[2018] Role of Magnification in Visual Evaluation of Manual and Automated HPV In-Situ Hybridization in Cell Blocks (CB) from Metastatic Oropharyngeal Carcinoma

Momin T Siddiqui, Cynthia Cohen, Nazneen Fatima. Emory University Hospital, Atlanta, GA

Background: Determination of Human papillomavirus (HPV) status has strong diagnostic, prognostic, and therapeutic implications in oropharyngeal cancers (OC). Determining the integration status of HPV by in situ hybridization (ISH) is cost effective and routinely utilized in clinical practice. However, the reliability of reporting is dependent on individual expertise and visual evaluation. In this study, we have compared the role of magnification in interpreting HPV ISH results of manual versus an automated method, using the Dako and the Enzo probe respectively.
Design: We evaluated 41 CB from fine needle aspiration of metastatic OC to neck lymph nodes. We performed manual HPV ISH with the Dako probe, and Leica Bond-III automation using the Enzo probe. Punctate dot-like nuclear positivity observed with low, intermediate or high magnifications, 10 x, 20 x or 40-60x, was assigned as 3+, 2+ and 1+ respectively. A single cell showing nuclear punctate dot-like staining was considered a positive result.
Results: Two of 41 CB (4%) with manual HPV ISH and 12 of 41 CB (29%) with the automated method positive at 10x magnification, were considered 3+. Fourteen of 41 CB (34%) with manual HPV ISH and 3 of 41 CB (7%) with automation positive at 20x magnification, were 2+. Nine of 41 CB (21%) with manual HPV ISH and 15 of 41 CB (36%) with automation positive at 40-60x magnification, were considered 1+.

HPV ISH (Positive)10X (3+)20X (2+)40-60X (1+)
Manual2/41 (4%)14/41 (34%)9/41 (21%)
Automated12/41 (29%)3/41 (7%)15/41 (36%)

Conclusions: The dot like punctate staining may be very focal which requires careful visual determination of all tumor cells. Low magnification (10x) reveals positivity in 4% of cases with the manual method, whereas the automated technique, being more sensitive, reveals a higher positivity rate (29%). Hence, automation provides a better and more efficient method for evaluating HPV ISH in cases of metastatic OC. Failure to evaluate under high magnification (40-60x) however, could give false negative results in 21% of cases with the manual method and 36% with automation. This could confound the analysis and effect therapeutic decisions.The authors conclude that magnification power plays a critical role in visual evaluation of HPV ISH. Additionally, automated technique of HPV ISH evaluation provided better quality assaurance for this type of testing in their laboratory.
Category: Quality Assurance

Tuesday, March 5, 2013 9:30 AM

Poster Session III # 288, Tuesday Morning


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