Purpose: KRAS mutation (MT) is a major oncogenic driver in pancreatic ductal adenocarcinoma (PDAC). A small subset of PDACs harbor KRAS wild-type (WT). We aim to characterize the molecular profiles of KRAS WT PDAC to uncover new pathogenic drivers and offer targeted treatments. Experimental Design: Tumor tissue obtained from surgical or biopsy material was subjected to next-generation DNA/RNA sequencing, microsatellite instability (MSI) and mismatch repair status determination. Results: Of the 2,483 patients (male 53.7%, median age 66 years) studied, 266 tumors (10.7%) were KRAS WT. The most frequently mutated gene in KRAS WT PDAC was TP53 (44.5%), followed by BRAF (13.0%). Multiple mutations within the DNA-damage repair (BRCA2, ATM, BAP1, RAD50, FANCE, PALB2), chromatin remodeling (ARID1A, PBRM1, ARID2, KMT2D, KMT2C, SMARCA4, SETD2), and cell-cycle control pathways (CDKN2A, CCND1, CCNE1) were detected frequently. There was no statistically significant difference in PD-L1 expression between KRAS WT (15.8%) and MT (17%) tumors. However, KRAS WT PDAC were more likely to be MSI-high (4.7% vs. 0.7%; P < 0.05), tumor mutational burden–high (4.5% vs. 1%; P < 0.05), and exhibit increased infiltration of CD8+ T cells, natural killer cells, and myeloid dendritic cells. KRAS WT PDACs exhibited gene fusions of BRAF (6.6%), FGFR2 (5.2%), ALK (2.6%), RET (1.3%), and NRG1 (1.3%), as well as amplification of FGF3 (3%), ERBB2 (2.2%), FGFR3 (1.8%), NTRK (1.8%), and MET (1.3%). Real-world evidence reveals a survival advantage of KRAS WT patients in overall cohorts as well as in patients treated with gemcitabine/nab-paclitaxel or 5-FU/oxaliplatin. Conclusions: KRAS WT PDAC represents 10.7% of PDAC and is enriched with targetable alterations, including immuno-oncologic markers. Identification of KRAS WT patients in clinical practice may expand therapeutic options in a clinically meaningful manner.

Background: Biliary tract cancers (BTCs) are a heterogeneous group of aggressive, rare malignancies with limited standard chemotherapeutic options for advanced disease. Recent studies have demonstrated potential novel biliary cancer targets and a possible role for immunotherapy in the treatment of patients with this disease. Intrahepatic cholangiocarcinoma (IHCC), extrahepatic cholangiocarcinoma (EHCC), and gallbladder carcinoma (GBC) are frequently grouped together in clinical trials despite differences in tumor biology. Methods: To further investigate tumor biology differences, we profiled 1,502 BTCs using next-generation sequencing (NGS), immunohistochemistry, in situ hybridization, and RNA sequencing. Results: IHCCs had higher rates of IDH1, BAP1, and PBRM1 mutations and FGFR2 fusions; EHCCs had higher rates of KRAS, CDKN2A, and BRCA1 mutations; and GBCs had higher rates of homologous recombination repair deficiency and Her2/neu overexpression and amplification. IHCCs and GBCs had higher rates of potential positive predictive biomarkers for immune checkpoint inhibition (PD-L1 expression, high microsatellite instability, and high tumor mutational burden) than EHCCs. Conclusions: These findings support clinical molecular profiling of BTCs to inform potential therapeutic selection and clinical trial design based on the primary tumor’s site of origin within the biliary tree.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, debilitating respiratory disease whose pathogenesis is poorly understood. In IPF, the lung parenchyma undergoes extensive remodeling. We hypothesized that lymphangiogenesis is part of lung remodeling and sought to characterize pathways leading to lymphangiogenesis in IPF. We found that the diameter of lymphatic vessels in alveolar spaces in IPF lung tissue correlated with disease severity, suggesting that the alveolar microenvironment plays a role in the lymphangiogenic process. In bronchoalveolar lavage fluid (BALF) from subjects with IPF, we found short-fragment hyaluronic acid, which induced migration and proliferation of lymphatic endothelial cells (LECs), processes required for lymphatic vessel formation. To determine the origin of LECs in IPF, we isolated macrophages from the alveolar spaces; CD11b ⁺ macrophages from subjects with IPF, but not those from healthy volunteers, formed lymphatic-like vessels in vitro. Our findings demonstrate that in the alveolar microenvironment of IPF, soluble factors such as short-fragment hyaluronic acid and cells such as CD11b ⁺ macrophages contribute to lymphangiogenesis. These results improve our understanding of lymphangiogenesis and tissue remodeling in IPF and perhaps other fibrotic diseases as well.

Purpose: Transcriptional profiling of pancreatic cancers has defined two main transcriptional subtypes: classical and basal. Initial data suggest shorter survival for patients with basal tumors and differing treatment sensitivity to FOLFIRINOX and gemcitabine plus nab-paclitaxel by transcriptional subtype. Experimental Design: We examined 8,743 patients with RNA sequencing from pancreatic cancers performed at Caris Life Sciences. Classical and basal subtypes were identified using purity independent subtyping algorithm on RNA sequencing, and two cohorts were analyzed: (i) the biomarker cohort included patients with complete molecular profiling data (n = 7,250) and (ii) the outcome cohort included patients with metastatic disease with available survival outcomes (n = 5,335). A total of 3,842 patients were shared between the two cohorts. Kaplan–Meier curves and Cox proportional hazards regression were used to assess patient survival. Results: In the biomarker cohort, 3,063 tumors (42.2%) were strongly classical (SC) and 2,015 tumors (27.8%) were strongly basal (SB). SC and SB tumors showed strong associations with histologic phenotypes and biopsy sites. SB tumors had higher rates of KRAS, TP53, and ARID1A mutations, lower rates of SMAD4 mutation, and transcriptional evidence of epithelial-mesenchymal transition. Sixty of 77 cases (78%) maintained their transcriptional subtype between temporally and/or spatially disparate lesions. In the outcome cohort, the SB subtype was associated with shorter overall survival time, regardless of whether they received FOLFIRINOX or gemcitabine plus nab-paclitaxel as first-line chemotherapy. The mutant KRAS allele type was prognostic of outcomes; however, this impact was restricted to SC tumors, whereas all mutant KRAS alleles had similarly poor outcomes in SB tumors. Conclusions: The SB subtype is a strong independent predictor of worse outcomes, regardless of the up-front chemotherapy regimen used. Clinical trials should further investigate pancreatic cancer transcriptional subtypes as a prognostic and predictive biomarker.

Purpose: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist.Experimental Design: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations.Results: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24–6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts.Conclusions: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.