. Anatomic subsites of PDAC tumors were grouped by primary tumor sites into H (N = 2058) or B/T (N = 1384). There were significantly more metastatic tumors profiled from B/T vs. H (57% vs. 44%, p < 0.001). KRAS mutations (93.8% vs. 90.2%), genomic loss of heterozygosity (12.7% vs. 9.1%), and several copy number alterations (FGF3, FGF4, FGF19, CCND1, ZNF703, FLT4, MUTYH, TNFRS14) trended higher in B/T when compared to H (p < 0.05 but q > 0.05). Expression analysis of immuno-oncology (IO)-related genes showed significantly higher expression of CTLA4 and PDCD1 in H (q < 0.05, fold change 1.2 and 1.3) and IDO1 and PDCD1LG2 expression trended higher in B/T (p < 0.05, fold change 0.95). To our knowledge, this is one of the largest cohorts of PDAC tumors subjected to broad molecular profiling. Differences in IO-related gene expression and TIME cell distribution suggest that response to IO therapies may differ in PDAC arising from H vs. B/T. Subtle differences in the genomic profiles of H vs. B/T tumors were observed., (© 2024. The Author(s).)

Background: CDC37 is a key determinant of client kinase recruitment to the HSP90 chaperoning system. We hypothesized that kinase-specific dependency on CDC37 alters the efficacy of targeted therapies for metastatic colorectal cancer (mCRC)., Material and Methods: Two independent mCRC cohorts were analyzed to compare the survival outcomes between CDC37-high and CDC37-low patients (stratified by the median cutoff values): the CALGB/SWOG 80405 trial (226 and 207 patients receiving first-line bevacizumab- and cetuximab-containing chemotherapies, respectively) and Japanese retrospective (50 refractory patients receiving regorafenib) cohorts. A dataset of specimens submitted to a commercial CLIA-certified laboratory was utilized to characterize molecular profiles of CDC37-high (top quartile, N = 5055) and CDC37-low (bottom quartile, N = 5055) CRCs., Results: In the bevacizumab-treated group, CDC37-high patients showed significantly better progression-free survival (PFS) (median 13.3 vs 9.6 months, hazard ratio [HR] 0.59, 95% confidence interval [CI] 0.44-0.79, p < 0.01) than CDC37-low patients. In the cetuximab-treated group, CDC37-high and CDC37-low patients had similar outcomes. In the regorafenib-treated group, CDC37-high patients showed significantly better overall survival (median 11.3 vs 6.0 months, HR 0.24, 95% CI 0.11-0.54, p < 0.01) and PFS (median 3.5 vs 1.9 months, HR 0.51, 95% CI 0.28-0.94, p = 0.03). Comprehensive molecular profiling revealed that CDC37-high CRCs were associated with higher VEGFA, FLT1, and KDR expressions and activated hypoxia signature., Conclusions: CDC37-high mCRC patients derived more benefit from anti-VEGF therapies, including bevacizumab and regorafenib, but not from cetuximab. Molecular profiles suggested that such tumors were dependent on angiogenesis-relating pathways., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: H-JL reports receiving honoraria from consultant/advisory board membership for Merck Serono, Bayer, and Genentech. EL reports research grants from the American Cancer Society (RSG-22–022-01-CDP) 2022–2026, and the Minnesota Ovarian Cancer Alliance in 2019, 2021, and 2022; The Randy Shaver Cancer Research and Community Fund; honoraria and travel expenses for lab-based research talks 2018–21, and equipment for laboratory-based research 2018-present, Novocure, Ltd; honorarium for panel discussion organized by Antidote Education for a CME module on diagnostics and treatment of HER2 + gastric and colorectal cancers, funded by Daiichi-Sankyo, 2021 (honorarium donated to lab); compensation for scientific review of proposed printed content, Elsevier Publishing and Johns Hopkins Press; consultant, Nomocan Pharmaceuticals (no financial compensation); Scientific Advisory Board Member, Minnetronix, LLC, 2018–2019 (no financial compensation); consultant and speaker honorarium, Boston Scientific US, 2019. Institutional Principal Investigator for clinical trials sponsored by Celgene, Novocure, Intima Biosciences, and the National Cancer Institute, and University of Minnesota membership in the Caris Life Sciences Precision Oncology Alliance (no financial compensation). YB, JX, and WMK are employees of Caris Life Sciences. All remaining authors have declared no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Oncogenic drivers such as KRAS extensively modulate the tumor inflammatory microenvironment (TIME) of colorectal cancer (CRC). The influence of KRAS on modulating immune cell composition remains unclear. The objective of this study was to identify signatures of infiltrative immune cells and distinctive patterns that differ between RAS wild-type (WT) and oncogenic mutant (MT) CRC that explain immune evasion in MT tumors. A total of 7,801 CRC specimens were analyzed using next-generation DNA sequencing, whole-exome sequencing, and/or whole transcriptome sequencing. Deficiency of mismatch repair (dMMR)/microsatellite instability (MSI) and tumor mutation burden (TMB) were also assessed. KRAS mutations were present in 48% of CRC, similarly distributed in patients younger than vs. 50 years and older. In microsatellite stable (MSS) KRAS MT tumors, composition of the TIME included higher neutrophil infiltration and lower infiltration of B cells. MSI-H/dMMR was significantly more prevalent in RAS WT (9.1%) than in KRAS MT (2.9%) CRC. In MSS CRC, TMB-high cases were significantly higher in RAS MT (3.1%) than in RAS WT (2.1%) tumors. KRAS and NRAS mutations are associated with increased neutrophil infiltration, with codon-specific differences. These results demonstrate significant differences in the TIME of RAS mutant CRC that match previous reports of immunoevasive characteristics of such tumors., Competing Interests: J.X., Y.B., C.N., and M.O. are employed by Caris Life Sciences. E.L. reports support from the University of Minnesota Clinical Center for the Study of Pancreatic Disease, part of The Chronic Pancreatitis Diabetes Pancreatic Cancer Research consortium funded by the National Institute of Diabetes and Digestive and Kidney Diseases (5U01DK126300-03). He reports support from research grants from the American Cancer Society (RSG-22-022-01-CDP) 2022–2026; the Minnesota Ovarian Cancer Alliance in 2019, 2021, and 2022; the American Association for Cancer Research (2019 AACR-Novocure Tumor-Treating Fields Research Grant 1-60-62-LOU); the Randy Shaver Cancer Research and Community Fund; honoraria and travel expenses for lab-based research talks 2018–2021 and equipment for laboratory-based research 2018–present from Novocure; honorarium for panel discussion organized by Antidote Education for a CME module on diagnostics and treatment of HER2+ gastric and CRCs, funded by Daiichi-Sankyo, 2021 (honorarium donated to lab); compensation for scientific review of proposed printed content from Elsevier Publishing and The Johns Hopkins University Press; institutional principal investigator for clinical trials sponsored by Celgene, Novocure, Intima Bioscience, and the National Cancer Institute; and University of Minnesota membership in the Caris Life Sciences Precision Oncology Alliance (no financial compensation). He acknowledges and thanks the following groups for donations in support of cancer research: friends and family of Gayle Huntington; the Mu Sigma Chapter of the Phi Gamma Delta Fraternity, University of Minnesota; the Litman Family Fund for Cancer Research; Dick and Lynnae Koats; Ms. Patricia Johnson; and the Love Like Laurie Legacy. A. Saeed reports research grants (to institution) from AstraZeneca, Bristol Myers Squibb, Merck, Clovis, Exelixis, Actuate Therapeutics, Incyte Corporation, Daiichi Sankyo, Five Prime Therapeutics, Amgen, Innovent Biologics, Dragonfly Therapeutics, KAHR Medical, Oxford Biotherapeutics, BioNTech, and Data Safety Monitoring Committee (DSMC); consulting fees from Arcus Therapeutics; and advisory board fees from AstraZeneca, Bristol Myers Squibb, Exelixis, Pfizer, and Daiichi Sankyo. E.F. reports the following disclosures: employee of Hospital Corporation of America (HCA) International; personal financial interest (conference attendance): Repair Therapeutics, CARIS Life Science, Seagen, and Sapience Pharma; funding to institution: Repair Therapeutics, Bicycle Therapeutics, Artios Pharma, Seagen, Amgen, Nurix Therapeutics, BioNTech SE, Relay Therapeutics, Tahio Pharmaceutical, Pfizer, Roche, Daiichi Sankyo, Gilead Science, Basilea Pharmaceutica, Jiangsu Hengrui Medicine, Mereo Biopharma, HUTCHMED, Merus, Crescendo Biologics, GSK plc, BeiGene, Turning Point Therapeutics, Sapience Pharma; and leadership role: European Organisation for Research and Treatment of Cancer. A.F.S. reports funding from Caris Life Sciences for research, travel, and the speaker’s bureau. H.J.L. reports advisory board/honoraria from Bayer, BMS, Merck, 3TBioscience, Fulgent, Boehringer-Ingelheim, UCB, Replimune, and Adagene, and is supported by SWOG Cancer Research Network, the National Cancer Institute, and the NIH. A. Seeber is on the speaker’s panel for Caris Life Sciences., (© 2024 The Author(s).)

Purpose: Non-small-cell lung cancer (NSCLC) with STK11 mut has inferior outcomes to immune checkpoint inhibitors (ICIs). Using multiomics, we evaluated whether a subtype of STK11 mut NSCLC with a uniquely inflamed tumor immune microenvironment (TIME) harboring TP53 comutations could have favorable outcomes to ICIs., Patients and Methods: NSCLC tumors (N = 16,896) were analyzed by next-generation sequencing (DNA-Seq/592 genes). A subset (n = 5,034) underwent gene expression profiling (RNA-Seq/whole transcriptome). Exome-level neoantigen load for STK11 mut NSCLC was obtained from published pan-immune analysis. Tumor immune cell content was obtained from transcriptome profiles using the microenvironment cell population (MCP) counter. ICI data from POPLAR/OAK (n = 34) and the study by Rizvi et al (n = 49) were used to model progression-free survival (PFS), and a separate ICI-treated cohort (n = 53) from Dana-Farber Cancer Institute (DFCI) was used to assess time to treatment failure (TTF) and tumor RECIST response for STK11 mut TP53 mut versus STK11 mut TP53 wt NSCLC., Results: Overall, 12.6% of NSCLC tumors had a STK11 mut with the proportions of tumor mutational burden (TMB)-high (≥10 mut/Mb), PD-L1 ≥50%, and microsatellite instability-high being 38.3%, 11.8%, and 0.72%, respectively. Unsupervised hierarchical clustering of STK11 mut (n = 463) for stimulator of interferon-gamma (STING) pathway genes identified a STING-high cluster, which was significantly enriched in TP53 mut NSCLC ( P < .01). Compared with STK11 mut TP53 wt , tumors with STK11 mut TP53 mut had higher CD8+T cells and natural killer cells ( P < .01), higher TMB ( P < .001) and neoantigen load ( P < .001), and increased expression of MYC and HIF-1A ( P < .01), along with higher expression ( P < .01) of glycolysis/glutamine metabolism genes. Meta-analysis of data from OAK/POPLAR and the study by Rizvi et al showed a trend toward improved PFS in patients with STK11 mut TP53 mut . In the DFCI cohort, compared with the STK11 mut TP53 wt cohort, the STK11 mut TP53 mut tumors had higher objective response rates (42.9% v 16.7%; P = .04) and also had longer TTF (14.5 v 4.5 months, P adj = .054) with ICI., Conclusion: STK11 mut NSCLC with TP53 comutation is a distinct subgroup with an immunologically active TIME and metabolic reprogramming. These properties should be exploited to guide patient selection for novel ICI-based combination approaches.

Background: The C-C motif chemokine receptor 5 (CCR5)/C-C motif chemokine ligand 5 (CCL5) axis plays a major role in colorectal cancer (CRC). We aimed to characterize the molecular features associated with CCR5 / CCL5 expression in CRC and to determine whether CCR5 / CCL5 levels could impact treatment outcomes., Methods: 7604 CRCs tested with NextGen Sequencing on DNA and RNA were analyzed. Molecular features were evaluated according to CCR5 and CCL5 tumor gene expression quartiles. The impact on treatment outcomes was assessed in two cohorts, including 6341 real-world patients and 429 patients from the Cancer and Leukemia Group B (CALGB)/SWOG 80405 trial., Results: CCR5 / CCL5 expression was higher in right-sided versus left-sided tumors, and positively associated with consensus molecular subtypes 1 and 4. Higher CCR5 / CCL5 expression was associated with higher tumor mutational burden, deficiency in mismatch repair and programmed cell death ligand 1 (PD-L1) levels. Additionally, high CCR5 / CCL5 were associated with higher immune cell infiltration in the tumor microenvironment (TME) of MMR proficient tumors. Ingenuity pathway analysis revealed upregulation of the programmed cell death protein 1 (PD-1)/PD-L1 cancer immunotherapy pathway, phosphatase and tensin homolog (PTEN) and peroxisome proliferator-activated receptors (PPAR) signaling, and cytotoxic T-lymphocyte antigen 4 (CTLA-4) signaling in cytotoxic T lymphocytes, whereas several inflammation-related pathways were downregulated. Low CCR5 / CCL5 expression was associated with increased benefit from cetuximab-FOLFOX treatment in the CALGB/SWOG 80405 trial, where significant treatment interaction was observed with biologic agents and chemotherapy backbone., Conclusions: Our data show a strong association between CCR5/CCL5 gene expression and distinct molecular features, gene expression profiles, TME cell infiltration, and treatment benefit in CRC. Targeting the CCR5/CCL5 axis may have clinical applications in selected CRC subgroups and may play a key role in developing and deploying strategies to modulate the immune TME for CRC treatment., Competing Interests: Competing interests: H-JL reports receiving honoraria from consultant/advisory board membership for Merck Serono, Bayer, and Genentech. JX, YB, JPA and WMK are employers of Caris Life Sciences. AFS reports funding for research, travel, and the speakers bureau from Caris Life Sciences. BAW reports receiving honoraria from Bayer, Sirtex, Lilly, Taiho, and HalioDx. RMG reports stock and other ownership interests from Advanced Chemotherapy Technologies and Compass Therapeutics, consulting/advisory role for AbbVie, G1 Therapeutics, GSK, Merck, Eisai, Compass Therapeutics, Inspirna, Taiho, Novartis, AstraZeneca, and Bayer, expert testimony from Taiho Pharmaceutical. FI is an AbbVie employee and receives stocks from the company. All remaining authors have declared no conflicts of interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Two ROS1 tyrosine kinase inhibitors have been approved for ROS1 fusion positive (ROS1+) non-small cell lung cancer (NSCLC) tumors. We performed a pan-tumor analysis of the incidence of ROS1 fusions to assess if more ROS1+ patients who could benefit from ROS1 TKIs could be identified., Methods: A retrospective analysis of ROS1 positive solid malignancies identified by targeted RNA sequencing and whole transcriptome sequencing of clinical tumor samples performed at Caris Life Science (Phoenix, AZ)., Results: A total of 259 ROS1+ solid malignancies were identified from approximately 175,350 tumors that underwent next-generation sequencing (12% from targeted RNA sequencing [Archer]; 88% from whole transcriptome sequencing). ROS1+ NSCLC constituted 78.8% of the ROS1+ solid malignancies, follow by glioblastoma (GBM) (6.9%), and breast cancer (2.7%). The frequency of ROS1 fusion was approximately 0.47% among NSCLC, 0.29% for GBM, 0.04% of breast cancer. The mean tumor mutation burden for all ROS1+ tumors was 4.8 mutations/megabase. The distribution of PD-L1 (22C3) expression among all ROS1+ malignancies were 0% (18.6%), 1%-49% (29.4%), and ≥ 50% (60.3%) [for NSCLC: 0% (17.8%); 1-49% (27.7%); ≥ 50% (53.9%). The most common genetic co-alterations of ROS1+ NSCLC were TP53 (29.1%), SETD2 (7.3%), ARIAD1A (6.3%), and U2AF1 (5.6%)., Conclusions: ROS1+ NSCLC tumors constituted the majority of ROS1+ solid malignancies with four major fusion partners. Given that > 20% of ROS1+ solid tumors may benefit from ROS1 TKIs treatment, comprehensive genomic profiling should be performed on all solid tumors., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: RET fusions are driver alterations in cancer and are most commonly found in non-small cell lung cancer and well-differentiated thyroid cancer. However, RET fusion have been reported in other solid tumors., Material and Methods: A retrospective analysis of RET+ solid malignancies identified by targeted RNA sequencing and whole transcriptome sequencing of clinical tumor samples performed at Caris Life Science (Phoenix, AZ)., Results: As of March 22, 2022, a total of 378 RET+ solid malignancies were identified in 15 different tumor types and carcinoma of unknown primary (CUP) that underwent next-generation RNA sequencing. RET+ NSCLC and RET+ thyroid cancer constituted 66.9% and 11.1% of the RET+ solid malignancies, respectively. RET+ colorectal adenocarcinoma and RET+ breast adenocarcinoma constituted 10.1% and 2.6%, respectively. The estimated frequency of RET fusions within specific tumor types were NSCLC 0.7%, thyroid cancer 3.1%, colorectal cancer 0.2% and breast cancer 0.1%. KIF5B (46.8%) was the most common fusion partner followed by CCDC6 (28.3%) and NCOA4 (13.8%) in RET+ solid tumors. KIF5B-RET was the dominant fusion variant in RET+ NSCLC, NCOA4-RET was the dominant variant in RET+ colorectal carcinoma, and CCDC6-RET was the dominant variant in thyroid cancer. The most common single gene alterations in RET+ tumors were TP53 (34.8%), RASA1 (14.3%) and ARIAD1A (11.6%). RET+ CRC had a high median TMB of 20.0 and were commonly MSI-H., Conclusions: RET fusions were identified in multiple tumor types. With a higher median TMB and commonly MSI-H, RET fusion positive CRC may be a unique molecular subset of CRC., Competing Interests: Declaration of Competing Interest There was no funding allocated for this research and there are no direct conflicts of interest. Potential COI from all authors are listed below. MN is on the advisory board for AstraZeneca, Daiichi Sankyo, Takeda, Novartis, EMD Serono, Janssen, Pfizer, Eli Lilly and Company, Bayer and Genentech; consultant for Caris Life Sciences (virtual tumor board); speaker for Blueprint Medicines, Janssen, Mirati and Takeda; and reports travel support from AnHeart Therapeutics.  DB has no disclosures. YB, JX, JJS and DS are employees and shareholders of Caris Life Sciences. MNA discloses the following: Speaker Bureau: IPSEN, AstraZeneca, Guardant Health. External advisory board: CTI-Facts (CRO company). CK served as a consultant for Novartis, Janssen, Astrazeneca, Sanofi, PierianDx, Diffuse pharmaceuticals, Mirati, Jazz Pharmaceuticals, and Arcus Biosciences, and received research funding (to institution) from AstraZeneca, Bristol-Myers Squibb, Novartis, Genentech, Janssen, Regeneron, Debiopharm, Karyopharm, and Blueprint Medicines. JN discloses the following: Consulting: Aadi Biosciences, Astra Zeneca, Bristol Myers Squibb, Fujirebio, G1 Therapeutics, Genentech, Mindmed, Naveris, Takeda, Western Oncolytics., Research Support: Genentech, Merck, Intellectual Property: Cansera and Ownership Interests: Cansera, Epic Sciences, Indee Bio, Quantgene. WMK has stock ownership of Caris Life Sciences. MS has received honoraria from AstraZeneca, Bayer, Roche, Celgene, BMS, Genentech, Novartis and Lilly. MS is a consultant for Genentech and Novartis and has received research support from AstraZeneca, Roche and Takeda. LER has received research support from BMS, Astra-Zeneca, Roche, Pfizer, Merck, Velos, Guardant Health, Natera, Genentech, Bio Alta. BH has grants or contracts from Boehringer Ingelheim, Astra Zeneca, Merck, BMS, Advaxis, Amgen, AbbVie, Daiichi, Pfizer, GSK, Beigene, Janssen, has received consulting fees from Veracyte and has been on monitoring or advisory boards for Astra Zeneca, Boehringer Ingelheim, Apollomics, Janssen, Takeda, Merck, BMS, Genentech, Pfizer, Eli-Lilly, TPT, Arcus and Merus. SHIO has stock ownership and was on the scientific advisory board of Turning Point Therapeutics Inc (until Feb 28, 2019), is a member of the SAB of Elevation Oncology, and has received speaker honorarium from Merck, Roche/Genentech, Astra Zeneca, Takeda/ARIAD and Pfizer; has received advisory fees from Roche/Genentech, Astra Zeneca, Takeda/ARIAD, Pfizer, Foundation Medicine Inc, Spectrum, Daiichi Sankyo, Jassen/JNJ, and X-Covery., (Copyright © 2023. Published by Elsevier Inc.)

Purpose: Using a real-world database with matched genomic-transcriptomic molecular data, we sought to characterize the distinct molecular correlates underlying clinical differences between patients with young-onset pancreatic cancer (YOPC; younger than 50 years) and patients with average-onset pancreatic cancer (AOPC; 70 years and older)., Methods: We analyzed matched whole-transcriptome and DNA sequencing data from 2,430 patient samples (YOPC, n = 292; AOPC, n = 2,138) from the Caris Life Sciences database (Phoenix, AZ). Immune deconvolution was performed using the quanTIseq pipeline. Overall survival (OS) data were obtained from insurance claims (n = 4,928); Kaplan-Meier estimates were calculated for age- and molecularly defined cohorts. Significance was determined as FDR-corrected P values ( Q ) < .05., Results: Patients with YOPC had higher proportions of mismatch repair-deficient/microsatellite instability-high, BRCA2 -mutant, and PALB2 -mutant tumors compared with patients with AOPC, but fewer SMAD4- , RNF43- , CDKN2A- , and SF3B1- mutant tumors. Notably, patients with YOPC demonstrated significantly lower incidence of KRAS mutations compared with patients with AOPC (81.3% v 90.9%; Q = .004). In the KRAS wild-type subset (n = 227), YOPC tumors demonstrated fewer TP53 mutations and were more likely driven by NRG1 and MET fusions, whereas BRAF fusions were exclusively observed in patients with AOPC. Immune deconvolution revealed significant enrichment of natural killer cells, CD8 + T cells, monocytes, and M2 macrophages in patients with YOPC relative to patients with AOPC, which corresponded with lower rates of HLA-DPA1 homozygosity. There was an association with improved OS in patients with YOPC compared with patients with AOPC with KRAS wild-type tumors (median, 16.2 [YOPC- KRAS WT ] v 10.6 [AOPC- KRAS WT ] months; P = .008) but not KRAS -mutant tumors ( P = .084)., Conclusion: In this large, real-world multiomic characterization of age-stratified molecular differences in pancreatic ductal adenocarcinoma, YOPC is associated with a distinct molecular landscape that has prognostic and therapeutic implications.

Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821-1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs., (© 2023. The Author(s).)

Background: Genomic signatures contributing to high tumour mutational burden (TMB-H) independent from mismatch-repair deficiency (dMMR) or microsatellite instability-high (MSI-H) status are not well studied. We aimed to characterise molecular features of microsatellite stable (MSS) TMB-H gastrointestinal tumours., Methods: Molecular alterations of 48 606 gastrointestinal tumours from Caris Life Sciences (CARIS) identified with next-generation sequencing were compared among MSS-TMB-H, dMMR/MSI-H, and MSS-TMB-low (L) tumours, using χ 2 or Fisher's exact tests. Antitumour immune response within the tumour environment was predicted by analysing the infiltration of immune cells and immune signatures using The Cancer Genome Atlas database. The Kaplan-Meier method and the log-rank test were used to evaluate the impact of gene alterations on the efficacy of immune checkpoint inhibitors in MSS gastrointestinal cancers from the CARIS database, a Memorial Sloan Kettering Cancer Center cohort, and a Peking University Cancer Hospital cohort., Findings: MSS-TMB-H was observed in 1600 (3·29%) of 48 606 tumours, dMMR/MSI-H in 2272 (4·67%), and MSS-TMB-L in 44 734 (92·03%). Gene mutations in SMAD2, MTOR, NFE2L2, RB1, KEAP1, TERT, and RASA1 might impair antitumour immune response despite TMB-H, while mutations in 16 other genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2, and XPO1) were related to TMB-H with enhanced antitumour immune response independent of dMMR/MSI-H, constructing a predictive model (modified TMB [mTMB]) for immune checkpoint inhibitor efficacy. Patients with any mutation in the mTMB gene signature, in comparison with patients with mTMB wildtype tumours, showed a superior survival benefit from immune checkpoint inhibitors in MSS gastrointestinal cancers in the CARIS cohort (n=95, median overall survival 18·77 months [95% CI 17·30-20·23] vs 7·03 months [5·73-8·34]; hazard ratio 0·55 [95% CI 0·31-0·99], p=0·044). In addition, copy number amplification in chromosome 11q13 (eg, CCND1, FGF genes) was more prevalent in MSS-TMB-H tumours than in the dMMR/MSI-H or MSS-TMB-L subgroups., Interpretation: Not all mutations related to TMB-H can enhance antitumour immune response. More composite biomarkers should be investigated (eg, mTMB signature) to tailor treatment with immune checkpoint inhibitors. Our data also provide novel insights for the combination of immune checkpoint inhibitors and drugs targeting cyclin D1 or FGFs., Funding: US National Cancer Institute, Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Gene Gregg Pancreas Research Fund, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Victoria and Philip Wilson Research Fund, Fong Research Project, Ming Hsieh Research Fund, Shanghai Sailing Program, China National Postdoctoral Program for Innovative Talents, China Postdoctoral Science Foundation, National Natural Science Foundation of China., Competing Interests: Declaration of interests H-JL reports receiving honoraria from serving as a consultant or from advisory board membership for Merck Serono, Bayer, and Genentech. JX, AF, YB, MO, DS, and WMK are employees of Caris Life Sciences. AFS reports funding for research, travel, and speakers bureau participation from Caris Life Sciences. BAW reports receiving honoraria from Bayer, Sirtex, Lilly, Taiho, and HalioDx. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels., Experimental Design: The Cancer Genome Atlas database was used to explore molecular and immunologic features associated with CXCR4 mRNA expression in PDAC. A large real-word dataset (n = 3,647) served for validation and further exploratory analyses. Single-cell RNA analyses on a publicly available dataset and in-house multiplex immunofluorescence (mIF) experiments were performed to elaborate cellular localization of CXCR4., Results: High CXCR4 mRNA expression (CXCR4high) was associated with increased infiltration of regulatory T cells, CD8+ T cells, and macrophages, and upregulation of several immune-related genes, including immune checkpoint transcripts (e.g., TIGIT, CD274, PDCD1). Analysis of the validation cohort confirmed the CXCR4-dependent immunologic TME composition in PDAC irrespective of microsatellite instability-high/mismatch repair-deficient or tumor mutational burden. Single-cell RNA analysis and mIF revealed that CXCR4 was mainly expressed by macrophages and T-cell subsets. Clinical relevance of our finding is supported by an improved survival of CXCR4high PDAC., Conclusions: High intratumoral CXCR4 mRNA expression is linked to a T cell- and macrophage-rich PDAC phenotype with high expression of inhibitory immune checkpoints. Thus, our findings might serve as a rationale to investigate CXCR4 as a predictive biomarker in patients with PDAC undergoing immune checkpoint inhibition., (©2022 The Authors; Published by the American Association for Cancer Research.)

Objectives: Low grade serous ovarian cancer (LGSOC) differs from high grade serous in terms of pathogenesis, molecular, genetic, and clinical features. Molecular studies have been hampered by small sample sizes, heterogenous histology, and lack of comprehensive testing. We sought to molecularly profile LGSOC in a homogenously tested, histologically confirmed cohort., Methods: Using hot-spot and whole exome next generation sequencing (NGS), fusion gene analysis interrogating RNA, fragment analysis, in situ hybridization and/or immunohistochemistry, 179 specimens were evaluated by Caris Life Sciences (Phoenix, AZ). A second independent histologic review confirmed histology in 153 specimens., Results: Most frequently mutated genes (5% or greater) were members of the mitogen-activated protein kinase (MAPK) pathway: KRAS (23.7%, n = 36), NRAS (11.2%, n = 19), NF1 (7.9%, n = 5), and BRAF (6.6%, n = 10). Class III mutations were seen in 3 of 10 BRAF mutations while 7 were Class I V600E. Overall, estrogen and progesterone receptor expression was 80.2% (n = 130) and 27.8% (n = 45), respectively. Of those that were hormone negative, nearly 50% contained KRAS or NF1 mutations. None were NRAS mutated. Markers of response to immunotherapy were low to absent., Conclusion: BRAF mutations were seen to be lower than those traditionally reported. With increased MAPK activation resulting in ligand independent activation of ERα, a role of combination therapy with hormonal and targeted therapy should be considered as 49.2% of hormone negative specimens were KRAS or NF1 mutated. Absence of immunotherapy biomarkers suggest limited benefit to immunotherapeutic agents., Competing Interests: Declaration of Competing Interest David Arguello and Yasmin Baca are employees of Caris Life Sciences., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objectives: Disparate outcomes exist between Black and White patients with endometrial cancer (EC). One contributing factor is the disproportionately low representation of Black patients in clinical trials and in tumor molecular profiling studies. Our objective was to investigate molecular profiles of ECs in a cohort with a high proportion of tumors from Black patients., Methods: A total of 248 EC samples and self-reported race data were collected from 6 institutions. Comprehensive tumor profiling and analyses were performed by Caris Life Sciences., Results: Tumors from 105 (42%) Black and 143 (58%) White patients were included. Serous histology (58% vs 36%) and carcinosarcoma (25% vs 16%), was more common among Black patients, and endometrioid was less common (17% vs 48%) (p < 0.01). Differences in gene mutations between cohorts corresponded to observed histologic differences between races. Specifically, TP53 mutations were predominant in serous tumors. In endometrioid tumors, mutations in ARID1A were the most common, and high rates of MSI-H, MMRd, and TMB-H were observed. In carcinosarcoma tumors, hormone receptor expression was high in tumors of Black patients (PR 23.4%, ER 30.8%). When stratified by histology, there were no significant differences between tumors from Black and White women., Conclusions: This cohort had a high proportion of tumors from Black women. Distinct molecular profiles were driven primarily by more aggressive histologic subtypes among Black women. Continued effort is needed to include Black women and other populations under-represented in EC molecular profiling studies as targeted therapies and personalized medicine become mainstream., (Copyright © 2022. Published by Elsevier Inc.)

Purpose: Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies., Experimental Design: A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings., Results: The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1-RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings., Conclusions: This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC., Experimental Design: To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations., Results: Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation., Conclusions: These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups., (©2022 American Association for Cancer Research.)

The prevalence of homologous recombination-DNA damage response (HR-DDR) genetic alterations is of therapeutic interest in gastroesophageal cancers. This study is a comprehensive assessment of HR-DDR mutation prevalence across gastroesophageal adenocarcinomas and squamous cell carcinomas. Here we investigate the association of HR-DDR mutations with known predictors for immune-checkpoint inhibition [deficiency in mismatch-repair (dMMRP), tumor mutational burden (TMB), and programmed death ligand 1 (PD-L1)]. We confirmed HR-DDR mutations are present in a subset of gastroesophageal adenocarcinomas (23%) and gastroesophageal squamous cell carcinomas (20%). Biomarker expression of dMMRP (18% vs. 1%) and TMB-high with a cutoff of ≥10 mt/MB (27% vs. 9%) was significantly more prevalent in the DDR-mutated cohort compared with the non-DDR-mutated cohort. Mean combined positive score for PD-L1 in the total adenocarcinoma cohort was significantly higher in the DDR-mutated cohort compared with the non-DDR-mutated cohort (10.1 vs. 5.8). We demonstrated that alterations in ARID1A , BRCA2 , PTEN , and ATM are correlated with dMMRP, TMB-high, and increased PD-L1 expression in gastroesophageal adenocarcinomas. Our findings show that a subset of gastroesophageal tumors harbor HR-DDR mutations correlated with established immune biomarkers. By better understanding the relationship between HR-DDR mutations and immune biomarkers, we may be able to develop better immunotherapy combination strategies to target these tumors., (©2021 American Association for Cancer Research.)

Background: HER2 alteration (mutation and/or amplification) is associated with poor survival in NSCLC and can mediate resistance to EGFR tyrosine kinase inhibitors., Methods: We retrospectively analyzed de-identified molecular information from 12,946 NSCLC samples that underwent next-generation sequencing (NGS) with Caris Life Sciences. The objectives were to determine the prevalence and type of HER2 alterations with and without EGFR as a co-mutation. Insurance claims were utilized to obtain outcomes data., Results: Three hundred and twenty-one patients (2.5%) had HER2 alteration: mutation in 197 patients and amplification in 134. Median age was 65 years and 62% were female. A total of 84% were adenocarcinoma. HER2 exon 20 insertion was most common (69%). A total of 1551 (12%) patients had EGFR mutations. Among samples with EGFR mutations, 24 (1.5%) had concurrent HER2 alteration (8 with HER2 mutation and 16 with amplification). Among 8 patients who had both EGFR and HER2 mutations, 3 had EGFR exon 19 deletions and exon 8 HER2 mutation (S310F). One-third of the patients (7/21) with HER2 extracellular domain (ECD) mutation had co-occurring EGFR mutations. All 7 were S310. Patients with concurrent EGFR mutation and HER2 amplification had longer median time on treatment with EGFR TKI(s) than those with EGFR mutation without HER2 amplification (HR 2.284, P =.004)., Conclusion: A minority of NSCLC samples with EGFR mutations had HER2 alterations. In patients with both mutations, exon 21 mutations for EGFR and exon 8 mutations for HER2 were common. It will be critical to continue to accumulate valuable clinical data for further real-world outcomes analysis., (Copyright © 2021. Published by Elsevier Inc.)

Cancer/testis antigens (CTAs) are strongly expressed in some solid tumors but minimally expressed in normal tissue, making them appealing therapeutic targets. KK-LC-1 (CXorf61) has cytoplasmic expression in gastric, breast, and lung cancer. We characterized the molecular subtypes of non-small cell lung cancer (NSCLC) expressing KK-LC-1 to inform rational clinical trials of T-cell receptor therapy (TCR-T) targeting KK-LC-1. 9790 NSCLC tumors that underwent whole transcriptome sequencing (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ) were analyzed. Tumors were split into quartiles based on KK-LC-1 expression and pathological and molecular differences were investigated. Adenocarcinoma had significantly higher KK-LC-1 expression than squamous cell carcinoma (median, 3.25 vs. 1.17 transcripts per million (TPM), p < 0.0001). Tumors with the highest quartile of KK-LC-1 expression had a greater proportion of tumors with high tumor mutation burden (TMB) (≥10 mutations per megabase; 44% vs. 28% in Q1, p < 0.001). Increased KK-LC-1 expression was associated with increased M1 macrophage abundance. Higher levels of KK-LC-1 expression were seen in pan-wild type and KRAS mutated tumors and associated with high TMB. TCR-T therapy directed against KK-LC-1 should be considered in patients whose clinical features reflect these characteristics., Competing Interests: CONFLICTS OF INTEREST Dr. Nieva receives personal fees from Astra Zeneca, Fujirebio and Naveris. He receives research support from Merck and Genentech. Dr. Hsu was a consultant for Targeted Healthcare Communications. Ms. Baca and Dr. Xiu are employees of Caris Life Sciences. Dr. Kim is a consultant or advisory board member for Novartis, Janssen, AstraZeneca, and PierianDx and has received research funding to his institution from AstraZeneca, Bristol-Myers Squibb, Novartis, Genentech, Regeneron, Spectrum, Mirati, Debiopharm, Karyopharm, and Janssen. Dr. Mamdani is a consultant for Zentalis and has been a consultant for AstraZeneca, Caris Life Sciences, and Takeda. Dr. Nagasaka is a consultant for Caris and a speaker for Blueprint. She has also participated in advisory boards for AstraZeneca, Daiichi, Takeda, Novartis, EMD Serono, JNJ, Pfizer, Lilly, Genentech, and has received travel support from AnHeart Therapuetics. Dr. Puri is a consultant/advisor for AstraZeneca and G1 Therapeutics. Dr. Liu is a consultant/serves on advisory board for Amgen, AstraZeneca, Bayer, Beigene, Blueprint, Bristol-Myers Squibb, Daiichi Sankyo, Eisai, Elevation Oncology, Genentech/Roche, Guardant Health, Janssen, Jazz Pharmaceuticals, Lilly, Merck/MSD, Novartis, Regeneron, Takeda, Turning Point Therapeutics. He also has received research funding to his institution from Alkermes, Bayer, Blueprint, Bristol-Myers Squibb, Elevation Oncology, Genentech, Lilly, Merck, Merus, Pfizer, Rain Therapeutics, RAPT, Turning Point Therapeutics. Dr. Korn is an employee of Caris Life Sciences and has stock ownership and stock options from Caris Life Sciences. He also receives consultant fees from Merck. All other authors have no conflicts of interests to disclose., (Copyright: © 2021 Hsu et al.)

KRAS is the most commonly mutated oncogene in NSCLC and development of direct KRAS inhibitors has renewed interest in this molecular variant. Different KRAS mutations may represent a unique biologic context with different prognostic and therapeutic impact. We sought to characterize genomic landscapes of advanced, KRAS -mutated non-small cell lung cancer (NSCLC) in a large national cohort to help guide future therapeutic development.Molecular profiles of 17,095 NSCLC specimens were obtained using DNA next-generation sequencing of 592 genes (Caris Life Sciences) and classified on the basis of presence and subtype of KRAS mutations. Co-occurring genomic alterations, tumor mutational burden (TMB), and PD-L1 expression [22C3, tumor proportion score (TPS) score] were analyzed by KRAS mutation type.Across the cohort, 4,706 (27.5%) samples harbored a KRAS mutation. The most common subtype was G12C (40%), followed by G12V (19%) and G12D (15%). The prevalence of KRAS mutations was 37.2% among adenocarcinomas and 4.4% in squamous cell carcinomas. Rates of high TMB (≥10 mutations/Mb) and PD-L1 expression varied across KRAS mutation subtypes. KRAS G12C was the most likely to be PD-L1 positive (65.5% TPS ≥ 1%) and PD-L1 high (41.3% TPS ≥ 50%). STK11 was mutated in 8.6% of KRAS wild-type NSCLC but more frequent in KRAS -mutant NSCLC, with the highest rate in G13 (36.2%). TP53 mutations were more frequent in KRAS wild-type NSCLC (73.6%). KRAS mutation subtypes have different co-occurring mutations and a distinct genomic landscape. The clinical relevance of these differences in the context of specific therapeutic interventions warrants investigation., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Nuclear protein transport is essential in guiding the traffic of important proteins and RNAs between the nucleus and cytoplasm. Export of proteins from the nucleus is mostly regulated by Exportin 1 (XPO1). In cancer, XPO1 is almost universally hyperactive and can promote the export of important tumor suppressors to the cytoplasm. Currently, there are no studies evaluating XPO1 amplifications and mutations in NSCLC and the impact on outcomes., Methods: Tumor samples were analyzed using next-generation sequencing (NGS) (NextSeq, 592 Genes), immunohistochemistry (IHC), and whole transcriptome sequencing (WTS, NovaSeq) (Caris Life Sciences, Phoenix, AZ). Survival was extracted from insurance claims data and calculated from time of tissue collection to last contact using Kaplan-Meier estimate., Results: Among 18,218 NSCLC tumors sequenced, 26 harbored XPO1 mutations and 24 had amplifications. XPO1 mutant tumors were more likely to have high TMB (79% vs. 52%, p = 0.007) and less likely to have high PD-L1 (32% vs. 68%, p = 0.03). KRAS co-mutations were seen in 19% (n = 5) and EGFR co-mutations were rare (n = 2). Among the 17,449 NSCLC tumors with clinical data, there were 24 XPO1 mutant. Comparison of survival between XPO1 mutant and WT showed a negative association with a hazard ratio (HR) of 1.932 (95% CI: 1.144-3.264 p = 0.012). XPO1 amplification was not associated with survival., Conclusions: XPO1 pathogenic mutations were associated with a poor survival in NSCLC. Although XPO1 mutations are rare in NSCLC, further studies to assess its associations with treatment responses are warranted., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Frequent mutations of genes in the histone-lysine N-methyltransferase 2 (KMT2) family members were identified in gastric cancers (GCs). Understanding how gene mutations of KMT2 family affect cancer progression and tumor immune microenvironment may provide new treatment strategies. A total of 1245 GCs were analyzed using next-generation sequencing, whole transcriptome sequencing, immunohistochemistry (Caris Life Sciences, Phoenix, AZ). The overall mutation rate of genes in the KMT2 family was 10.6%. Compared to KMT2-wild-type GCs, genes involved in epigenetic modification, receptor tyrosine kinases/MAPK/PI3K, and DNA damage repair (DDR) pathways had higher mutation rates in KMT2-mutant GCs (p < 0.05). Significantly higher rates of high tumor mutational burden, microsatellite instability-high/mismatch-repair deficiency (dMMR), and PD-L1 positivity were observed in KMT2-mutant GCs (p < 0.01), compared to KMT2-wild-type GCs. The association between PD-L1 positivity and KMT2 mutations remained significant in the proficient-MMR and microsatellite stable subgroup. Based on transcriptome data from the TCGA, cell cycle, metabolism, and interferon-α/β response pathways were significantly upregulated in KMT2-mutant GCs than in KMT2-wild-type GCs. Patients with KMT2 mutation treated with immune checkpoint inhibitors had longer median overall survival compared to KMT2-wild-type patients with metastatic solid tumors (35 vs. 16 months, HR = 0.73, 95% CI: 0.62-0.87, p = 0.0003). In conclusion, this is the largest study to investigate the distinct molecular features between KMT2-mutant and KMT2-wild-type GCs to date. Our data indicate that GC patients with KMT2 mutations may benefit from ICIs and drugs targeting DDR, MAPK/PI3K, metabolism, and cell cycle pathways., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The molecular heterogeneity of KRAS is well established, with a pool of variants comprising >75% of all known mutations; this pool includes mutations in classic codons 12, 13, and 61, as well as 146 and 117. In addition, there are rare variants that are more frequently encountered clinically due to the advances in next-generation sequencing and more widespread implementation of All- RAS sequencing over the past five years. We have previously identified a missense variant of KRAS , A59T, in a patient with CRC that was associated with a response to an epidermal growth factor inhibitor when added to chemotherapy, supporting the hypothesis that distinct biochemical impacts of different KRAS mutations may produce varied responses to targeted therapy. In this study, we explored a large genomic database comprising 17,909 cases of CRC to determine the prevalence of the A59T mutation and characterized the concurrent genomic alterations associated with this variant in more detail, particularly in relation to the expanding set of potential predictive immuno-oncologic biomarkers. We identified 14 cases of A59 mutations in this dataset (0.08% prevalence). We evaluated the prevalence of high tumor mutation burden (TMB), positive PD-L1 expression, and microsatellite instability-high/mismatch repair-deficiency (MSI-H/dMMR) using both next generation sequencing (NGS) and immunohistochemistry (IHC). The genomic features of pertinent signaling pathways were also described, including RAS pathway, chromatin remodeling, DDR, hedgehog signaling, PI3K, receptor tyrosine kinases, signal transduction, TGF-beta, TP53, and WNT. We uncovered a high level of association of predictive markers of responsiveness to checkpoint inhibition and potentially other forms of immunotherapy, with nearly half of all cases harboring microsatellite instability as assessed using NGS. A59T was also detected in 11 additional cancer types, most prominently in cases of gynecologic or other gastrointestinal sites of origin. This study provides supportive evidence that A59T, and possibly other similarly rare KRAS variants, co-occur with predictive biomarkers of response to immunotherapy.

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, has been implicated in the pathogenesis of many cancers, with involvement in cell-cycle regulation, apoptosis, and immune response. Small-molecule GSK-3β inhibitors are currently undergoing clinical investigation. Tumor sequencing has revealed genomic alterations in GSK-3β , yet an assessment of the genomic landscape in malignancies is lacking. This study assessed >100,000 tumors from two databases to analyze GSK-3β alterations. GSK-3β expression and immune cell infiltrate data were analyzed across cancer types, and programmed death-ligand 1 (PD-L1) expression was compared between GSK-3β -mutated and wild-type tumors. GSK-3β was mutated at a rate of 1%. The majority of mutated residues were in the kinase domain, with frequent mutations occurring in a GSK-3β substrate binding pocket. Uterine endometrioid carcinoma was the most commonly mutated (4%) tumor, and copy-number variations were most commonly observed in squamous histologies. Significant differences across cancer types for GSK-3β -mutated tumors were observed for B cells ( P = 0.018), monocytes ( P = 0.002), dendritic cells ( P = 0.005), neutrophils ( P = 0.0003), and endothelial cells ( P = 0.014). GSK-3β mRNA expression was highest in melanoma. The frequency of PD-L1 expression was higher among GSK-3β -mutated tumors compared with wild type in colorectal cancer ( P = 0.03), endometrial cancer ( P = 0.05), melanoma ( P = 0.02), ovarian carcinoma ( P = 0.0001), and uterine sarcoma ( P = 0.002). Overall, GSK-3β molecular alterations were detected in approximately 1% of solid tumors, tumors with GSK-3β mutations displayed a microenvironment with increased infiltration of B cells, and GSK-3β mutations were associated with increased PD-L1 expression in selected histologies. These results advance the understanding of GSK-3β complex signaling network interfacing with key pathways involved in carcinogenesis and immune response., (©2020 American Association for Cancer Research.)