P16-Negative, High-Risk Human Papilloma Virus-Associated Penile Squamous Cell Carcinomas

To the Editor: Immunohistochemistry (IHC) for p16 (INK4a/CDKN2A) is generally considered a reliable surrogate marker for high-risk human papilloma virus (HR-HPV)-associated penile squamous cell carcinomas (pSCC).1 Indeed, block-type p16 positivity is recommended as the “most practical and reliable method where morphological association is not straightforward and/or molecular methods are unavailable” in the current edition of the World Health Organization Classification of Urinary and Male Genital Tumors.2 However, p16 IHC has limitations, with false-positive and false-negative results described in a subset of squamous cell carcinomas, both extragenital and genital.1,3–6 For example, in the study by Poetsch et al3, 7 of the 17 pSCC harboring HPV 16 showed negative p16 staining. In our prior study that included 37 HR-HPV–positive pSCC, we had encountered 1 case with discordant results (p16-negative; HPV E6/E7 RNA in situ hybridization [ISH]-positive). Panviral metagenomic sequencing (PVMS) demonstrated HPV16 in this case.1 Herein, we report 2 additional cases of p16-negative pSCCs that harbored HR-HPV (Fig. 1). We repeated p16 IHC to confirm the negative result and used similar methodology (p16 IHC, HPV E6/E7 RNA ISH, and PVMS) to evaluate these cases, as previously described.1,7 In all cases, HPV E6/E7 RNA ISH was positive, with further documentation of HPV genotypes through PVMS available in 2 cases. Of note, all 3 cases exhibited morphologic features typically associated with HR-HPV (Table 1).FIGURE 1.: Representative images of p16-negative high-risk human papilloma virus-associated penile squamous cell carcinomas (A–C, 40× magnification; D–F, 200× magnification; insets, 600× magnification). TABLE 1. - Summary of the p16-Negative, High-Risk HPV-Associated Penile Squamous Cell Carcinomas Included in the Study Case Age at the Time of Procedure (Years) Morphologic Subtype p16 IHC HPV in Situ Hybridization (ISH) Studies Panviral Metagenomic Sequencing Low-Risk DNA ISH (With Probes Targeting HPV 6 and 11) HPV High-Risk DNA ISH (With Probes Targeting HPV 16, 18, 31, 33, and 51) HPV RNA ISH (Targeting E6/E7 Transcripts in High-Risk Genotypes) 1 33 Warty Negative Not performed Not performed Positive HPV 39 2 71 Basaloid Negative Not performed Positive Positive Failed* 3† 65 Medullary Negative Negative Negative Positive HPV 16 *Panviral metagenomic sequencing resulted in insufficient number of reads for interpretation.†Previously reported.1 The reasons behind the false-negative p16 results in the setting of HR-HPV–associated tumors remain to be elucidated. In HR-HPV infection, the viral oncoprotein E7 disrupts the function of the retinoblastoma protein (pRb), which abrogates the inhibitory effect of pRb on p16 transcription.8 However, HPV-independent mechanisms also regulate p16 expression. A study on pSCCs identified HPV-unrelated mechanisms that can interfere with the p16/cyclin D/Rb pathway, such as overexpression of the product of BMI-1 polycomb.8 Another study demonstrated promoter hypermethylation and/or biallelic loss involving the p16INK4A region in a subset of p16-negative HPV16-positive pSCCs.3 A false-negative HPV result is highly unlikely given that the HPV status was confirmed using 2 different methodologies in all 3 cases (Table 1). In 2 cases, PVMS, a highly sensitive technique offering broad and targeted coverage of the viral genome, demonstrated presence of HR-HPV genotypes.1,7 PVMS was performed, but failed in 1 case (case 2) because of insufficient number of reads for interpretation. In this context, possible explanations for the negative p16 IHC results include biallelic loss of function alterations, post-translational events or concomitant non-HPV–mediated, possibly epigenetic, mechanisms that result in downregulation of p16. Several studies from North America1,9,10 demonstrated a survival benefit for HR-HPV–associated pSCCs, which underscores the importance of accurate HPV status determination. False-negative p16 IHC, as illustrated in some case series, can occur in up to 30%–40% of HR-HPV–associated penile tumors.3,4 Conversely, false-positive p16 results may also be observed through upregulation of the p16/cyclin D/Rb pathway through HPV-independent mechanisms.8 Therefore, caution is warranted when p16 IHC is used in isolation as a surrogate for HR-HPV status in pSCCs, because this approach may result in misclassification of HPV status and affect prognostication in a subset of cases. In our opinion, there is utility in reflex testing with HPV E6/E7 RNA ISH or molecular methods in cases where there is a discrepancy between morphology and p16 IHC. Further studies are needed to determine the optimal approach to HPV status assessment in pSCCs.