Utility of UroVysion Fluorescence in situ Hybridization in Improving the Diagnostic Performance of Urine Cytology.

INTRODUCTION: The atypical urothelial cell (AUC) category in The Paris System (TPS) in urine cytology (UrCy) is a challenging area. This study aimed to evaluate the UroVysion fluorescence in situ hybridization (U-FISH) assay in predicting the outcome of AUC. Additionally, we explored the association of abnormal U-FISH results in high-grade urothelial carcinoma (HGUC) concerning muscularis propria invasion (MPI). METHODS: This is a retrospective study, and U-FISH was done on archived Papanicolaou-stained smears. Four cohorts were included: non-neoplastic AUC (AUC-NN), neoplastic AUC (AUC-N), muscle-invasive HGUC (HGUC-MI), and muscle-free HGUC (HGUC-MF) outcome on histopathology (HPE) and with clinical follow-up of 12-29 months. U-FISH was evaluated for diagnostic purposes, and MPI and tumor stage prediction by urine FISH score (UFS; high vs. low) based on copy number gain of chromosomes (Chr). RESULTS: U-FISH was performed on 70 cases (20 AUC-NN, 20 AUC-N, 15 HGUC-MI, and 15 HGUC-MF) and was successful in 58/70 (82.85%) cases. All UC cases showed polysomy of ≥2Chr, and all the AUC-NN cases reported non-neoplastic on HPE were negative for U-FISH. U-FISH picked up all carcinoma cases in the AUC-N cohort. Chr 3 polysomy was statistically significant in differentiating HGUC-MI from HGUC-MF and low-grade urothelial carcinoma cases. Chr 3 signals with a cut-off of 6 signals could identify MPI with a sensitivity of 80.95% and specificity of 41.94%. The UFS of the HGUC-MI group was significantly higher than HGUC-MF. CONCLUSIONS: U-FISH successfully identified all cases of AUC with neoplastic outcomes. In the HGUC group, there was a difference in cases with and without MPI, which requires further confirmation in a larger prospective cohort.

HER2/ERBB2 overexpression in advanced gallbladder carcinoma: comprehensive evaluation by immunocytochemistry and fluorescence in situ hybridisation on fine-needle aspiration cytology samples.

AIMS: Advanced gallbladder carcinoma (AGBC) carries a poor prognosis with dismal survival. There are no data regarding HER2/ERBB2 expression in AGBC. This study evaluated the overexpression of HER2/ERBB2 in cytological aspirates from AGBCs to identify potential patients for whom anti-HER2 targeted therapies can benefit. METHODS: This prospective, case-control study was performed on 50 primary AGBC cases. A detailed cytomorphological assessment, followed by immunocytochemistry (ICC) for HER2/ERBB2, was performed on AGBC cell blocks. A similar number of age-matched and gender-matched resected chronic cholecystitis specimens were included as controls. Fluorescence in situ hybridisation (FISH) was performed in equivocal cases. RESULTS: A total of 10 (20%) cases showed positive (3+), 19 (38%) equivocal (2+) expression and 21 (42%) were negative on HER2/ERBB2 ICC. None of the equivocal cases demonstrated HER2 amplification by FISH. Among the controls, none showed positive (3+) immunoexpression, 23 (46%) demonstrated equivocal expression and 27 (54%) were negative. On statistical analysis, HER2/ERBB2 overexpression was significantly associated with AGBC compared with the controls. Of all the clinical, radiological and cytomorphological parameters, the predominant papillary or acinar arrangements of the tumour cells were significantly associated with HER2/ERBB2 overexpression. CONCLUSIONS: This is the first study to evaluate the expression of HER2/ERBB2 on cytological aspirates in AGBC using ICC and FISH. HER2/ERBB2 overexpression(20%) was significantly associated with AGBC. Furthermore, predominant papillary or acinar arrangements of tumour cells in the cytological smears were significantly associated with HER2/ERBB2 overexpression. They can serve as potential predictors of HER2/ERBB2 overexpression to select AGBC patients for anti-HER2 targeted therapies.

Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis.

BACKGROUND: Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation - plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. DESIGN: A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ2 test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. RESULTS: CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). CONCLUSION: SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

Cell blocks from scraping of cytology smear: comparison with conventional cell block.

OBJECTIVE: To compare cytomorphology preservation and immunohistochemistry results between conventional cell blocks (CCB) and cytoscrape cell blocks (SCB). STUDY DESIGN: Fine needle aspiration (FNAC) was done in 17 consecutive cases. Air-dried smears for May-Grünwald-Giemsa stain and wet-fixed smear for hematoxylin-eosin (H-E) stain were prepared. Simultaneously another pass was made in each case for preparation of material for CCB. One of the H-E-stained smears was spared for SCB. SCB was compared with CCB for cell morphology. Immunostaining was performed both cell blocks, as well as on FNA smears in 8 cases. Results were evaluated for intensity of staining and percentage of cells showing positivity. RESULTS: CCB and SCB sections showed adequate cellularity in all cases. Morphologic preservation was good in SCB sections. There was good architectural and nuclear preservation in all cases of SCB. Immunostaining results showed better and clear intensity of staining with little background in all cell block cases. CONCLUSION: SCB is a valuable technique in cell blocks from stained FNA smears. The cytomorphologic details are equally good in SCB and CCB. Additional panels of immunostaining can be done on SCB for better diagnosis and classification, particularly in cases in which repeat FNA is not possible.

Analysis of morphological markers of chromosomal instability in ascitic fluid.

Chromosomal instability (CI) plays a major role in the carcinogenesis. Micronuclei, nuclear budding, chromatin bridges,and multipolar mitoses are the morphological markers of CI and have never been studied in routine cytological specimens. Aims of the study is to analyze the significance of morphological markers of CI in malignant and benign ascitic fluid smears. A total of sixty benign and 40 malignant ascitic fluid samples were selected for this study. All the cases with malignant ascitic fluid showed histopathological evidence of malignancy in ovary and omentum. Chromatin bridges, multipolar mitosis (MPM), micronuclei and nuclear budding were counted in 1000 cells in representative May Grunwald Giemsa (MGG) stained smears. The CI markers were correlated with the cytological diagnosis of effusion. The mean number of micronuclei, nuclear budding, chromatin bridge and multipolar mitoses found in malignant effusions were 13.2611.79, 10.1067.07, 2.5362.67, 1.964.5, respectively. The mean number of micronuclei, nuclear budding, anaphase bridges, and MPM found in benign effusion cases were 0.566761.07934, 0.516761.33, 0.66760.25, and 0, respectively. The student t test showed significant differences between malignant and benign ascitic fluid samples for each marker of CI. This is the first comprehensive study of morphological markers of CI in ascitic fluid smears. This study has shown strong correlation between markers of CI and cytological diagnosis of malignancy. In future, the knowledge of these markers can be applied to diagnose malignancy in suspected cases of effusion in difficult situations.

Protein p 16INK4A expression in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of uterine cervix.

UNLABELLED: The association of human papilloma virus (HPV) infection and cervical intraepithelial neoplasia (CIN) is well recognized. Interaction of HPV oncogenic proteins with cellular regulatory proteins leads to up regulation of p16 INK4A , a CDK inhibitor, which is a biomarker for HPV infection. We investigated p16 expression in CIN and invasive squamous cell carcinoma (SCC) which has not been reported in the Indian population previously. MATERIALS AND METHODS: Retrospective analysis of 100 cases with 20 cases each of histologically normal cervical epithelium, CIN1, 2, 3 and invasive SCC for p16 expression was performed by immunohistochemistry using commercially available mouse monoclonal antibody to p16 (clone 6H12). STATISTICAL ANALYSIS: For differences in expression among groups, statistical analysis was carried out using ANOVA and post hoc test of Scheffe. RESULTS: p16 immunoreactivity was found to be both nuclear and/or cytoplasmic. The normal cervical epithelium was predominantly negative for p16 (18/20). There was a progressive increase of p16 expression with the grade of CIN. In CIN 1, two cases (20%) showed nuclear and nucleocytoplasmic positivity respectively. In contrast, diffuse strong nuclear or nucleocytoplasmic expression was observed in 45 and 55% cases of CIN 2 and CIN 3 respectively. All except one squamous cell carcinoma stained strongly positive for p16. The difference in expression between CIN 2/3 and SCC versus normal cervix was found highly significant (p is equal to 0.008 and p less than 0.001). CONCLUSIONS: p16 expression correlates excellently with the grade of CIN and is a sensitive marker of cervical intraepithelial neoplasia.