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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. [ABSTRACT FROM AUTHOR]

An abstract of the article "Routine Cytology and Reflex Fluorescence In-Situ Hybridization Analysis of Bronchial Brushing Specimens for Central and Peripheral Lung Lesions," by Jesse S. Voss, Benjamin R. Kipp, Amy C. Clayton, Otis B. Rickman, James R. Jett, Michael R. Henry, and Kevin C. Halling is presented.

Purpose Molecular diagnostic testing has become an integral part of the evaluation of patients with metastatic colorectal cancer (CRC). Expanded mutational testing, such as next-generation sequencing (NGS), often identifies mutations with unclear clinical or prognostic implications. One such example is BRAF mutations that occur outside of codon 600 ( non-V600 BRAF mutations). Methods We conducted this multicenter, retrospective cohort study to characterize the clinical, pathologic, and survival implications of non-V600 BRAF mutations in metastatic CRC. We pooled patients in whom non-V600 BRAF mutations were identified from NGS databases at three large molecular genetics reference laboratories. Results A total of 9,643 patients with metastatic CRC underwent NGS testing. We identified 208 patients with non-V600 BRAF mutations, which occurred in 2.2% of all patients tested and accounted for 22% of all BRAF mutations identified. Cancers with non-V600 BRAF mutations, compared with cancers with V600E BRAF ( V600E BRAF) mutations, were found in patients who were significantly younger (58 v 68 years, respectively), fewer female patients (46% v 65%, respectively), and patients who had fewer high-grade tumors (13% v 64%, respectively) or right-sided primary tumors (36% v 81%, respectively). Median overall survival was significantly longer in patients with non-V600 BRAF-mutant metastatic CRC compared with those with both V600E BRAF-mutant and wild-type BRAF metastatic CRC (60.7 v 11.4 v 43.0 months, respectively; P < .001). In multivariable analysis, non-V600 BRAF mutation was independently associated with improved overall survival (hazard ratio, 0.18; P < .001). Conclusion Non-V600 BRAF mutations occur in approximately 2.2% of patients with metastatic CRC and define a clinically distinct subtype of CRC with an excellent prognosis.

Objective: We demonstrate the feasibility of detecting EC by combining minimally-invasive specimen collection techniques with sensitive molecular testing., Methods: Prior to hysterectomy for EC or benign indications, women collected vaginal pool samples with intravaginal tampons and underwent endometrial brushing. Specimens underwent pyrosequencing for DNA methylation of genes reported to be hypermethylated in gynecologic cancers and recently identified markers discovered by profiling over 200 ECs. Methylation was evaluated individually across CpGs and averaged across genes. Differences between EC and benign endometrium (BE) were assessed using two-sample t-tests and area under the curve (AUC)., Results: Thirty-eight ECs and 28 BEs were included. We evaluated 97 CpGs within 12 genes, including previously reported markers (RASSF1, HSP2A, HOXA9, CDH13, HAAO, and GTF2A1) and those identified in discovery work (ASCL2, HTR1B, NPY, HS3ST2, MME, ADCYAP1, and additional CDH13 CpG sites). Mean methylation was higher in tampon specimens from EC v. BE for 9 of 12 genes (ADCYAP1, ASCL2, CDH13, HS3ST2, HTR1B, MME, HAAO, HOXA9, and RASSF1) (all p<0.05). Among these genes, relative hypermethylation was observed in EC v. BE across CpGs. Endometrial brush and tampon results were similar. Within tampon specimens, AUC was highest for HTR1B (0.82), RASSF1 (0.75), and HOXA9 (0.74). This is the first report of HOXA9 hypermethylation in EC., Conclusion: DNA hypermethylation in EC tissues can also be identified in vaginal pool DNA collected via intravaginal tampon. Identification of additional EC biomarkers and refined collection methods are needed to develop an early detection tool for EC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Determination of tumor genetic architecture based on tissue analysis yields important information on signaling pathways involved in cancer pathogenesis and plays a growing role in choosing the optimal medical management of malignancies. Specifically, the advent of next-generation sequencing has led to a rapidly evolving era of relatively inexpensive, high-throughput DNA sequencing of tumors. One such example is multiplexed tumor genotyping (ie, panel testing) of more than 2800 mutations across 50 commonly mutated cancer-associated genes. This resulting mutational landscape shows medically actionable pathogenic alterations to optimize antitumor therapy. We recently assessed the performance and outcome of targeted next-generation sequencing with archived endoscopic ultrasound fine-needle aspirates across a broad range of primary and metastatic sites with encouraging accuracy. As a result, endoscopic ultrasound has the potential to move from a test for diagnosis or confirmation of malignancy, to one in which it could facilitate the personalization of cancer-directed therapy., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Grading criteria for biliary dysplasia associated with primary sclerosing cholangitis (PSC) have been recently described. Although a dysplasia to cholangiocarcinoma (CCA) sequence is implied, supportive data are lacking. Seventeen liver explants with biliary dysplasia from patients with PSC were selected. Formalin-fixed, paraffin-embedded blocks from each patient were evaluated to identify areas of normal/reactive biliary epithelium, intestinal metaplasia, low-grade dysplasia, high-grade dysplasia, and CCA. Areas of interest were assessed for chromosomal alteration with fluorescence in situ hybridization using probes directed to locus 9p21 and centromeres 3, 7, and 17. The cutoffs for calling probe copy number abnormalities for polysomy, single locus gain, and homozygous 9p21 loss were established by receiver operating characteristic curve analysis. Of 4 areas of intestinal metaplasia, 19 low-grade dysplasias, 19 high-grade dysplasias, and 5 CCAs, 0%, 11%, 58%, and 40% displayed polysomy and 0%, 0%, 16%, and 40% exhibited homozygous 9p21 loss as the most severe abnormality, respectively. Patients with prior or current CCA were more likely to display polysomy in dysplasia than patients without CCA (70% versus 14%; P = .05); however, high-grade dysplasia was proportionally more common in the CCA-associated dysplasia group. Polysomy and homozygous 9p21 loss are detected in biliary dysplasia and CCA. These findings support a dysplasia-carcinoma sequence in PSC patients and suggest that fluorescence in situ hybridization analysis could help refine the grading of biliary dysplasia in these patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Studies have shown that fluorescence in situ hybridization (FISH) testing increases lung cancer detection on cytology specimens in peripheral nodules. The goal of this study was to determine whether a predictive model using clinical features and routine cytology with FISH results could predict lung malignancy after a nondiagnostic bronchoscopic evaluation. Patients with an indeterminate peripheral lung nodule that had a nondiagnostic bronchoscopic evaluation were included in this study (N = 220). FISH was performed on residual bronchial brushing cytology specimens diagnosed as negative (n = 195), atypical (n = 16), or suspicious (n = 9). FISH results included hypertetrasomy (n = 30) and negative (n = 190). Primary study end points included lung cancer status along with time to diagnosis of lung cancer or date of last clinical follow-up. Hazard ratios (HRs) were calculated using Cox proportional hazards regression model analyses, and P values < .05 were considered statistically significant. The mean age of the 220 patients was 66.7 years (range, 35-91), and most (58%) were men. Most patients (79%) were current or former smokers with a mean pack year history of 43.2 years (median, 40; range, 1-200). After multivariate analysis, hypertetrasomy FISH (HR = 2.96, P < .001), pack years (HR = 1.03 per pack year up to 50, P = .001), age (HR = 1.04 per year, P = .02), atypical or suspicious cytology (HR = 2.02, P = .04), and nodule spiculation (HR = 2.36, P = .003) were independent predictors of malignancy over time and were used to create a prediction model (C-statistic = 0.78). These results suggest that this multivariate model including test results and clinical features may be useful following a nondiagnostic bronchoscopic examination., (© 2013.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Recent discoveries of inherited glioma risk loci and acquired IDH mutations are providing new insights into glioma etiology. IDH mutations are common in lower grade gliomas and secondary glioblastomas and uncommon in primary glioblastomas. Because the inherited variant in 11q23 has been associated with risk of lower grade glioma and not with glioblastomas, we hypothesized that this variant increases susceptibility to IDH-mutated gliomas, but not to IDH-wild-type gliomas., Methods: We tested this hypothesis in patients with glioma and controls from the San Francisco Adult Glioma Study, the Mayo Clinic, and Illumina controls (1102 total patients, 5299 total controls). Case-control additive associations of 11q23 risk alleles (rs498872, T allele) were calculated using logistic regression, stratified by tumor IDH status (mutated or wild-type) and by histology and grade. We also adjusted for the recently discovered 8q24 glioma risk locus rs55705857 G allele., Results: The 11q23 glioma risk locus was associated with increased risk of IDH-mutated gliomas of all histologies and grades (odds ratio [OR] = 1.50; 95% confidence interval [CI] = 1.29-1.74; P = 1.3X10(-7)) but not with IDH-wild-type gliomas of any histology or grade (OR = 0.91; 95% CI = 0.81-1.03; P = 0.14). The associations were independent of the rs55705857 G allele., Conclusion: A variant at the 11q23 locus increases risk for IDH-mutated but not IDH-wild-type gliomas, regardless of grade or histology.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: To assess Hybrid Capture 2 (HC2) and fluorescence in situ hybridization (FISH) for the detection of cervical intraepithelial neoplasia 2 or worse (CIN 2+) in patients with a cytologic diagnosis of low grade squamous intraepithelial lesion (LSIL)., Study Design: Residual samples from 115 LSIL-diagnosed cervical cytology specimens were evaluated by high-risk human papillomavirus (HR-HPV) HC2 testing and FISH using biotin-labeled probes to HR-HPV and chromosomal probes to 3q26 (TERC) and 8q24 (CMYC). A cervical biopsy diagnosis of CIN 2+ was considered as evidence of high grade disease., Results: The positive and negative predictive values of HC2 and FISH for detecting patients with CIN 2+ were 32% vs. 37% and 100% vs. 93%, respectively. The sensitivities of HC2 and FISH for CIN 2+ were not significantly different (100% vs. 90%, p = 0.25), while the specificity of HC2 was significantly lower than that of FISH (28% vs. 48%, p=0.003). FISH diagnosed fewer specimens as positive as compared to HC2 (62% vs. 79%)., Conclusion: These preliminary data suggest that FISH testing may be useful for determining which patients with LSIL are most likely to have CIN 2+ on clinical follow-up.

Objective: To compare a recently developed fluorescence in situ hybridization (FISH) high-risk human papillomavirus (HR-HPV) assay to Hybrid Capture 2 (HC2) (Digene Corporation, Gaithersburg, Maryland, U.S.A.) and polymerase chain reaction (PCR) for the detection of HR-HPV subtypes in cervical cytology specimens., Study Design: One hundred forty-one liquid-based cytology specimens were used to produce a thin-layer slide for FISH analysis. The remaining material was sent for HC2 and PCR HR-HPV testing. Thin-layer slides were hybridized with a FISH probe set containing a biotin-labeled HR-HPV cocktail and were manually screened for HR-HPV-infected cells. Specimens with > or = 1 HPV-positive cell by FISH were considered positive for HR-HPV infection., Results: There was complete concordance between HC2, FISH and PCR in 104 (75%) specimens. FISH was concordant with HC2 and PCR in 120 (85%) and 115 (82%) specimens, respectively. HC2 and PCR were concordant in 118 (84%) specimens., Conclusion: The concordance of HR-HPV detection between FISH and HC2/PCR appears similar to concordances between HC2 and PCR. This suggests that FISH may be another method of detecting HR-HPV while having the potential to add additional information such as integrated/episomal staining and the ability to detect chromosomal abnormalities in individual cells.