Distinctive Association of p16 INK4a Overexpression With Penile Intraepithelial Neoplasia Depicting Warty and/or Basaloid Features: A Study of 141 Cases Evaluating a New Nomenclature Alcides Chaux, MD,* Rolf Pfannl, MD,w Bele´n Lloveras, MD, PhD,zy Marı´a Alejo, MD, PhD,yJ Omar Clavero, MD,y Cecilia Lezcano, MD,* Nubia Mun ˜oz, MPh, MD, PhD,y Silvia de Sanjose´, MD, PhD,yz Xavier Bosch, MPh, MD, PhD,y Marier Herna ´ndez-Pe´rez, MD,# Elsa F. Velazquez, MD,** and Antonio L. Cubilla, MD* Abstract: From the pathogenic point of view, penile cancers may be grouped in human papillomavirus-related and unrelated tumors, each one of them with distinctive morphologic features. The former are predominantly composed of small, undiffer- entiated basaloid cells, with more or less prominent koilocytic changes, and the latter of keratinizing differentiated squamous cells. The same cellular types are observed in precancerous lesions. On the basis of these observations, we constructed a novel nomenclature for penile precancerous lesions and classified them as penile intraepithelial neoplasia (PeIN) of differentiated, warty, basaloid, and warty-basaloid types. The aim of this study was to test the usefulness of immunohisto- chemical p16 INK4a overexpression, considered as a surrogate for high-risk human papillomavirus infection, using this classifica- tion system. We pathologically evaluated 141 patients with PeIN, associated (123 cases) and unassociated (18 cases) with invasive cancer. Distribution of PeIN types was: differentiated, 72%; basaloid, 9%; warty-basaloid, 7%; warty, 4%; and mixed, 7%. There was a striking similarity in the morphology of in situ and invasive squamous cell carcinomas. Differentiated PeIN was commonly associated with usual, verrucous, papillary, and other low-grade keratinizing variants of squamous cell carcinoma whereas in basaloid and warty carcinomas the presence of in situ lesions with similar morphology was habitual. We evaluated p16 INK4a overexpression using a 4-tiered (0, 1, 2, and 3) pattern- based system. To properly distinguish differentiated PeIN from in situ lesions with warty and/or basaloid features only pattern 3, which requires full-thickness staining in all epithelial cells, was considered positive. Using this approach, there was a significant association of the negative patterns and differentiated PeIN and of the positive pattern and warty, basaloid, and warty-basaloid PeIN (P <0.0001). Basaloid variant had the strongest associa- tion. The sensitivity rate of p16 INK4a positivity for discriminat- ing types of PeIN was of 82%, with a specificity of 100% and an accuracy of 95%. Lichen sclerosus was identified in 42 cases and their epithelial component was p16 INK4a negative in all cases. Although more studies are necessary to confirm these observations, p16 INK4a overexpression seems to be a useful tool for discriminating differentiated from warty, basaloid, and warty-basaloid PeIN. Key Words: penile intraepithelial neoplasia, penile cancer, p16 INK4a , HPV (Am J Surg Pathol 2010;34:385–392) I t has been suggested that penile carcinogenesis follows a bimodal pathway, one associated with human papillo- mavirus (HPV) infection and the other related to nonviral factors such as phimosis, chronic inflammation, and lichen sclerosus (LS). 8,43 This hypothesis is based on the frequent association of HPV with basaloid and warty carcinomas and the low or even null HPV detection rate in other well-differentiated squamous cell carcinoma (SCC) subtypes. 10,17,43 HPV-related tumors are partly or entirely composed of small to intermediate basophilic, undifferentiated cells (‘‘basaloid cells’’), with more or less prominent koilocytic changes, whereas HPV-negative tumors are predominantly composed of highly kerati- nized, differentiated squamous cells. 10,27 We have ob- served that these cells are also present in precursor lesions of the penis. On the basis of these findings, we constructed a simplified classification of precancerous lesions based on the morphology and cell differentiation and accord- ingly proposed a subdivision of penile intraepithelial neoplasia (PeIN) in differentiated, warty, basaloid, and warty-basaloid types. 13 This scheme is in consonance with the recently revised classification system for vulvar intraepithelial neoplasia proposed by the International Copyright r 2010 by Lippincott Williams & Wilkins From the *Instituto de Patologı´a e Investigacio´n, Asuncio´n, Paraguay; wTufts Medical Center; #Boston Medical Center; **Brigham’s and Women Hospital, Harvard Medical School, Boston, MA; zHospital de Mar, IMAS-IMIN; yInstituto Catalan de Oncologia, Barcelona; JHospital General de l’Hospitalet, L’Hospitalet de Llobregat; and zCIBER Epidemiologı´a y Salud Pu´blica (CIBERESP), Spain. Disclosure: No financial support was received for this work. Correspondence: Antonio L. Cubilla, MD, Instituto de Patologı´a e Investigacio´n, Martin Brizuela 325, Asuncion, Paraguay (e-mail: acubilla@institutodepatologia.com.py). ORIGINAL ARTICLE Am J Surg Pathol  Volume 34, Number 3, March 2010 www.ajsp.com | 385