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Introduction The hepatocyte growth factor receptor mesenchymal-epithelial transition (MET) is reported to be a negative prognostic marker in EGFR-mutant NSCLC and involved in resistance to EGFR inhibitors. Emibetuzumab, a humanized immunoglobulin G4 monoclonal bivalent MET antibody, blocks ligand-dependent and ligand-independent hepatocyte growth factor/MET signaling. This phase 2 study compared erlotinib with and without emibetuzumab in first-line treatment of EGFR-mutant metastatic NSCLC. Methods Patients with stage IV EGFR-mutant NSCLC and disease control after an 8-week lead-in with erlotinib (150 mg daily) were randomized to continue taking erlotinib with or without emibetuzumab (750 mg every 2 weeks). The primary end point was progression-free survival (PFS). Additional end points included overall survival, overall response rate, safety, pharmacokinetics, and exploratory analysis of MET expression. Results No significant difference in median PFS was observed in the intent-to-treat population (9.3 months with emibetuzumab + erlotinib versus 9.5 months with erlotinib monotherapy [hazard ratio (HR) = 0.89, 90% confidence interval (CI): 0.64–1.23]). The median overall survival was 34.3 months with emibetuzumab plus erlotinib versus 25.4 months with erlotinib (HR = 0.74, 90% CI: 0.49–1.11). Emibetuzumab plus erlotinib was well tolerated, with peripheral edema and mucositis as the only adverse events occurring 10% or more frequently relative to erlotinib. Exploratory post hoc analysis showed an improvement of 15.3 months in median PFS for the 24 patients with the highest MET expression (MET expression level of 3+ in ≥90% of tumor cells) (20.7 with emibetuzumab + erlotinib versus 5.4 months with erlotinib [HR = 0.39, 90% CI: 0.17–0.91]). Conclusions No statistically significant difference in PFS was noted in the intent-to-treat population. Exploratory analysis confirmed that high MET expression is a negative prognostic marker for patients treated with erlotinib, indicating that emibetuzumab plus erlotinib may provide clinically meaningful benefit.

Ectodomain shedding unleashes the aggressive nature of the MET oncogene product. Using specific C- and N-terminal MET antibodies (D1C2 and A2H2-3), MET protein status (i.e., no MET, decoy MET, transmembranous C-terminal MET with or without the ectodomain) was investigated in oral squamous cell carcinoma. For the cancers showing transmembranous C-terminal MET, the impact of ectodomain shedding on prognosis was investigated. To examine ectodomain shedding, reduced lysates of oral squamous cell carcinoma cell lines were immunoblotted using D1C2 and an ELISA was performed on culture media using A2H2-3. In addition, reduced lysates of fresh frozen tissues of 30 oral squamous cell carcinoma were immunoblotted using D1C2 and immunohistochemistry was performed on corresponding formalin-fixed paraffin-embedded tissues using both antibodies on parallel sections. To examine MET protein status, differences between membranous D1C2 and A2H2-3 immunoreactivities were scored using parallel tissue microarray sections representing 156 oral squamous cell carcinoma. The prognostic value of ectodomain shedding was examined using Cox regression analysis for disease-free survival and overall survival. Ectodomain shedding was observed in all cell lines, 43% (n = 13) of fresh frozen and 50% (n = 15) of formalin-fixed paraffin-embedded cancers (27% overlap, n = 8). The tissue microarray showed no MET in 23% (n = 36), decoy MET in 9% (n = 14), and transmembranous C-terminal MET in 68% (n = 106) of examined cancers. Within the latter group, ectodomain shedding occurs in 36% (n = 38) of the cases and is independently associated with poor disease-free survival (HR = 2.41; 95% CI, 1.35–4.30 and P = 0.003)—though not overall survival (HR = 1.64; 95% CI, 0.92–2.94 and P = 0.095)—after correcting for factors known to influence survival. In conclusion, MET ectodomain shedding occurs in transmembranous C-terminal MET positive oral squamous cell carcinoma and is independently associated with disease-free survival. These findings might aid in designing companion diagnostics for targeted therapies directed against MET.

Using tissue microarrays, it was shown that membranous C-terminal MET immunoreactivity and ectodomain (ECD) shedding are associated with poor prognosis in oral cancer. Seen the potential diagnostic value, extrapolation of these results to whole-tissue sections was investigated. Because MET orchestrates epithelial-to-mesenchymal transition (EMT), the results were benchmarked to loss of E-cadherin, a readout for EMT known to be associated with poor prognosis. C-terminal MET, N-terminal MET, and E-cadherin immunoreactivities were examined on formalin-fixed paraffin-embedded parallel sections of 203 oral cancers using antibody clones D1C2, A2H2-3, and NCH-38. Interantibody and intra-antibody relations were examined using a novel scoring system, nonparametric distribution, and median tests. Survival analyses were used to examine the prognostic value of the observed immunoreactivities. Assessment of the three clones revealed MET protein status (no, decoy, transmembranous C-terminal positive), ECD shedding, and EMT. For C-terminal MET-positive cancers, D1C2 immunoreactivity is independently associated with poor overall survival (hazard ratio [HR] = 2.40; 95% confidence interval [CI] = 1.25 to 4.61; and P = 0.008) and disease-free survival (HR = 1.83; 95% CI = 1.07-3.14; P = 0.027). For both survival measures, this is also the case for ECD shedding (43.4%, with HR = 2.30; 95% CI = 1.38 to 3.83; and P = 0.001 versus HR = 1.87; 95% CI = 1.19-2.92; P = 0.006) and loss of E-cadherin (55.3%, with HR = 2.21; 95% CI = 1.30 to 3.77; and P = 0.004 versus HR = 1.90; 95% CI = 1.20-3.01; P = 0.007). The developed scoring system accounts for MET protein status, ECD shedding, and EMT and is prognostically informative. These findings may contribute to development of companion diagnostics for MET-based targeted therapy.

Simple Summary Pancreatic cancer is one of the most lethal types of cancer, and immunotherapy treatment options for these patients are limited by the characteristically “cold” tumor microenvironment. In this work, we analyze the expression levels and prognostic value of stromal tumor-infiltrating lymphocyte (CD4, CD8, and CD20) and cancer-associated fibroblast (Thy-1, FAP, and SMA) subpopulations in a cohort of pancreatic ductal adenocarcinoma patients. We additionally characterize the expression and prognostic value of CD200, a potential target for immune checkpoint blockade in these patients. CD8 and FAP were found to have prognostic significance for progression-free survival and overall survival after multivariate analysis. CD200 expression was heterogeneous in tumor and stromal cells and did not demonstrate prognostic value in this cohort. Our results point to CD8 and FAP as potential prognostic biomarkers and demonstrate the heterogeneous expression pattern of CD200 in patients with pancreatic ductal adenocarcinoma. Abstract Pancreatic cancer is marked by a desmoplastic tumor microenvironment and low tumor immunogenicity, making it difficult for immunotherapy drugs to improve outcomes for patients. Tumor-infiltrating lymphocytes (TILs) and cancer-associated fibroblasts (CAFs) are seen in the tumor microenvironment of patients with pancreatic ductal adenocarcinoma (PDAC). In this work, we sought to characterize the expression levels and potential prognostic value of TILs (CD4, CD8, and CD20) and CAFs (Thy-1, FAP, and SMA) in a large retrospective cohort of PDAC patients. Additionally, we investigated the expression levels and prognostic significance of CD200, an immunoinhibitory protein that has shown interest as a potential target for immune checkpoint blockade. We measured the expression levels of these seven proteins with multiplexed immunofluorescence staining and quantitative immunofluorescence (QIF). We found CD8 and FAP to be independent predictors of progression-free survival and overall survival. CD200 was found to be heterogeneously expressed in both the tumor and stromal compartments of PDAC, with the majority of patients having positive stromal expression and negative tumor expression. This work demonstrates the potential clinical utility of CD8 and FAP in PDAC patients, and it sheds light on the expression patterns of CD200 in pancreatic cancer as the protein is being tested as a target for immune checkpoint blockade.

Highlights • PD-L1 expression was assessed for heterogeneity in 107 NSCLC patients. • Intra-tumoural heterogeneity was observed in 78% of cases. • Inter-tumoural heterogeneity was observed in 53% of cases. • 23% of cases had clinically relevant changes between primary and secondary tumours. • Sample site selection is an important consideration for testing PD-L1., Objectives PD-L1 expression on tumour cells can guide the use of anti-PD-1/PD-L1 immune modulators to treat patients with non-small cell lung cancer (NSCLC). Heterogeneity of PD-L1 expression both within and between tumour sites is a well-documented phenomenon that compromises its predictive power. Our aim was to better characterise the pattern and extent of PD-L1 heterogeneity with a view to optimising tumour sampling and improve its accuracy as a biomarker. Materials and methods Expression of PD-L1 was assessed by immunochemistry using the SP263 clone in 107 resected primary NSCLCs and their nodal metastases. Intra-tumoural heterogeneity, defined as ‘small-scale’ (mm²), ‘medium-scale’ (cm²) and ‘large-scale’ (between tumour blocks), was assessed by digital imaging using a novel ‘squares method’. Inter-tumoural heterogeneity between the primary tumours and their nodal metastases and between N1 and N2 nodal stages was also assessed. Results The majority of tumours demonstrated intra-tumoural heterogeneity (small-scale 78%, medium-scale 50%, large-scale 46%). Inter-tumoural heterogeneity between the primary and nodal metastases was present in 53% of cases and, in 17%, between N1 and N2 disease. These differences were occasionally sufficient to lead to discrepancy across the ≥1%, ≥25% and ≥50% cut-offs used to guide therapy. Conclusion Heterogeneity of PD-L1 expression is common, variable in scale and extent, and carries significant implications for its accuracy as a predictive biomarker. Extensive sampling reduces, but cannot eliminate, this inaccuracy.

CD200/CD200R is an immune checkpoint with broad expression patterns and a potential target for immune therapy. In this study, we assess both CD200 and CD200R expression in solid tumors, with a focus on lung cancer, and evaluate their association with clinicopathologic characteristics, mutation status, outcome, and programmed death-ligand 1 (PD-L1) expression. We used multiplexed quantitative immunofluorescence (QIF) to measure the expression of CD200 and CD200R in a total of 455 patients from three lung cancer cohorts. Using carefully validated antibodies, we performed target measurement with tyramide-based QIF panels and analyzed the data using the PM2000 microscope and AQUA software. CD200 tumor positivity was found in 29.7% of non-small cell lung cancer (NSCLC) patients and 33.3% of lung large cell neuroendocrine carcinoma (LCNEC) patients. CD200 demonstrated notable intratumoral heterogeneity. CD200R was expressed in immune cells in 25% of NSCLC and 41.3% of LCNEC patients. While CD200R is predominantly expressed in immune cells, rare tumor cell staining was seen in a highly heterogeneous pattern. CD200R expression in the stromal compartment was significantly higher in patients with squamous differentiation (p &lt, 0.0001). Neither CD200 nor CD200R were associated with other clinicopathologic characteristics or mutation status. Both biomarkers were not prognostic for disease-free or overall survival in NSCLC. CD200 showed moderate correlation with PD-L1. CD200/CD200R pathway is frequently expressed in lung cancer patients. Differential expression patterns of CD200 and CD200R with PD-L1 suggest a potential role for targeting this pathway alone in patients with NSCLC.

Background With increased understanding of the unique entities, subtype-specific approaches for peripheral T-cell lymphoma (PTCL) are emerging, and more precise diagnoses are becoming increasingly important. Patients and Methods We analyzed the approach to the histopathologic diagnosis of PTCL using data from the comprehensive oncology measures of peripheral T-cell lymphoma (COMPLETE) study. The COMPLETE trial is a large prospective cohort study of patients with newly diagnosed PTCL in the United States. Results A total of 499 patients were enrolled from 40 academic and 15 community-based centers. Baseline assessment forms were collected for 493 patients, of which 435 (88%) were available for analysis. The most common diagnoses were PTCL, not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma (AITL). A mean of 10 markers (range, 0-21) was assessed per patient. CD30 was assessed frequently but not uniformly in cases that were not anaplastic large cell lymphoma. Only 17% of PTCL-NOS cases were assessed for PD1. CXCL13 was a relatively sensitive marker in AITL, expressed in 84% of tested cases; however, only 3% of PTCL-NOS cases were tested. T follicular helper cell marker assessment differed between academic and community practices, with PD1 more often evaluated by academic centers in cases of AITL (62% vs. 12%; P = .01). Conclusion The diagnostic workup for PTCL in the United States varies widely and often lacks important phenotypic information to fully characterize the lymphoma. Gaps in testing of selected markers should be filled, given the impending revision to the World Health Organization classification. The accuracy of diagnosis will become increasingly important as we enter the era of targeted treatment for PTCL.

Background: CD200 is a membrane-bound glycoprotein expressed on normal tissue, tumor and immune cells. The interaction of CD200 with its receptor leads to attenuation of the inflammatory process, resulting in tumor cell mediated immune suppression. CD200 has been characterized in hematologic malignancies and consists of an independent negative prognostic factor for chronic lymphocytic leukemia, acute myeloid leukemia and multiple myeloma patients. Here we assess both CD200 and CD200R expression in lung cancer and evaluate its association with clinicopathologic characteristics, mutation status, outcome and PD-L1 expression. Methods: We used quantitative multiplexed immunofluorescence (QIF) to measure the expression of CD200 and CD200R in 287 non-small cell lung cancer (NSCLC) patients and 30 patients with large cell neuroendocrine carcinomas (LCNEC) of the lung. We performed target measurement with tyramide-based QIF panels and analyzed the data using the PM2000 microscope and AQUA software. Targets were measured in cytokeratin-positive (CK+) tumor compartment and cytokeratin-negative (CK-) stromal compartment. Results: CD200 tumor positivity was found in 29.7% of NSCLC patients and 33.3% of LCNEC patients. CD200 demonstrated notable intratumoral heterogeneity (R2=0.09-0.31 between different TMA blocks versus 0.46-0.60 for CD200R). CD200R was expressed in immune cells in 25% of NSCLC and 41.3% of LCNEC patients. While CD200R is predominantly expressed in immune cells, rare tumor cell staining was seen in a highly heterogeneous pattern. CD200R expression in the stromal compartment was significantly higher in patients with squamous differentiation (p Conclusions: CD200 and CD200R pathway represents a novel immune checkpoint, frequently expressed in lung cancer patients. Co-expression patterns of CD200 and CD200R with PD-L1 suggest a potential role for targeting this pathway alone or in combination with PD-1/PD-L1 axis inhibitors. Citation Format: Ioannis A. Vathiotis, Tyler MacNeil, Jon Zugazagoitia, Sandra Martinez-Morilla, Fahad Ahmed, Konstantinos N. Syrigos, Aaron M. Gruver, Kyla Driscoll, David L. Rimm. Quantitative assessment of CD200 and CD200R expression in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 937.

Background and objectives Although MET amplification/overexpression was observed in a subset of gastric cancer (GC) patients, the relationship between MET amplification/overexpression in primary GC and liver metastasis was unclear. Methods GC samples and matched liver metastases (N = 47) were analyzed by fluorescence/silver in-situ hybridization (FISH/SISH) and by immunohistochemistry for MET amplification and MET expression, respectively. MET-copy number (CN) and Met expression data from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD, N = 356) were also analyzed. Results Significant overlap existed between MET amplification and Met expression in both primary stomach tumors (P = 0.013) and liver metastasis (P = 0.001). In TCGA-STAD, MET-CN (≥4 copies) and MET expression were also positively correlated (r = 0.761; P = 0.017). Comparative analysis revealed a strong association between MET expression and MET amplification (85% concurrence) in primary stomach tumors and matched liver metastasis. MET status in synchronous liver metastasis (N = 36) was correlated with primary stomach tumors. However, a significant correlation between primary tumors and liver metastases was not observed in patients with metachronous liver metastasis. Survival analyses revealed that both MET amplification and MET overexpression were prognostic of poor outcomes. Conclusions MET amplification and Met overexpression were positively correlated in GC. MET status should be re-evaluated in GC patients with liver metastasis, especially for metachronous metastasis.

// Bryce P Portier 1 , Eugen C Minca 2 , Zhen Wang 2 , Christopher Lanigan 2 , Aaron M Gruver 2 , Erinn Downs-Kelly 2 , G Thomas Budd 3 , and Raymond R Tubbs 2 1 Department of Pathology and Genomic Medicine, Houston Methodist, Houston, TX 2 Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH 3 Department of Solid Tumor Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH Correspondence: Bryce P. Portier, email: // Keywords : Breast carcinoma, HER2, HER4, Immunohistochemistry, RT-qPCR, Trastuzumab, H-Score Received : July 31, 2013 Accepted : August 24, 2013 Published : August 26, 2013 Abstract Prognostic and predictive markers utilized in invasive breast carcinoma are limited and include ER, PR, Ki67, and ERBB2 (HER2). In the case of HER2, over-expression or amplification serves as eligibility for anti-HER2 based therapy, including trastuzumab (Herceptin®, Genentech). While clinical trials have shown trastuzumab improves overall survival and time to progression, an individual’s response to anti-HER2 based therapy is highly variable. This suggests that, in a “uniform” HER2 positive population, additional markers could help in predicting patient outcome to therapy. Here we utilized a recently validated high-specificity HER4 antibody (E200) and generated a standard clinical HER4 scoring algorithm (HER4 H-Score) utilizing two breast carcinoma cohorts: 1) patients receiving neoadjuvant trastuzumab (n=47) and 2) patients receiving trastuzumab for metastatic disease (n=33). Our HER4 H-Score showed significant correlation with high sensitivity RT-qPCR performed on matched patients (p=

Background: Breast carcinomas that demonstrate a heterogeneous ERBB2 (HER2) status or equivocal results by both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) present diagnostic challenges for which there is neither a standard methodology to achieve resolution in the clinical laboratory nor a uniform approach to management. We assessed the feasibility of using a novel automated and quantitative HER2 mRNA bright field in situ hybridization (ISH) assay capable of single molecule detection to determine HER2 status in invasive breast carcinomas that demonstrated significant tumor heterogeneity or failed to be resolved by standard IHC and FISH algorithmic testing. Design: Formalin-fixed, paraffin-embedded (FFPE) breast carcinomas from a non-consecutive series of 163 patients were analyzed for HER2 mRNA using a fully automated bright field RNA ISH assay (RNAscope, Advanced Cell Diagnostics, Hayward, CA). Cases were assigned into either a training set (n = 34) or a validation set (n = 129) and analyzed by both Q-RT-PCR and RNAscope. automated image analysis was used to numerate the punctate signal dots per cell in RNAscope-stained slides. A HER2 mRNA score based on single-cell quantification by RNAscope was developed and correlated to HER2 FISH and HER2 mRNA Q-RT-PCR results. A simple cutoff value was derived using the training set and applied to the validation set. Results: Evaluable HER2 results were obtained for 154 cases (94.5%) by RNAscope and 163 cases (100%) by Q-RT-PCR. In the training set, both FISH/IHC positive and negative cases were definitively separated by both Q-RT-PCR and RNAscope. HER2 mRNA dots per cell correlated strongly to FISH (Spearman r=0.77) and Q-RT-PCR (r = 0.81). Application of both methods to the validation set resulted in correct identification of 31/31 positive cases and 41/43 negative (overall concordance=97.3%) for both RNAscope and Q-RT-PCR. RNAscope showed a significant advantage over Q-RT-PCR in correctly identifying cases equivocal by FISH that were resolved by reflex IHC testing. RNAscope classified 7 of 26 (26.9%) FISH/IHC double equivocal cases as positives. In cases with HER2 protein heterogeneity, RNAscope showed a 100% concordance with FISH results, whereas Q-RT-PCR showed a 42.9% concordance. Conclusion: RNAscope analysis of HER2 mRNA is an effective means to resolve HER2 status in double equivocal cases and cases that demonstrate heterogeneity. Automation and image analysis-based quantification minimize analytical and post-analytical variability. Quantification of single RNA transcripts in situ at single-cell level demonstrates superiority over qRTPCR and great potential in predictive biomarker analysis. Further studies of larger cohorts correlating clinical response with HER2 mRNA expression in situ are warranted. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-04.

9019 Background: MET expression is a mechanism of resistance to EGFR inhibition in EGFRmt NSCLC and correlated with poor prognosis. Emi (LY2875358) is a humanized IgG4 monoclonal bivalent MET antibody that blocks ligand dependent and independent HGF/MET signaling. This Phase 2 study compared the clinical activity of Emi + E versus single agent E in 1st line EGFRmt metastatic NSCLC. Methods: Stage IV, EGFRmt NSCLC pts with disease control following an 8-week lead-in E (150 mg PO QD) treatment were randomized 1:1 to receive Emi (750 mg IV Q2W) + E or E alone. Pts were stratified by ECOG PS, ethnicity, MET expression status, and response at the end of the lead-in. The primary endpoint was PFS from randomization. Additional endpoints included safety, OS, PK, and exploratory analysis of MET-expressing populations. Results: Out of181 pts enrolled, 141 pts were randomized (Emi+E: 71; E: 70). In the ITT population, median PFS for EMI+E was 9.3 months (m) compared with 9.5 m for E (HR = 0.89: 90% CI 0.64-1.23; p = 0.534). Exploratory analysis of MET-high expressing pts (MET 3+ expression in ≥90% of tumor cells; n = 24 pts) showed a 15.3 m improvement in PFS (EMI+E: 20.7 m; E: 5.4 m [HR: 0.39; 90% CI: 0.17-0.91]). No difference in PFS was observed in the complementary population (HR: 1.1 [90% CI: 0.7-1.7]). Similar frequencies of related AEs were reported for both treatment arms. Drug-related TEAEs that were more frequent ( > 10%) for Emi+E were peripheral edema and fatigue (all grade 1 or 2). Emi serum concentrations were consistent with previously obtained PK results, and no apparent exposure-response was observed. Median OS in the ITT population was not achieved (NA) for either arm. In MET-high expressing pts, median OS was 20.6 m for E (90% CI: 8.87, NA) whereas it was not achieved for Emi+E (90% CI: NA, NA). Conclusions: No statistically significant difference in PFS was noted in the ITT population.Exploratory analysis confirmed that high MET expression is a negative prognostic marker for pts treated with E and indicated that these pts may receive clinically meaningful benefit from Emi+E. Clinical trial information: NCT01897480.

Homologous recombination is a double-strand break repair pathway required for resistance to DNA damage and maintaining genomic integrity. In mitotically dividing vertebrate cells, the primary proteins involved in homologous recombination repair are RAD51 and the five RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. In the absence of Rad51d, human and mouse cells fail to proliferate, and mice defective for Rad51d die before birth, likely as a result of genomic instability and p53 activation. Here, we report that a p53 deletion is sufficient to extend the life span of Rad51d-deficient embryos by up to 6 days and rescue the cell lethal phenotype. The Rad51d−/− Trp53−/− mouse embryo–derived fibroblasts were sensitive to DNA-damaging agents, particularly interstrand cross-links, and exhibited extensive chromosome instability including aneuploidy, chromosome fragments, deletions, and complex rearrangements. Additionally, loss of Rad51d resulted in increased centrosome fragmentation and reduced levels of radiation-induced RAD51-focus formation. Spontaneous frequencies of sister chromatid exchange were not affected by the absence of Rad51d, but sister chromatid exchange frequencies did fail to be induced upon challenge with the DNA cross-linking agent mitomycin C. These findings support a crucial role for mammalian RAD51D in normal development, recombination, and maintaining mammalian genome stability.

Cholangiocarcinoma (CCA) originates from biliary tract epithelium and can be classified anatomically into intrahepatic or extrahepatic CCA. Although a relatively rare disease in the United States and Europe, the incidence of intrahepatic CCA is on the rise with unknown causes. CCA incidence is higher in Asia and the etiology is associated with infections such as liver fluke and hepatitis B/C. Prognosis at diagnosis is poor with median survival time of < 1 year, and only 10-20% of patients are eligible for tumor resection at diagnosis. This study examined the prevalence of MET over-expression, activating single point mutations of KRAS and IDH1/2, and ROS1 gene fusions in ∼100 intrahepatic and extrahepatic CCA FFPE tumor tissues obtained from non-Asian (n=40) and Asian (n=60) patients. Immunohistochemistry performed with an anti-MET-specific antibody (A2) demonstrated that MET is expressed in the majority of all intrahepatic CCA samples analyzed, with ∼50% of samples reported with membranocytoplasmic scores of 2+ (moderate intensity) or 3+ (strong intensity) on a 0-3+ scale. Interestingly, most non-malignant bile ducts and much vascular endothelia also stained lightly positive for MET. To determine mutation frequencies of KRAS and IDH1/2, competitive allele-specific Taqman® PCR (castPCR) was performed on DNA extracted from FFPE CCA tissue (limit of mutation detection 0.1%). Fourteen KRAS activating mutations (G12S, G12R, G12A, G13C, G13S, G13R, G12D, G13D, G12V, G12C, Q61R, Q61L, Q61H (c.183A>C and c.183A>T for Q61H), 5 IDH1 mutations (R132G, R132S, R132H, R132C, R132L), and 5 IDH2 mutations (R172G, R172M, R172K, R172W, R172S) were analyzed. Overall, 25% of analyzed samples were positive for KRAS mutation, and G12D was the predominant mutation (∼60%). One-third of Asian samples were positive for KRAS mutation, whereas less than one-fifth of non-Asian samples contained KRAS mutations. For IDH1, the frequency of mutation was less than 10% overall, and the majority of patients with IDH1 mutations were non-Asian. The R132C mutation was the predominant IDH1 mutation, and all tissues that were positive for IDH1 mutations were of intrahepatic origin. Interestingly, 2 out of the 7 samples positive for IDH1 mutations (R132C) were also positive for G12D KRAS mutation. There is no trend of MET expression correlating with either KRAS or IDH1 mutations. IDH2 analyses by castPCR and FISH studies examining ROS1 gene fusion are ongoing. Based on these data, inhibitors of receptor tyrosine kinases and their signaling pathways such as MET and ROS1 may merit clinical evaluation in CCA patients. LY2801653, a MET inhibitor which also has inhibitory activity against ROS1 and MKNK1/2 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). Citation Format: Megan N. Thobe, S. Betty Yan, Kelly M. Credille, Aejaz Nasir, Jessica A. Roseberry Baker, Mary Lajiness, Nathan A. Brooks, Darryl W. Ballard, Donna M. Farley, Victoria L. Peek, Suzane L. Um, G. Jason Jin, Raymond Gilmour, Christoph Reinhard, Jeremy R. Graff, Andrew E. Schade, Aaron M. Gruver, Bruce Colligan, Larry Douglass, Julia Carter, Richard A. Walgren. Prevalence of MET expression, activating mutations of KRAS and IDH1/2, and ROS1 fusions in cholangiocarcinoma patient tumor samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2013-2339