p16/Ki-67 Dual Labeling and Urinary Cytology Results According to the New Paris System for Reporting Urinary Cytology: Impact of Extended Follow-Up Eric Piaton, MD 1,2 ; Anne-Sophie Advenier, MD 2,3 ; Christian Carr e, ScD 4 ; Myriam Decaussin-Petrucci, MD 5,6 ; Florence Me `ge-Lechevallier, MD 2,7 ; Karine Hutin, CT(IAC) 1 ; Cindy Nennig, CT(IAC) 1 ; Marc Colombel, MD 2,8 ; and Alain Ruffion, MD 2,9 BACKGROUND: Overexpression of p16 INK4a has been identified in urothelial malignancies both cytologically and histologi- cally. In addition, p16/Ki-67 dual labeling has been shown to identify high-grade urothelial cancer cells and some progression cases within a 12-month delay. The Paris System for Reporting Urinary Cytology (TPS) was published in late 2015. Its aim is to clarify the criteria for diagnosing or, conversely, excluding high-grade urothelial carcinoma (HGUC). METHODS: Dual label- ing was performed on archived ThinPrep-based Papanicolaou slides. A total of 208 samples (negative for high-grade uro- thelial carcinoma [NHGUC], 59; consistent with low-grade urothelial neoplasia [LGUN], 24; atypical urothelial cells [AUC], 15; and suspicious for or showing HGUC, 110) were analyzed for p16/Ki-67 after reclassification according to TPS. We assessed the oncologic status of the patients with cystoscopy, urinary cytology, histology, and prolonged 36-month fol- low-up data. RESULTS: The sensitivity of p16/Ki-67 for life-threatening lesions was not different from that of urinary cytol- ogy (82.8% vs 83.6%; P 5 1). However, among patients with samples classified as NHGUC and AUC, disease-free survival was significantly shorter for dual-labeled cases versus cases with negative dual labeling (P < .0001). The same tendency was observed in patients with histologically proven LGUN (P < .0001). As for specificity in patients with negative cystos- copy and cytology combined, prolonged follow-up showed 90% overall survival at 24 months. CONCLUSIONS: A long- term evaluation of p16/Ki-67 dual labeling may identify HGUC and progression in cases with negative/low-grade urinary cytology results, and there are potential implications for the clinical management of patients after the conservative treat- ment of non–muscle-invasive urothelial carcinoma. Cancer Cytopathol 2017;000:000–000. V C 2017 American Cancer Society . KEY WORDS: bladder; disease-free survival; Ki-67; p16 INK4a ; prognosis; urinary cytology; urothelial carcinoma. INTRODUCTION The p16 (INK4a) gene, located on chromosome 9p21, is also known as CDKN2A. Overexpression of the p16 INK4a protein has been documented in almost all preneoplastic and carcinomatous lesions of the uterine cervix secondary to interference between the E6 and E7 oncoproteins of high-risk papillomaviruses with the retinoblas- toma (Rb) gene product. 1,2 In urothelial carcinomas, p16 INK4a is most often undetectable 3,4 as it is reported in many nongynecologic carcinomas. 5,6 Homozygote deletions, methylation of 5 0 CpG islands, and mutations of CDKN2A have been reported as possible causes of a loss of p16 INK4a expression. 7–10 Conversely, p16 overexpres- sion has been reported in urothelial lesions, 11–13 particularly in 80% of high-grade tumors and approximately Corresponding author: Eric Piaton, MD, Centre de Pathologie Est, Hospices Civils de Lyon, H^ opital Femme-Me `re-Enfant, 59 Boulevard Pinel, 69677 Bron Cedex, France; Fax: (011) 033 472-35-70-67; eric.piaton@chu-lyon.fr 1 Centre de Pathologie Est, H^ opitaux de Lyon, H^ opital Femme-Me `re-Enfant, Bron, France; 2 Universit e Claude Bernard Lyon 1, Lyon, France; 3 Service de M edecine L egale, Lyon, France; 4 Roche Diagnostics France, Meylan, France; 5 UMR INSERM U 1052/CNRS 5286, Universit e Claude Bernard Lyon 1, France; 6 Centre de Pathologie Sud, Centre Hospitalier Lyon Sud, Pierre B enite, France; 7 Service d’Anatomie et Cytologie Pathologiques, H^ opital Edouard Herriot, Lyon, France; 8 Service d’Urologie, H^ opital Edouard Herriot, Lyon, France; 9 Service d’Urologie, Centre Hospitalier Lyon Sud, Pierre B enite, France. Received: November 25, 2016; Revised: January 4, 2017; Accepted: January 4, 2017 Published online Month 00, 2017 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21853, wileyonlinelibrary.com Cancer Cytopathology Month 2017 1 Original Article