Diagnostic utility of high-risk human papillomavirus mRNA in situ hybridisation in squamous cell carcinoma of unknown primary in the head and neck and implementing American Society of Clinical Oncology guideline recommendations.

INTRODUCTION: The American Society of Clinical Oncology (ASCO)-endorsed College of American Pathologists guideline recommends high-risk human papillomavirus (HPV) testing for metastatic squamous cell carcinoma (SCC) of lymph nodes level II/III of unknown primary. Herein, the performance of HPV-RNA in situ hybridisation (ISH) in detection of HPV-related SCC is evaluated implementing the ASCO guideline recommendations. METHODS: Eighty head and neck (HN) SCC fine needle aspirations, which utilized HPV-RNA ISH/P16, were evaluated at Johns Hopkins Hospital (2015-2018) to investigate their performance and concordance with histology. The results were compared to a prior study of 59 HNSCCs, which HPV-DNA ISH. RESULTS: Of the 80 reviewed fine needle aspirations, 65 (50 male, 15 female) were included. The mean age was 63.2 ± 14.0 years. The most common site was neck lymph nodes (47, 72.3%). Fifty-five cases (84.6%) were accompanied by concurrent core biopsy, and 48 cases (59.4%) had surgical follow-ups. HPV-RNA ISH was positive in 44 (67.7%), and P16 was strongly positive in 46 (70.8%). The HPV-RNA ISH/ P16 concordance rate was 92.3% on cytology material. The cytology/surgical concordance rate for HPV-RNA ISH was 88.9% (16/18). There was a discordance between the results in five cases (7.7%; HPV-RNA ISH-/P16+). CONCLUSION: HPV-RNA ISH is a robust and reliable method for detecting HPV-related HNSCC on cytology material showing concordance rate of 92.3% between HPV-RNA ISH and P16, which is a sensitive but non-specific marker. Compared to HPV-DNA ISH, HPV-RNA ISH reproducibly identifies HPV-related HNSCC with fewer discrepancies between cytology and histology. The findings of this study are in agreement with the ASCO recommendations.

Assessing the diagnostic accuracy for pleomorphic adenoma and Warthin tumor by employing the Milan System for Reporting Salivary Gland Cytopathology: An international, multi-institutional study.

BACKGROUND: The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) has established distinct diagnostic categories for reporting cytopathological findings, and each is associated with a defined risk of malignancy (ROM). However, the ROM is applied at the overall category level and is not specific for particular morphological entities within a category. Here, the diagnostic performance of the MSRSGC for pleomorphic adenoma (PA) and Warthin tumor (WT) is reported. METHODS: The pathology archives of 11 institutions from 4 countries were retrospectively searched to identify all salivary gland fine-needle aspiration (FNA) biopsies with a differential or definitive diagnosis of PA or WT and all resection specimens with a diagnosis of PA or WT; only paired cases were included. All FNA diagnoses were retrospectively classified according to the MSRSGC. RESULTS: A total of 1250 cases met the inclusion criteria, and they included 898 PA cases and 352 WT cases. The ROM in the benign neoplasm category was 3.0% and 1.3% for cases with a differential or definitive diagnosis of PA and WT, respectively. The ROM in the salivary gland neoplasm with uncertain malignant potential (SUMP) category was 2.7% and 18.8% for PA and WT, respectively (P = .0277). The diagnostic accuracy for PA and WT was 95.1% and 96.1%, respectively. CONCLUSIONS: The diagnostic accuracy for PA and WT on FNA is high. Furthermore, these findings highlight the difference in the ROMs associated with 2 specific differential diagnoses in the SUMP category: basaloid neoplasms and oncocytoid neoplasms.

"Suspicious" salivary gland FNA: Risk of malignancy and interinstitutional variability.

BACKGROUND: Fine-needle aspiration (FNA) cytology is well accepted as a safe, reliable, minimally invasive, and cost-effective method for the diagnosis of salivary gland lesions. Salivary gland neoplasms are often difficult to diagnose because of morphologic heterogeneity and a variety of epithelial metaplastic changes. Hence, a number of salivary gland FNA specimens yield indeterminate results. For indeterminate FNA specimens, the suspicious-for-malignancy (SFM) category is used when a specific neoplasm falls short in quantity or quality for the criteria for malignancy. Therefore, the findings are not sufficient for a conclusive diagnosis of malignancy. METHODS: This study was designed to evaluate the risk of malignancy (ROM) for the SFM group at 5 tertiary medical centers worldwide with the aforementioned criteria. Among 12,606 salivary gland FNA cases between 1997 and 2014, 276 (2.2%) were reported to be SFN. Specifically, 114 suspicious cases (41%) had histological follow-up. RESULTS: Histological follow-up of the 114 suspicious cases showed 95 malignant tumors indicating a risk of malignancy (ROM) of 83.3%. The ROM varied between 74% and 88% for the 5 participating institutions, and a Fisher's exact test with significance set to p<.05 showed no significant difference in ROM among the institutions (p = .78). CONCLUSIONS: Overall, 83.3% of SFM salivary gland FNA specimens turned out to be malignant; there was no significant interinstitutional variability in the ROMs. The SFM category for salivary gland FNA is very homogeneous, and the ROMs are quite similar worldwide. Cancer Cytopathol 2018;126:94-100. © 2017 American Cancer Society.

Refractory inflammatory bowel disease: is there a role for Epstein-Barr virus? A case-controlled study using highly sensitive Epstein-Barr virus-encoded small RNA1 in situ hybridization.

A potential role for viral infections has been implicated in inflammatory bowel disease (IBD) unresponsive to medical treatment. It is well known that Epstein-Barr virus (EBV) infection can elicit a brisk mononuclear response in the gastrointestinal tract. The aim of this study was to further evaluate the role of EBV in patients with refractory IBD and compare them with nonrefractory IBD cases. Surgically resected colonic specimens from 67 patients with refractory IBD (62 with ulcerative colitis, 3 patients with Crohn disease, and 2 patients with indeterminate colitis) were retrieved. Twelve colectomy specimens from patients with ulcerative colitis who had undergone resections for dysplasia or endometriosis were included as controls. Highly sensitive EBV-encoded small RNA1 (EBER-1) in situ hybridization was performed on a representative block from each specimen. EBER-1 reactivity was graded as absent, focal, or diffuse. EBV was detected in 60% (40/67) of patients with refractory IBD compared with 25% (3/12) of the control group (P < .05). Focal EBER-1 positivity was present in 45% of cases of refractory IBD compared with 25% of controls. Diffuse EBER-1 reactivity was seen in 15% of cases of refractory IBD (10/67); none of the samples from the control group contained diffuse EBER-1 positivity. There was a positive correlation between EBER positivity and depth of inflammation and mucosal ulceration in patients with refractory IBD. Our findings suggest a potential role for EBV infection in patients with refractory IBD.

Interpretation of HPV DNA in situ hybridization in HPV-related head and neck squamous cell carcinoma: an achievable task in cell block and small biopsy material.

INTRODUCTION: Human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is a distinct entity with a better prognosis than conventional disease. Therefore, an accurate and reproducible HPV test is needed. Herein, the analytical factors and interpretation of the HPV DNA in-situ hybridization (ISH) test are investigated. MATERIALS AND METHODS: We evaluated 63 ultrasound-guided fine-needle aspiration (FNA) HNSCC cases for a semi-quantitative assessment of the ease of interpretation, staining pattern, and highest magnification needed for HPV DNA ISH on cell block and core biopsy. RESULTS: A total of 72 HPV DNA ISH tests were performed in 59 (93.6%) cases. Of these, 17 had more than one HPV DNA ISH assays and 4 (6.4%) had no HPV tests. At least one HPV stain was positive in 38 (62.2%) cases. Eleven (28.95%) ISH tests were rated as difficult or moderately difficult to interpret, and 27 (78.05%) were rated as easy or moderately easy. Twenty-four (63.2%) ISH tests demonstrated strong staining and 14 (36.8%) demonstrated weak staining. Twenty-seven (71.1%) stained diffusely, and 11 (29.0%) focally. Twenty-seven ISH tests required 400× or higher magnification for interpretation. Background debris and nonspecific staining were present in 25 (35.7%) and 15 (21.4%) HPV DNA ISH cases, respectively. p16/HPV ISH was discrepant in 4 (7.3%) cases (3 P16+/HPV-, and 1 p16-/HPV+). CONCLUSIONS: HPV DNA ISH interpretation can be challenging because of focal or weak staining, which requires careful examination at high magnification. An alternate method is needed for DNA ISH-/p16+ cases.

Molecular Profiling of Malignant Pleural Effusion in Metastatic Non-Small-Cell Lung Carcinoma. The Effect of Preanalytical Factors.

RATIONALE: Non-small-cell lung cancer (NSCLC)-associated malignant pleural effusions (MPEs) are sometimes the only available specimens for molecular analysis. OBJECTIVES: This study evaluates diagnostic yield of NSCLC-associated MPE, its adequacy for molecular profiling and the potential influence of MPE volume/cellularity on the analytic sensitivity of our assays. METHODS: Molecular results of 50 NSCLC-associated MPE cases during a 5-year period were evaluated. Molecular profiling was performed on cell blocks and consisted of fluorescent in situ hybridization (FISH) for ALK gene rearrangements and the following sequencing platforms: Sanger sequencing (for EGFR) and high-throughput pyrosequencing (for KRAS and BRAF) during the first 4 years of the study period, and targeted next-generation sequencing performed thereafter. RESULTS: A total of 50 NSCLC-associated MPE cases were identified where molecular testing was requested. Of these, 17 cases were excluded: 14 cases (28%) due to inadequate tumor cellularity and 3 cases due to unavailability of the slides to review. A total of 27 out of 50 MPE cases (54%) underwent at least EGFR and KRAS sequencing and FISH for ALK rearrangement. Of the 27 cases with molecular testing results available, a genetic abnormality was detected in 16 cases (59%). The most common genetic aberrations identified involved EGFR ( 9 ) and KRAS ( 7 ). Six cases had ALK FISH only, of which one showed rearrangement. MPE volume was not associated with overall cellularity or tumor cellularity (P = 0.360). CONCLUSIONS: Molecular profiling of MPE is a viable alternative to testing solid tissue in NSCLC. This study shows successful detection of genetic aberrations in 59% of samples with minimal risk of false negative.