[360] HPV In-Situ Hybridization: Does Magnification Play a Role in Visual Evaluation?

Nazneen Fatima, Cynthia Cohen, Momin T Siddiqui. Emory University School of Medicine, Atlanta

Background: Oropharyngeal cancers (OC) are rapidly increasing in incidence world-wide. Seventy percent of these cases are attributable to Human papillomavirus (HPV) comprising squamous cell carcinomas occurring mainly in non-smokers and non-drinkers. Determination of HPV status has strong diagnostic, prognostic, and therapeutic implications. Detection of HPV in metastatic cancer in neck lymph nodes may also be used to localize the primary within oropharynx, with a high degree of certainty. 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 analyzed the role of magnification in interpreting HPV ISH results.
Design: We retrospectively evaluated 63 small biopsies of primary OC and 34 FNA cell blocks of metastatic OC by HPV ISH for punctuate dot like nuclear positivity observed with low, intermediate or high magnifications, 10x, 20x or 40-60x. Positivity was assigned as 3+, 2+ and 1+ respectively. A single cell showing nuclear punctuate dot like staining was considered a positive result. A comparison was also made between initial interpretation and our review.
Results: Nineteen of 63 (30%) biopsies and 5 of 34 (14%) CB, positive at 10x magnification were considered 3+. Thirty one of 63 (49%) biopsies and 17 of 34 (50%) CB were positive at 20x magnification and were considered 2+. Thirteen of 63 (20%) biopsies and 12 of 34 (35%) CB showed positivity at 40-60x magnification and were considered 1+.

	10X (3+)	20X (2+)	40X -60X (1+)
Biopsies	19/63 (30%)	31/63 (49%)	13/63 (20%)
CB	5/34 (14%)	17/34 (50%)	12/34 (35%)

CB: Cell Block

A total of 22 biopsies and 16 CB were initially interpreted as negative but our review found them to be positive at 1+ magnification.
Conclusions: Magnification power plays a critical role in visual evaluation of HPV ISH. Although intermediate magnification (20x) reveals the most positivity (50%), it gives false negative results in a significant number of cases (20% biopsies and 35% CB). Failure to evaluate under higher magnification (1+) could confound the analysis and effect therapeutic decisions. The dot like punctuate staining may be very focal which requires careful visual determination of all tumor cells and this may have a learning curve for pathologists performing this evaluation. We recommend that HPV ISH slides should be interpreted at a high microscopic magnification, especially when most tumor cells appear negative.
Category: Cytopathology

Wednesday, March 21, 2012 1:00 PM

Poster Session VI # 94, Wednesday Afternoon