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International audience; Biliary tract carcinomas are divided into intrahepatic, perihilar, distal extrahepatic cholangiocarcinomas, and gallbladder adenocarcinomas. Therapies targeting ROS1, ALK, MET, and HER2 alterations are currently evaluated in clinical trials. We assessed ROS1 and ALK translocations/amplifications as well as MET and HER2 amplifications for each tumor subtype by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) in 73 intrahepatic, 40 perihilar bile duct, 36 distal extrahepatic cholangiocarcinomas, and 45 gallbladder adenocarcinomas (n = 194). By FISH, we detected targetable alterations in 5.2% of cases (n = 10): HER2 and MET amplifications were found in 4.1% (n = 8) and 1.0% (n = 2), respectively. The HER2-amplified cases were mostly gallbladder adenocarcinomas (n = 5). The MET- and HER2-amplified cases were all positive by IHC. Fourteen cases without MET amplification were positive by IHC, whereas HER2 over-expression was detected by IHC only in HER2-amplified cases. We detected no ALK or ROS1 translocation or amplification. Several alterations were consistent with aneuploidy: 24 cases showed only one copy of ROS1 gene, 4 cases displayed a profile of chromosomal instability, and an over-representation of centromeric alpha-satellite sequences was found in five cases. We confirm a relatively high rate of HER2 amplifications in gallbladder adenocarcinomas and the efficacy of IHC to screen these cases. Our results also suggest the value of IHC to screen MET amplification. Contrary to initial publications, ROS1 rearrangements seem to be very rare in biliary tract adenocarcinomas. We confirm a relatively high frequency of aneuploidy and chromosomal instability and reveal the over-representation of centromeric alpha-satellite sequences in intrahepatic cholangiocarcinomas.

Aims Clusterin (CLU) is a sulphated glycoprotein implicated in many physiological and pathological processes, including tumorigenesis. We have previously demonstrated that CLU is highly expressed in pancreatic neuroendocrine tumours (NETs). The aims of this study were: to investigate CLU expression in gastrointestinal NETs; the potential correlation between this expression and different clinicopathological parameters; and its usefulness in the differential diagnosis of liver metastases. Methods and results Immunohistochemistry using an anti-CLU antibody was performed on paraffin sections from 108 primary NETs [G3 (13 cases), G2 (18 cases), and G1 (77 cases), according to the 2010 WHO classification] and 60 metastases. Cytoplasmic positivity was scored qualitatively and quantitatively. The pattern of staining was also assessed. Two-step statistical analyses (univariate and multivariate logistic regression) were performed. More than 90% of small-intestine NETs were completely negative. The probability of obtaining a positive CLU score was higher for the appendix, the stomach, the duodenum and the rectum than for the small intestine and colon. All G3 NETs and most G2 NETs were negative as compared with G1. CLU expression in the metastatic foci was identical to that of the primary tumour. Conclusions Clusterin expression in gastrointestinal NETs is highly correlated with location and probably also with grading, in both the primary tumour and metastases. Underexpression of CLU in small-intestine NETs is helpful for identifying the origin of liver metastases: a strong CLU score in a liver biopsy makes the small intestine highly unlikely as a primary site.

Background HER2 is overexpressed in 10 to 20% of gastro-esophageal adenocarcinoma (GE-ADK), and is a target for trastuzumab in metastatic patients. We conducted a study to compare HER2 expression between diagnostic biopsies (DBs) and surgical specimens (SSs) of GE-ADK, and to determine the influence of non-trastuzumab containing neoadjuvant chemotherapy (NAC) on this expression. Patients and methods Pathological specimens from biopsies of 228 patients operated on between 2004 and 2011 were collected. Two cohorts treated (n = 141) or not (n = 87) with a NAC were constituted. Two blind independent pathological HER2 analyses on DB and on SS were carried out using immunohistochemistry (IHC) and colorimetric in situ hybridization (CISH). HER-2 overexpression (HER2+) was defined by a score 3+ in IHC, or 2+ with a positive CISH test, according to the specific HER2 scoring guidelines for GE-ADK. Results Paired HER2 status could be determined for 218 out of the 228 patients (95.6%). HER2+ rates were 13.3% on DB (29/218) and 14.7% on SS (32/218). HER2+ tumors were mainly cardial or esophageal adenocarcinomas, with a well-differentiated, intestinal histological type. HER2 status differed between DB and SS in 6% of cases. When DB analyses were added to SS analyses, the relative increase in HER2+ cases was 13.5% (17.1% for patients with NAC and 23.5% for patients with histological response to NAC, versus 7.1% for patients without NAC, P = 0.4, NS). Differences between DB and SS HER2 expression could be explained by intratumoral heterogeneity and by a HER2 expression decrease in SS after NAC in responding patients possibly due to a higher chemosensitivity of HER2-positive clones. Conclusion The determination of HER2 status on DB provides results that complete those obtained with SS. Combining the analysis of DB and of SS enables to optimize the selection of trastuzumab-eligible patients in case of metastatic relapse, and particularly in previously NAC-responding patients.

18 Background: HER-2 is overexpressed in 15 to 20% of gastric and esophageal adenocarcinoma (ADK), and is a target for trastuzumab in metastatic patients (Bang YJ et al, Lancet 2010). The main objective of this study was to compare HER-2 expression between diagnostic biopsies and surgical specimens of gastric ADK, and to determine the influence of non-trastuzumab containing neoadjuvant chemotherapy (NAC) on this expression. Methods: Pathological specimens and biopsies of patients operated on with a curative aim in two French hospitals (Institut Mutualiste Montsouris and Saint-Antoine hospital) between 2004 and 2011 were collected; two cohorts treated or not with a NAC were constituted. Blind HER-2 analyses on biopsies and on surgical specimens were performed using IHC and ISH methods, with two independent pathological readings. HER-2 overexpression was defined by a score 3+ in IHC, or by a score 2+ in IHC with a positive ISH test. Results: Paired samples of 201 patients wereanalyzed. HER-2 overexpression rates were 16.4% on biopsies and 19.4% on surgical specimens. HER-2 positive tumors were mainly cardial or esophageal tumors, with a well-differentiated, intestinal histological type. Surgical specimens seemed to allow better identification of HER-2 positive tumors than diagnostic biopsies for patients that had not received NAC (13.5% of HER-2 positivity on biopsies versus 24.3% on surgical specimens), whereas this trend was reversed for patients with NAC (18.1% of HER-2 positivity on biopsies versus 16.5% on surgical specimens). HER-2 status differed between biopsies and surgical specimens in 10% of patients. Conclusions: The determination of HER-2 status on biopsies provides results that completed those obtained with the analysis of surgical specimens. Analyzed only on surgical specimens, HER-2 status is underestimated in particular after NAC. This study suggests the interest of repeating HER-2 evaluations to optimize the identification of patients that can benefit from trastuzumab treatment.