Emily G. Barr Fritcher

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Long-term outcomes of positive fluorescence in situ hybridization tests in primary sclerosing cholangitis

Patients with primary sclerosing cholangitis (PSC) are at increased risk for developing cholangiocarcinoma (CCA). Fluorescence in situ hybridization (FISH) is a cytological test designed to enhance early CCA diagnosis. The long‐term outcome of PSC patients with a positive FISH test (polysomy, trisomy/tetrasomy) are unclear. All PSC patients with at least one FISH test were identified and defined to have CCA if they had a positive tissue biopsy, positive cytology, or evidence of cancer in the explant after liver transplantation. A total of 235 PSC patients had at least one FISH test performed, and 56 patients had CCA on histopathology (n = 35) or cytology (n = 21). Overall, 120 of 235 (51%) of PSC patients tested for FISH were positive, but only one third of these positive patients had CCA. Sensitivity and specificity for FISH polysomy were 46% and 88%, and for trisomy/tetrasomy they were 25% and 67%, respectively. Survival analysis showed that patients with FISH polysomy had an outcome similar to patients with CCA; whereas FISH trisomy/tetrasomy patients had an outcome similar to patients with negative FISH tests. The FISH polysomy patients without cancer compared with those with CCA had lower serum bilirubin, lower carbohydrate antigen 19‐9 (CA 19‐9), lower Mayo risk score, and lower occurrence of dominant strictures. Conclusion: In PSC patients, the presence of a dominant stricture plus FISH polysomy has a specificity of 88% for CCA. Patients with FISH showing trisomy or tetrasomy have a similar outcome to patients with negative FISH. FISH testing should be used selectively in patients with other signs indicating CCA and not as a screening tool in all PSC patients undergoing endoscopic retrograde cholangiopancreatography (ERCP). (HEPATOLOGY 2009.)

A Multivariable Model Using Advanced Cytologic Methods for the Evaluation of Indeterminate Pancreatobiliary Strictures

BACKGROUND & AIMS Ancillary cytologic tests including digital image analysis (DIA) and fluorescence in situ hybridization (FISH) have been developed to improve the sensitivity of routine cytology (RC) for the diagnosis of malignancy in pancreatobiliary strictures. The goal of this study was to retrospectively compare the performance of RC, DIA, and FISH on clinical brushing specimens. METHODS Endoscopic retrograde cholangiopancreatography brushings were obtained from 498 consecutive patients with pancreatobiliary strictures and analyzed by RC, DIA, and FISH as per standard practice. RC diagnostic categories included negative, atypical, suspicious, or positive. Aneuploid/tetraploid histograms were considered positive for DIA. FISH was performed using UroVysion (Abbott Molecular, Inc, Des Plaines, IL) and classified as negative, trisomy, tetrasomy, or polysomy. RESULTS The sensitivity of polysomy FISH (42.9%) was significantly higher than RC (20.1%) when equivocal RC results were considered negative (P < .001) with identical specificity (99.6%). There was a significant difference in time for diagnosis of carcinoma between FISH diagnostic categories (P < .001) and between RC diagnostic categories (P < .001). Logistic regression analysis revealed that polysomy FISH, trisomy FISH, suspicious cytology, primary sclerosing cholangitis status, and age were associated with carcinoma (P < .05). CONCLUSIONS Polysomy FISH had high sensitivity without compromise to specificity. DIA was not a significant independent predictor of malignancy. Multivariable modeling using RC, FISH, age, and primary sclerosing cholangitis status can be used to estimate the probability of carcinoma for an individual patient. We recommend including FISH as a routine test where available, along with RC, in the evaluation of indeterminate pancreatobiliary strictures.

Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma

Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases.

Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions

Cholangiocarcinoma is a highly lethal cancer of the biliary tract. The intrahepatic subtype of cholangiocarcinoma is increasing in incidence globally. Despite technologic advancements over the past decade, little is known about the somatic changes that occur in these tumors. The goal of this study was to determine the frequency of common oncogenes in resected cholangiocarcinoma specimens that could provide potential therapeutic targets for patients diagnosed with cholangiocarcinoma. Formalin-fixed, paraffin-embedded tissue blocks from 94 resected cholangiocarcinomas were used to extract DNA from areas comprising more than 20% tumor. Specimens were evaluated using the Sequenom MassARRAY OncoCarta Mutation Profiler Panel (San Diego, CA). This matrix-assisted laser desorption/ionization-time of flight mass spectrometry single genotyping panel evaluates 19 oncogenes for 238 somatic mutations. Twenty-five mutations were identified in 23 of the 94 cholangiocarcinomas within the following oncogenes: KRAS (n = 12), PIK3CA (n = 5), MET (n = 4), EGFR (n = 1), BRAF (n = 2), and NRAS (n = 1). Mutations were identified in 7 (26%) of 27 extrahepatic cholangiocarcinomas and 16 (24%) of 67 intrahepatic cholangiocarcinomas. When combined with IDH1/2 testing, 40 (43%) of the 94 cholangiocarcinomas had a detectable mutation. MassARRAY technology can be used to detect mutations in a wide variety of oncogenes using paraffin-embedded tissue. Clinical testing for somatic mutations may drive personalized therapy selection for cholangiocarcinomas in the future. The variety of mutations detected suggests that a multiplexed mutation detection approach may be necessary for managing patients with biliary tract malignancy.

Primary Sclerosing Cholangitis Patients With Serial Polysomy Fluorescence In Situ Hybridization Results Are at Increased Risk of Cholangiocarcinoma

OBJECTIVES:A polysomy fluorescence in situ hybridization (FISH) result in a pancreatobiliary brushing from a patient with primary sclerosing cholangitis (PSC) is very worrisome for carcinoma. However, treatment is not recommended unless verified by corroborative evidence of malignancy because of less than perfect test specificity in this population. The aim of this study was to evaluate the clinical outcome of PSC patients with serial polysomy FISH results.METHODS:Patients with PSC underwent endoscopic retrograde cholangiopancreatography with brushings when clinically indicated per standard practice. Brushings were evaluated by routine cytology and FISH. Retrospective review identified patients with a polysomy FISH result without definitive imaging or pathological evidence of malignancy at the time of the first polysomy, who underwent follow-up examinations including subsequent FISH testing (n=30). Patient records were reviewed to determine clinical outcome.RESULTS:In all, 9 of 13 patients (69%) with a subsequent polysomy result (i.e., serial polysomy) were diagnosed with cholangiocarcinoma (CCA) compared with 3 of 17 patients (18%) with subsequent non-polysomy results (P=0.008). There was a significant difference in time to a diagnosis of CCA between PSC patients with serial polysomy compared with those with subsequent non-polysomy (P=0.01). In four patients with serial polysomy results, imaging/pathological evidence of CCA was not found until 1–2.7 years after the initial polysomy FISH result.CONCLUSIONS:FISH may detect polysomic cells in pancreatobiliary brushings before other pathological or imaging techniques identify CCA. Patients with serial polysomy FISH results are at higher risk for having CCA than those with subsequent non-polysomy FISH results.

Utility of Biomarkers in Prediction of Response to Ablative Therapy in Barrett's Esophagus

BACKGROUND & AIMS Photodynamic therapy (PDT) has been shown to be effective in the treatment of high-grade dysplasia (HGD)/mucosal carcinoma in Barretts esophagus (BE). Substantial proportions of patients do not respond to PDT or progress to carcinoma despite PDT. The role of biomarkers in predicting response to PDT is unknown. We aimed to determine if biomarkers known to be associated with neoplasia in BE can predict loss of dysplasia in patients treated with ablative therapy for HGD/intramucosal cancer. METHODS Patients with BE and HGD/intramucosal cancer were studied prospectively from 2002 to 2006. Biomarkers were assessed using fluorescence in situ hybridization performed on cytology specimens, for region-specific and centromeric probes. Patients were treated with PDT using cylindric diffusing fibers (wavelength, 630 nm; energy, 200 J/cm fiber). Univariate and multiple variable logistic regression was performed to determine predictors of response to PDT. RESULTS A total of 126 consecutive patients (71 who underwent PDT and 55 patients who did not undergo PDT and were under surveillance, to adjust for the natural history of HGD), were included in this study. Fifty (40%) patients were responders (no dysplasia or carcinoma) at 3 months after PDT. On multiple variable analysis, P16 allelic loss (odds ratio [OR], 0.32; 95% confidence interval [CI], 0.10-0.96) predicted decreased response to PDT. BE segment length (OR, 0.71; 95% CI, 0.59-0.85), and performance of PDT (OR, 7.17; 95% CI, 2.50-20.53) were other independent predictors of loss of dysplasia. CONCLUSIONS p16 loss detected by fluorescence in situ hybridization can help predict loss of dysplasia in patients with BE and HGD/mucosal cancer. Biomarkers may help in the selection of appropriate therapy for patients and improve treatment outcomes.

Fibroblast growth factor receptor 2 translocations in intrahepatic cholangiocarcinoma

Patients with cholangiocarcinoma often present with locally advanced or metastatic disease. There is a need for effective therapeutic strategies for advanced stage cholangiocarcinoma. Recently, FGFR2 translocations have been identified as a potential target for tyrosine kinase inhibitor therapies. This study evaluated 152 cholangiocarcinomas and 4 intraductal papillary biliary neoplasms of the bile duct for presence of FGFR2 translocations by fluorescence in situ hybridization and characterized the clinicopathologic features of cases with FGFR2 translocations. Thirteen (10 women, 3 men; 8%) of 156 biliary tumors harbored FGFR2 translocations, including 12 intrahepatic cholangiocarcinomas (12/96; 13%) and 1 intraductal papillary neoplasm of the bile duct. Histologically, cholangiocarcinomas with FGFR2 translocations displayed prominent intraductal growth (62%) or anastomosing tubular glands with desmoplasia (38%). Immunohistochemically, the tumors with FGFR2 translocations frequently showed weak and patchy expression of CK19 (77%). Markers of the stem cell phenotype in cholangiocarcinoma, HepPar1 and CK20, were negative in all cases. The median cancer-specific survival for patients whose tumors harbored FGFR2 translocations was 123 months compared to 37 months for cases without FGFR2 translocations (P = .039). This study also assessed 100 cholangiocarcinomas for ERBB2 amplification and ROS1 translocations. Of the cases tested, 3% and 1% were positive for ERBB2 amplification and ROS1 translocation, respectively. These results confirm that FGFR2, ERRB2, and ROS1 alterations are potential therapeutic targets for intrahepatic cholangiocarcinoma.

Correlating Routine Cytology, Quantitative Nuclear Morphometry by Digital Image Analysis, and Genetic Alterations by Fluorescence In Situ Hybridization to Assess the Sensitivity of Cytology for Detecting Pancreatobiliary Tract Malignancy

Routine cytologic (RC), fluorescence in situ hybridization (FISH), digital image analysis (DIA), and quantifiable morphometric results from 284 pancreatobiliary stricture brushings were compared. We chose specific DIA nuclear features assessed by pathologists in evaluating RC specimens, such as area and shape. A visual nuclear morphometric score (VNMS) was calculated. There was a difference (P < .001) in the mean VNMS when RC results were classified as negative (11.5), atypical (12.5), suspicious (13.8), and positive (16.5). The mean VNMS of specimens diagnosed as disomy (11.3), trisomy 7 (12.1), and polysomy (14.7) by FISH was also different (P < .001). There was no difference in the VNMS of false-negative and true-negative cytologic specimens (P = .225). Our findings substantiate the relationship between cell nuclear visual alterations and genetic FISH abnormalities. The low sensitivity of cytologic examination for pancreatobiliary carcinoma is due to an absence of tumor cells or the presence of well-differentiated tumor lacking recognizable nuclear atypia.

A comparison of conventional cytology, DNA ploidy analysis, and fluorescence in situ hybridization for the detection of dysplasia and adenocarcinoma in patients with Barrett's esophagus ☆ ☆☆

New detection methods with prognostic power are needed for early identification of dysplasia and esophageal adenocarcinoma (EA) in patients with Barretts esophagus (BE). This study assessed the relative sensitivity and specificity of conventional cytology, DNA ploidy analysis with digital image analysis (DIA), and fluorescence in situ hybridization (FISH) for the detection of dysplasia and adenocarcinoma in endoscopic brushing specimens from 92 patients undergoing endoscopic surveillance for BE. FISH used probes to 8q24 (C-MYC), 9p21 (P16), 17q12 (HER2), and 20q13. Four-quadrant biopsies taken every centimeter throughout visible Barretts mucosa were used as the gold standard. The sensitivity of cytology, DIA, and FISH for low-grade dysplasia was 5%, 5%, and 50%, respectively; for high-grade dysplasia (HGD), 32%, 45%, and 82%, respectively; and for EA, 45%, 45%, and 100%, respectively. FISH was more sensitive (P < .05) than cytology and DIA for low-grade dysplasia, HGD, and EA. The specificity of cytology, DIA, and FISH among patients (n = 14) with tissue showing only benign squamous mucosa was 93%, 86%, and 100% (P = .22), respectively. All patients with a polysomic FISH result had HGD and/or EA within 6 months (n = 33). There was a significant difference between FISH categories (negative, 9p21 loss, gain of a single locus, and polysomy) for progression to HGD/EA (P < .001). These findings suggest that FISH has high sensitivity for the detection of dysplasia and EA in BE patients, with the power to stratify patients by FISH abnormality for progression to HGD/EA. Additional studies are needed to further evaluate the clinical use of FISH.