An Optimized Set of Fluorescence In Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples.

BACKGROUND & AIMS: Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. METHODS: We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. RESULTS: The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) (P < .001) or routine cytology analysis (18.8%) (P < .001), but similar specificity (92.9%, 90.8%, and 100.0% respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH (P < .001), a mass by cross-sectional imaging (P < .001), cancer cells by routine cytology (overall P = .003), as well as absence of primary sclerosing cholangitis (P = .011). CONCLUSIONS: We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers.

Prediction of response to endoscopic therapy of Barrett's dysplasia by using genetic biomarkers.

BACKGROUND: Endoscopic therapy for the treatment of high-grade dysplasia (HGD) and intramucosal cancer (IMC) in Barrett's esophagus (BE) may not always result in complete remission of dysplasia (CRD). OBJECTIVE: To determine whether genetic alterations in the Barrett's mucosa can predict response to endoscopic therapy. DESIGN: Retrospective cohort study. SETTING: Tertiary-care institution. PATIENTS: Selected patients who underwent endoscopic therapy for BE containing HGD/IMC between 2003 and 2010. INTERVENTIONS: Endoscopic therapy combining mucosal resection and different ablation modalities was performed based on patient characteristics, endoscopic findings, and technique evolution. Fluorescence in situ hybridization was used to evaluate genetic alterations on baseline endoscopic cytology brushings by using probes directed to loci 8q24 (MYC), 9p21 (CDKN2A; alias P16), 17q12 (ERBB2; alias Her-2/neu), and 20q13.2 (ZNF217). MAIN OUTCOME MEASUREMENTS: Genetic biomarkers predicting achievement of CRD after endoscopic therapy. RESULTS: A total of 181 patients were included (145 men; 66 ± 10 years of age). There were 130 patients (72%) who responded to endoscopic therapy with CRD. Multiple gains detected by fluorescence in situ hybridization was found to be a negative predictor (hazard ratio 0.57; 95% confidence interval, 0.40-0.82) after adjusting for potential clinical confounders. Similar results were found when analyses were restricted to patients (n = 66) undergoing radiofrequency ablation (hazard ratio 0.58; 95% confidence interval, 0.31-1.09). LIMITATIONS: Retrospective study, heterogeneity of treatment modalities. CONCLUSION: Patients with multiple gains detected by brush cytology specimens may have a lower response rate to endoscopic therapy. The presence of multiple gains can be an adjunct to standard histology in prognosticating BE patients with HGD/IMC undergoing endoscopic therapy.

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 Barrett's 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.

The development of a fluorescence in situ hybridization assay for the detection of dysplasia and adenocarcinoma in Barrett's esophagus.

The goal of this study was to identify a set of fluorescence in situ hybridization probes for the detection of dysplasia and adenocarcinoma in patients with Barrett's esophagus. We examined 170 brushing specimens from 138 patients with Barrett's esophagus or a history of Barrett's esophagus using fluorescence in situ hybridization with probes to 5p15, 5q21-22, centromere 7, 7p12, 8q24.12-13, centromere 9, 9p21, centromere 17, 17p13.1, 17q11.2-12, 20q13.2, and centromere Y. Receiver-operator curves were used to determine the sensitivity and specificity of various four-probe combinations for detecting low-grade dysplasia, high-grade dysplasia, and esophageal adenocarcinoma. Endoscopic biopsy results were used as the gold standard. Numerous four-probe combinations provided a similarly high sensitivity and specificity. Of these, a set consisting of probes to 8q24, 9p21, 17q11.2, and 20q13.2 was found to have a sensitivity and specificity, respectively, of 70% and 89% for low-grade dysplasia, 84% and 93% for high-grade dysplasia, and 94% and 93% for esophageal adenocarcinoma. This probe set was chosen for future prospective clinical evaluations based on its high sensitivity and specificity, its ability to distinguish adenocarcinoma and high-grade or low-grade dysplasia from lesser diagnostic categories, and the favorable signal quality for each of the probes.

Diagnostic accuracy of bronchial brush cytology and the added value of immunohistochemistry and fluorescence in situ hybridization of pulmonary neuroendocrine tumors.

BACKGROUND: Bronchial brush (BB) cytology carries low sensitivity for detecting neuroendocrine carcinomas (NECs), including typical carcinoid (TC) tumors of the lung. We aimed to investigate the detection of neuroendocrine tumors including TC through BB routine cytology cell block (CB), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH). MATERIALS AND METHODS: A SNOMED search showed 187 lung biopsy or resection specimens from 2008 through 2011 containing neuroendocrine or carcinoid in the diagnosis. Residual BB specimens retained in PreservCyt were used to prepare a ThinPrep slide for FISH analysis. CBs were stained with H and E and IHC for chromogranin and synaptophysin. RESULTS: Of the 187 cases, 16 had residual BB material available within 1 year of diagnosis and were used in CB preparation for IHC and FISH slides. Cytologic evaluation determined 1 case positive for malignancy (small cell lung carcinoma [SCLC]), 1 suspicious for adenocarcinoma, and 14 negative for malignancy. On the basis of histologic diagnosis, FISH was performed. SCLC showed polysomy (86% abnormal cells); 2 TC tumors showed a gain of 7p12 (15% abnormal cells) and a gain of 5q15 (72% abnormal cells), respectively. Two cases had CBs with positive immunoreactivity for chromogranin and synaptophysin. The sensitivity for detection of NEC was 18.8%, 15.4%, and 25% for cytologic evaluation, CB, and FISH, respectively. CONCLUSION: Neuroendocrine tumors, including TC are difficult to detect with BB cytologic evaluation, most likely because tumor cells lack in the specimen. Assessment of further studies is needed to explore the role of cytology and ancillary methods for detection of these tumors.

Fluorescence in situ hybridization mapping of esophagectomy specimens from patients with Barrett's esophagus with high-grade dysplasia or adenocarcinoma.

The progression of intestinal metaplasia to esophageal adenocarcinoma in patients with Barrett's esophagus is partly driven by chromosomal alterations that activate oncogenes and inactivate tumor suppressor genes. The goal of this study was to determine how alterations of 4 frequently affected genes correlate with the range of histopathologic lesions observed in resected esophagi of patients with Barrett's esophagus. Fluorescence in situ hybridization was used to assess 83 tissue sections from 10 Barrett's esophagus esophagogastrectomy specimens for chromosomal alterations of 8q24 (MYC), 9p21 (CDKN2A; alias P16), 17q12 (ERBB2), and 20q13.2 (ZNF217). Histologic lesions assessed included gastric metaplasia (n = 8), intestinal metaplasia (n = 43), low-grade dysplasia (n = 28), high-grade dysplasia (n = 25), and adenocarcinoma (n = 16). Histologic maps showing the correlation between fluorescence in situ hybridization abnormalities and corresponding histology were created for all patients. Chromosomal abnormalities included 9p21 loss, single locus gain, and polysomy. A greater number of chromosomal alterations were detected as the severity of histologic diagnosis increased from intestinal metaplasia to adenocarcinoma. All patients had alterations involving the CDKN2A gene. CDKN2A loss was the only abnormality detected in 20 (47%) of 43 areas of intestinal metaplasia. Polysomy, the most common abnormality in dysplastic epithelium and adenocarcinoma, was observed in 16 (57%) of 28 low-grade dysplasia, 22 (88%) of 25 high-grade dysplasia, and 16 (100%) of 16 adenocarcinoma. The findings of this study improve our understanding of the role that chromosomal instability and alterations of tumor suppressor genes such as CDKN2A and oncogenes such as ERBB2 play in the progression of intestinal metaplasia to adenocarcinoma in patients with Barrett's esophagus.

Correlation of histology with biomarker status after photodynamic therapy in Barrett esophagus.

BACKGROUND: Currently, histology is used as the endpoint to define success with photodynamic therapy (PDT) in patients with high-grade dysplasia (HGD). Recurrences despite 'successful' ablation are common. The role of biomarkers in assessing response to PDT remains undefined. The objectives of the current study were 1) to assess biomarkers in a prospective cohort of patients with HGD/mucosal cancer before and after PDT and 2) to correlate biomarker status after PDT with histology. METHODS: Patients who underwent PDT for HGD/mucosal cancer were studied prospectively. All patients underwent esophagogastroduodenoscopy, 4-quadrant biopsies every centimeter, endoscopic mucosal resection of visible nodules, and endoscopic ultrasound. Cytology samples were obtained by using standard cytology brushes. Biomarkers were assessed by using fluorescence in situ hybridization (FISH). The biomarkers that were assessed included loss of 9p21 (site of the p16 gene) and 17p13.1 (site of the p53 gene) loci; gains of the 8q24(c-myc), 17q (HER2-neu), and 20q13 loci; and multiple gains. Patients received PDT 48 hours after the administration of sodium porfimer. Demographic and clinical variables were collected prospectively. Patients were followed with endoscopy and repeat cytology for biomarkers. The McNemar test was used to compare biomarker proportions before and after PDT. RESULTS: Thirty-one patients were studied. The median patient age was 66 years (interquartile range [IQR], 56-73 years), and 28 patients (88%) were men. The mean Barrett segment length was 5 cm (standard error of the mean, 0.5 cm). Post-PDT biomarkers were obtained after a median duration of 9 months (IQR, 3-12 months). There was a statistically significant decrease in the proportion of several biomarkers assessed after PDT. Six patients without HGD after PDT still had positive FISH results for 1 or more biomarkers: of these, 2 patients (33%) developed recurrent HGD. CONCLUSIONS: In this initial study, histologic downgrading of dysplasia after PDT was associated with the loss of biomarkers that have been associated with progression of neoplasia in Barrett esophagus. Patients with persistently positive biomarkers appeared to be at a higher risk of recurrent HGD. These findings should be confirmed in a larger study.

A study of the reproducibility of a fluorescence in situ hybridization bladder cancer detection assay.

OBJECTIVE: To assess the reproducibility of the UroVysion fluorescence in situ hybridization (FISH) bladder cancer detection assay. STUDY DESIGN: Thirteen specimens (2 negative, 3 low-level positive [1-10% abnormal cells], 5 mid-level positive [11-75%], and 3 high-level positive [>75%]) were analyzed by 7 cytotechnologists. Each cytotechnologist rendered an overall diagnosis of positive or negative and determined the percentage of abnormal urothelial cells for all positive specimens. RESULTS: The interobserver reproducibility of the assay was 100% for mid-level and high-level positive specimens, 93% for negative specimens, and 78% for low-level positive specimens. The range of percent abnormal determinations was highest for mid-level positive specimens, with mean SDs of 1.8%, 16.4% and 10.1% for the low-, mid-, and high-level positives, respectively. CONCLUSION: There was a high level of reproducibility among the mid- and high-level positive specimens. The reproducibility for low-level positive specimens was lowest, suggesting that such specimens should be reviewed by a second technologist to ensure an accurate diagnosis. The findings of this study are important for further elucidating the clinical value of quantitative FISH analysis in the management of patients undergoing FISH testing for bladder cancer.

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 Barrett's 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 Barrett's 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.

Monitoring intravesical therapy for superficial bladder cancer using fluorescence in situ hybridization.

PURPOSE: We evaluated fluorescence in situ hybridization (FISH) for assessing the response to therapy in patients with superficial bladder cancer receiving bacillus Calmette-Guerin or other intravesical therapies. MATERIALS AND METHODS: A total of 37 patients receiving intravesical therapy for superficial bladder cancer were enrolled in this study. Urine specimens were collected for FISH analysis just prior to the first intravesical therapy in 31 cases and just prior to or within 2 months following the last intravesical therapy in 37. FISH was done using the UroVysion probe set (Abbott Laboratories, Abbott Park, Illinois) with results considered positive if 5 or more cells demonstrated polysomy. Biopsy, cystoscopy and/or cytology results were then compared to FISH results to evaluate the usefulness of the test for monitoring intravesical therapy. RESULTS: Of the patients 25 had a negative and 12 had a positive post-therapy FISH result. All 12 patients with a positive post-therapy FISH result had tumor recurrence, while tumor recurrence was observed in 13 of the 25 with a negative post-therapy FISH result (HR 4.6, 95% CI 1.9 to 11.1, p <0.001). Of the patients with tumor recurrence 7 of 12 with a positive post-therapy FISH result had muscle invasive tumor and 2 of 25 with a negative post-therapy FISH result had muscle invasive tumor (HR 9.4, 95% CI 1.9 to 45.3, p = 0.001). CONCLUSIONS: FISH appears to be useful for monitoring patients with superficial bladder cancer for the response to intravesical therapy. Patients with a positive FISH result at the end of treatment are at high risk for progression to muscle invasive bladder cancer.