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Fluorescence in situ hybridization (FISH) testing is used to detect bladder cancer in urine specimens. The purpose of this study was to determine whether there are associations between the percentage of chromosomally abnormal cells by FISH and time to bladder cancer recurrence and progression to metastasis. Clinical records were searched to identify patients with urine FISH results, history of non-invasive bladder cancer, and at least one follow-up pathological diagnosis. Covariates analyzed included age, gender, smoking status, treatment after FISH, cystoscopy result, and prior stage of bladder cancer. The percentage of abnormal cells (hazard ratio [HR] 1.03, 95% CI 1.02-1.03; P < 0.001), age (HR 1.02, 95% CI 1.00-1.03; P = 0.033), male gender (HR 0.60, 95% CI 0.41-0.87; P < 0.001), treatment (HR 0.37, 95% CI 0.25-0.55; P < 0.001), and history of TIS/T1-stage tumors (HR 1.66, 95% CI 1.23-2.24; P = 0.001) were significantly associated with time to cancer recurrence. Time to invasive cancer was significantly associated with the percentage of abnormal cells (HR 1.02, 95% CI 1.01, 1.03; P < 0.001), history of TIS/T1 tumor (HR 3.73, 95% CI 1.88, 7.38; P = 0.001), and treatment (HR 0.33, 95% CI 0.13, 0.83; P = 0.019), suggesting that the percentage of abnormal cells independently predicts cancer recurrence and progression to invasive disease in patients with a history of non-invasive bladder cancer.

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., ((c) 2008 American Cancer Society)

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.

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.

Background: Laboratory testing in suspected alpha-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency., Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis., Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result., Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

The purpose of this study was to assess the performance of ThinPrep UroCyte filters, which were designed specifically for the preparation of slides for fluorescence in situ hybridization (FISH) analysis of urine specimens. One hundred urine specimens were evenly split, and one portion was utilized to prepare a slide with the UroCyte filter method and the other portion was used to prepare a slide with a manual dropping method. All 17 of the 100 specimens identified as positive by the manual method were also identified as positive with the UroCyte method. No significant differences were noted in the percentage of chromosomally abnormal cells (P = 0.227), cellularity (P = 0.857), signal quality (P = 0.816), and DAPI counterstain quality (P = 0.369) between the two methodologies. The average time taken to prepare a batch of 10 slides using the UroCyte method, and that using manual method was 103 min (10.3 min/case) and 194 min (19.4 min/case), respectively. This study suggests that the UroCyte filter method of preparing slides for FISH analysis reduces the time required to prepare these slides with overall results that are similar to the currently utilized manual dropping method., (Copyright 2006 Wiley-Liss, Inc.)

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.

Objective: To determine which nuclear morphologic and background features are most associated with normal and abnormal chromosome patterns in urine cells evaluated by fluorescence in situ hybridization (FISH) with Vysis UroVysion (Downers Grove, Illinois, USA)., Study Design: One hundred specimens were analyzed and the nuclear morphologic and background features compared between FISH-negative (disomic) and -positive (polysomic) cases., Results: Our data show that polysomic urothelial cells have significantly (p < 0.001) weaker DAPI nuclear counterstaining, nuclear enlargement, more irregular nuclear shape and more chromatin clumping when compared to disomic cells., Conclusion: These findings indicate that there are specific staining and nuclear cytologic features that can help identify cells that have a high probability of being polysomic by FISH. This information may help cytologists decrease the time required to evaluate urine specimens by FISH and may aid in the development of instruments that automate FISH analysis.

Background and Aim: The aim of this study was to assess the relative sensitivities and specificities of fluorescence in situ hybridization (FISH) and routine cytology for the detection of malignancy in biliary tract strictures., Methods: Bile duct brushing and aspirate specimens were collected from 131 patients being evaluated for possible malignant bile duct strictures. Both specimen types were assessed by FISH but only brushing specimens were assessed by cytology. The FISH assay used a mixture of fluorescently-labeled probes to the centromeres of chromosomes 3, 7, and 17 and chromosomal band 9p21 (Vysis UroVysion) to identify cells having chromosomal abnormalities. A case was considered positive for malignancy if five or more cells exhibited polysomy., Results: Sixty-six of the 131 patients had surgical pathologic and/or clinical evidence of malignancy. Thirty-nine patients had cholangiocarcinoma, 19 had pancreatic carcinoma, and 8 had other types of malignancy. The sensitivity of cytology and FISH for the detection of malignancy in bile duct brushing specimens in these patients was 15% and 34% (p < 0.01), respectively. The sensitivity of FISH for the bile aspirate specimens was 23%, and the combined sensitivity of FISH for aspirate and brushing specimens was 35%. The specificity of FISH and cytology brushings were 91% and 98% (p= 0.06), respectively., Conclusions: FISH is significantly more sensitive than and nearly as specific as conventional cytology for the detection of malignant biliary strictures in biliary brushing specimens. FISH may improve the clinical management of patients who are being evaluated for malignancy in bile duct strictures.

For decades, cystoscopy and urine cytology have been the mainstay for monitoring superficial urothelial carcinoma patients for tumor recurrence. However, urine cytology has poor sensitivity for urothelial carcinoma and, consequently, numerous investigators have been endeavoring to develop more sensitive assays for the detection of urothelial carcinoma. This article presents an overview of the types of new assays that have been developed for urothelial carcinoma detection but focuses primarily on the features and performance of a new fluorescence in situ hybridization assay for urothelial carcinoma detection known as Vysis UroVysion.

Lymphocyte-rich gastric carcinomas may have a better prognosis than cancers without a pronounced host inflammatory response. Two subsets of gastric cancer-Epstein-Barr virus-positive and microsatellite instability high-have been associated with a lymphocyte-rich phenotype. We assessed relationships between tumor-infiltrating lymphocytes, Epstein-Barr virus status, microsatellite instability status, and cancer-specific survival in 110 resected gastric cancers. Seven patients had Epstein-Barr virus-positive cancer, including 4 (3.7%) of 107 consecutive patients. Tumors from 17 patients (16%) were designated microsatellite instability high on the basis of negative immunohistochemical staining for MLH1; all tumors had intact expression of MSH2 and MSH6. Epstein-Barr virus-positive cancers had increased tumor-infiltrating lymphocytes compared with Epstein-Barr virus-negative cancers (median 450/10 HPF versus 21/10 HPF, P <.001). Microsatellite instability-high cancers also had increased tumor-infiltrating lymphocytes compared with non-microsatellite instability-high cancers (median 150/10 HPF versus 20/HPF, P <.001). Microsatellite instability-high cancers affected older patients and were more likely to be intestinal in the Lauren classification and expanding in the Ming classification. By univariate analysis, decreased risk of death from gastric cancer was significantly associated with low tumor stage, expanding growth pattern, increasing tumor-infiltrating lymphocyte count, and microsatellite instability-high status. High tumor-infiltrating lymphocyte count and microsatellite instability-high status retained statistical significance as favorable prognostic factors after adjustment for tumor stage in multivariate analysis. Tumor-infiltrating lymphocyte count retained statistical significance as a favorable prognostic factor after adjustment for microsatellite instability-high status; but microsatellite instability-high status did not remain a significant independent prognosticator after adjustment for tumor-infiltrating lymphocyte count. The association between microsatellite instability-high cancers and high tumor-infiltrating lymphocyte counts may account for the association of microsatellite instability-high gastric cancers with improved survival.

Purpose: We determine the sensitivity and specificity of various assays for the detection of urothelial carcinoma., Materials and Methods: A total of 280 voided urine specimens from 265 patients were obtained immediately before cystoscopy for BTA stat, (Bard Diagnostic, Redmond, Washington) hemoglobin dipstick, (Bayer, Elkhart, Indiana) telomerase and UroVysion (Vysis, a wholly owned subsidiary of Abbott Laboratories, Abbott Park, Illinois) analysis., Results: Of the 265 patients 75 had biopsy proven urothelial carcinoma, and the sensitivity of the assays was determined from these patients. From most sensitive to least sensitive, the overall sensitivity of UroVysion (73 cases), BTA stat (72), hemoglobin dipstick (73) and telomerase (70) was 81%, 78%, 74%, and 46%, respectively. Each of the first 3 tests was statistically significantly more sensitive than the telomerase assay (p <0.05). However, the differences in overall sensitivity of UroVysion, BTA stat and hemoglobin dipstick were not statistically significant. The specificity of the tests was calculated for 80 of the 265 patients in this study who had no history of urothelial carcinoma and negative cystoscopy findings despite common urological complaints. From most specific to least specific, the specificity of UroVysion, telomerase, BTA stat and hemoglobin dipstick was 96%, 91%, 74% and 51%, respectively. UroVysion and telomerase were statistically significantly (p <0.01) more specific than the BTA stat and hemoglobin dipstick assays, and all of the assays were more specific than hemoglobin dipstick testing (p <0.001)., Conclusions: Our study reveals that UroVysion is the most sensitive and specific assay among those tested for the detection of urothelial carcinoma. Telomerase testing had good specificity but poor sensitivity. The BTA stat and hemoglobin dipstick tests had good sensitivity but relatively poor specificity. UroVysion is a promising new assay for the detection of urothelial carcinoma in urine specimens. However, further studies are needed to explore the role of the various assays in the treatment of patients with superficial urothelial carcinoma.

Objective: To evaluate amplification of the HER-2/neu gene by fluorescence ir situ hybridization (FISH) in tumors with weakly positive (2+) immunohistochemical staining., Methods: A total of 1556 breast tumor biopsy specimens were referred to Mayo Medical Laboratories, Rochester, Minn, for HER2 testing between August and December 2000. Immunohistochemical (IHC) analysis was performed with use of a diagnostic test for the assessment of HER2 overexpression, the HercepTest. The IHC-stained slides were interpreted and scored on a scale ranging from 0 to 3+ according to Food and Drug Administration-approved guidelines. All specimens scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA probe kit (PathVysion). Specimens were determined to be amplified if the ratio of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than 2.0., Results: Thirty-eight percent of the specimens evaluated with the HercepTest were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17., Conclusion: We recommend that all specimens with a 2+ HercepTest result be evaluated by FISH for HER-2/neu gene amplification. The results of both assays should be considered before making a decision to recommend anti-HER2 therapy.