Human Papillomavirus Detection in Scrotal Squamous Cell Carcinoma: Case Series from a Population-Based Cancer Registry

Abstract

Introduction:

Scrotal squamous cell carcinomas (SCCs) are rare malignancies that are not considered to be associated with the human papillomavirus (HPV) by the International Agency for Research on Cancer. However, recent studies have detected HPV in these cancers. We sought to determine the presence of HPV types among scrotal cancer cases identified through population-based cancer registries.

Methods:

Primary scrotal SCCs diagnosed from 2014 to 2015 were identified, and tissue sections from formalin-fixed, paraffin-embedded tissue blocks were obtained for laboratory testing. A pathology review was performed to confirm morphology. HPV testing was performed using L1 consensus polymerase chain reaction analysis. Immunohistochemistry was used to evaluate p16INK4a (p16) expression.

Results:

Five cases of scrotal SCC were identified from 1 cancer registry. Age at diagnosis ranged from 34 to 75 years (median, 56 years). Four cases were non-Hispanic White, and 1 was non-Hispanic Black. The morphologic subtype of 4 cases was keratinizing (usual), and 1 case was verrucous (warty) histologic subtype. Two of the usual cases of SCC were HPV-negative and p16-negative, and 2 were positive for HPV16 and p16. The verrucous (warty) SCC subtype case was HPV6-positive and p16-negative.

Conclusions:

The presence of HPV16 and p16 overexpression in the examined tissue specimens lends additional support for the role of HPV in the etiology of scrotal SCC.

Introduction

Oncogenic types of human papillomavirus (HPV) are known to cause cancers of the cervix, vagina, vulva, anus, penis, and oropharynx.¹ Studies with small sample sizes have reported detection of oncogenic HPV DNA in scrotal squamous cell carcinomas (SCCs)^2-5; however, scrotal SCCs are not currently considered an HPV-associated cancer by the International Agency for Research on Cancer.⁶

Scrotal cancers are rare malignancies. From 2015 to 2019, an average of approximately 260 cases per year were reported in the United States.⁷ From 1973 to 2002, scrotal cancer incidence rates nearly doubled from 0.049 to 0.095 per 100,000.⁸ Although scrotal cancer incidence rates have remained stable in recent years, the magnitude of rates is higher than previously reported, with an incidence rate of 0.21 per 100,000 in 2019.

Routine population-based tracking of HPV types among HPV-associated cancers is not currently conducted in the United States. Most studies of HPV type prevalence in HPV-associated cancers have been performed in limited geographic areas without population-based sampling strategies. The Centers for Disease Control and Prevention (CDC) has supported 2 special studies covering data from 2004 and 2005 (study 1) and 2014 and 2015 (study 2) that facilitated the development of the Cancer Registry Sentinel Surveillance System (CRSSS), which used central cancer registries to obtain tissue samples from HPV-associated cancers to determine HPV type prevalence.^1,9 CDC's CRSSS provides a novel framework for population-based sampling of HPV-associated tissue for genotyping and monitoring HPV prevalence. By using data collected from scrotal SCC cases identified in the second study during 2014 and 2015, our objectives were to: (1) identify the HPV types present in scrotal SCC cancers derived from cancer registries; (2) determine the p16INK4a (p16) overexpression in the same scrotal tissues by using immunohistochemistry; and (3) confirm the histologic subtypes of the scrotal SCC cases. This report will add information to the limited data on the etiologic role of HPV in scrotal cancers.

Materials and Methods

Study Population and Design

We analyzed data from CDC's CRSSS, which included data from 3 central cancer registries: Iowa, Kentucky, and Louisiana. Scrotal SCC cancers diagnosed in 2014 and 2015 (years covered by the CRSSS) were identified by the cancer registries using the International Classification of Diseases for Oncology, Third Edition (ICD-O-3) site (C63.2) and histology codes. All malignant cases (with behavior code 3) were included, except for melanomas (8720–8790), sarcomas (8800–8991), mesotheliomas (9050–9055), Kaposi sarcomas (9140), and leukemias and lymphomas (9590–9992). Eligible cases were tracked back to the pathology laboratories where the tumor tissues were stored. Eleven histologically eligible cases were identified from 2 cancer registries: 1 case from Kentucky and 10 cases from Louisiana. No eligible cases were identified in Iowa. The case from Kentucky could not be retrieved from the pathology laboratory. Among the remaining 10 eligible cases, 3 cases could not be shared by the pathology laboratories, and 1 case was ineligible because of out-of-state residence. Paraffin-embedded tissue from the remaining 6 samples was sent to the CDC HPV laboratory for slide preparation and HPV typing. One of the samples sent to the CDC HPV laboratory contained the wrong tissue type and was therefore excluded. All protocols were reviewed and approved by the institutional review boards (IRBs) of all participating organizations and CDC. Tissue collection was performed as part of cancer registry operations with all patient identifiers removed; therefore, no written informed consent was required by the IRBs.

Pathology and Laboratory Methods

Tissue processing, histology review, and laboratory methods have been described previously.^1,10 Briefly, central pathology laboratories associated with the cancer registries were asked to select 1 representative-archived, formalin-fixed, paraffin-embedded (FFPE) tissue block from each scrotal cancer case. Tissue sections were prepared by taking eight 5-µm sections from each block and performing hematoxylin and eosin staining on the first and last sections. Two unstained sections were placed in each of 2 tubes for DNA extraction, and 2 unstained slides were prepared for immunohistochemistry. All tissue blocks were processed with a standardized protocol to prevent contamination of samples.

Both high-risk (including HPV 16 and 18) and low-risk (including HPV 6 and 11) HPV types were tested. Primary testing was conducted with Linear Array (LA, Roche Diagnostics), which detects 37 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52 [XR], 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, 89, IS39) and human β-globin. HPV negative and inadequate (HPV negative and β-globin negative) tissues were retested with the RHA kit HPV SPF-10-LiPA25, version 1 (Labo Biomedical Products B.V.) that detects 25 types (HPV 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 70, 74). P16 immunohistochemistry was performed by using Ventana BenchMark XT automated system with monoclonal anti-p16INK4a (clone E6H4 in CINtec p16 assay, Ventana/Roche) and ultraView Universal DAB Detection kit (Ventana/Roche). A positive tissue control (FFPE cell pellet of HPV-positive cancer cell line) was included with each assay.

All HPV typing and p16 immunohistochemistry were conducted at the CDC HPV laboratory using standardized procedures. Cancer registries provided demographic and clinical data about each case, including age at diagnosis, race and ethnicity, and SEER Summary tumor stage at diagnosis. Interpretation of p16 results (p16 positive, p16 negative, or inadequate) was performed by a pathologist using light microscopic examination of slides processed with and without primary antibody using established criteria.¹¹

The original pathology review was performed by pathology laboratories and hospitals associated with the cancer registries. Two additional board-certified anatomic pathologists examined the tissue samples collected by the central pathology laboratory to confirm the reporting facility's tumor histology diagnosis. The reviewers also reached consensus and further classified SCCs into 1 of 3 common histologic subtypes (usual, warty, or basaloid).

Results

A total of 5 scrotal cancer tissue samples were analyzed. Patient age ranged from 34 to 75 years, with a median age of 56 years. Four cases were in non-Hispanic White men, and 1 was in a non-Hispanic Black man (Table 1).

Table 1.

Characteristics of Scrotal Squamous Cell Carcinoma Cases (n = 5), Centers for Disease Control and Prevention Cancer Registry Sentinel Surveillance System, 2014–2015

	Scrotal SCC cases
Characteristic	1	2	3	4	5
Age, y	30–39	40–49	50–59	70–79	70–79
Race/ethnicity	NH White	NH Black	NH White	NH White	NH White
Histologic classification	Keratinizing SCC, usual	Keratinizing SCC, usual	Verrucous SCC, warty	Keratinizing SCC, usual	Keratinizing SCC, usual
HPV genotype	HPV16	HPV16	HPV6	HPV-negative	HPV-negative
p16 overexpression	Positive	Positive	p16 negative	p16 negative	p16 negative

HPV, human papillomavirus; NH, non-Hispanic; SCC, squamous cell carcinoma

Representative images of histopathology and immunohistochemistry are presented in Figure 1 (A–E). Two of the scrotal SCCs were given morphologic code 8070 (SCC, not otherwise specified [NOS]) by the central pathology laboratory. These cases were reclassified by our study reviewers as SCC, keratinizing type (ICD-0-3 code, 8071). Review by the central pathology laboratory resulted in classification of 4 of the 5 scrotal SCCs as keratinizing SCC, usual type and 1 as warty/verrucous.

Figure 1.

Scrotal Squamous Cell Carcinoma Cases Hematoxylin and Eosin (H&E) Histological Images with Associated p16INK4a (p16) Expression Pattern on Immunohistochemistry, Centers for Disease Control and Prevention Cancer Registry Sentinel Surveillance System, 2014–2015

HPV-Positive Cases

Three SCCs (60%) were positive for HPV DNA. All had single-type infections. Of the cases positive for HPV, 2 were positive for HPV16 and 1 was positive for HPV6. Both cases positive for HPV16 were keratinizing SCCs and had p16 overexpression with diffuse nuclear and cytoplasmic p16 staining (cases 1 and 2). Of note, these 2 HPV16 SCC cases occurred in men aged younger than 50 years. The SCC classified as warty/verrucous was positive for HPV6 and was p16 negative.

HPV-Negative Cases

Two keratinizing SCCs, usual type were negative for HPV DNA (cases 4 and 5). Both cases were negative for p16 overexpression.

Discussion

HPV has been previously detected in scrotal SCC tissue, but its rarity among the population limits most studies to small sample sizes or case series. In our study of 5 scrotal SCCs, 2 cases of keratinizing SCC with usual histological subtype tested positive for HPV16 and demon-strated p16 overexpression. Although a small sample, our findings add further support for considering scrotal SCCs as HPV-associated cancers, similar to HPV-associated vulvar cancer.¹²

A retrospective chart review conducted in 3 Rhode Island institutions identified 10 invasive scrotal SCC cases diagnosed during 1993 to 2003 and detected oncogenic HPV in 3 of the samples, all with p16 overexpression.⁵ The same authors conducted a follow-up study to determine the specific HPV types present. They found that 5 in 10 cases of invasive scrotal SCCs were positive for oncogenic HPV, 2 of whom were positive for HPV16.⁴ In another series of 6 scrotal SCCs from various geographic locations (Australia, Nigeria, and Spain), HPV16 was detected in basaloid and warty scrotal SCCs, but not usual SCC.² The authors hypothesized that usual SCC appeared to be associated with a p53 mutation—the protein associated with the TP53 tumor suppressor gene—where warty and basaloid subtypes were related to HPV infection.²

In our case series, we found that 1 case with verrucous (warty) carcinoma tested positive for HPV6, an HPV type associated with genital warts. HPV6 has been found in some anogenital SCCs, and its potential oncogenic role requires further study.^13-15 In addition, it is possible that some of the tumors gave false-negative HPV results due to inadequate preservation, nonrepresentative sampling, copy number below the limit of detection, or HPV types not included in the assay. Similar to vulvar cancer, HPV-negative scrotal cases could originate from alternative etiologic pathways.

Scrotal SCC was once most commonly associated with occupational exposure to carcinogens, including oil, soot, tar, and paraffin.¹⁶ More recently, scrotal SCC is less associated with occupational hazards. Instead, scrotal SCC is linked with HPV exposure, immunocompromised states, and chronic skin infections.¹⁶ The pathobiology of scrotal SCC is not well described, but the etiology of scrotal SCC is thought to resemble penile and vulvar SCC. According to the 2022 World Health Organization Classification of Tumors for Urinary System and Male Genital Organs, the skin of the penis and scrotum share similar skin histology and potential risk factors for skin damage or cancer.¹⁷ The possible dual pathway of oncogenesis via (1) HPV16-related carcinogenesis, by which the viral proteins E6 and E7 alter normal tumor suppression by the TP53 and retinoblastoma genes (RB), or (2) via direct p53 mutation, would align with observed pathologic characteristics of penile cancers, perhaps reflecting unique skin histology in this anatomic region. Both HPV16-positive and HPV-negative cancers of the scrotum have been associated with overexpression of p53, indicating dysregulation of normal tumor suppression and possibly serving as a prognostic marker.³ Some researchers have posited a potential association between HPV16 infection and these cancer subtypes, speculating that the squamous epithelium in the scrotal region has unique histological characteristics that may predispose to high-risk HPV infection (ie, stem cell-like or genital-like embryology despite the anatomic location on the skin).^2,17

The introduction of the HPV vaccine has had an impact on the occurrence of cervical precancers¹⁸ as well as vulvar, vaginal, and anal precancers.¹⁹ Since 2006, the 4-valent vaccine, which protects against HPV types 6/11/16/18, has been available in the United States. As of 2015, the 9-valent vaccine is the only vaccine being distributed in the United States, which includes the types included in the 4-valent vaccine, plus 5 additional oncogenic types. In 2006, routine vaccination with the HPV vaccine among girls aged 11 to 12 years was recommended to prevent infection from HPV16 and HPV18, which cause nearly 70% of cervical cancers and 90% of HPV-associated anogenital cancers.²⁰ Current recommendations include routine vaccination with the 9-valent HPV vaccine for all persons aged 11 to 12 years, and catch-up vaccination for those who have not been adequately vaccinated through age 26 years.²¹ Shared clinical decision-making is recommended for persons aged 26 to 45 years.²¹ HPV vaccination coverage in the United States has gradually increased since its introduction. The percentage of adolescents aged 13 to 17 years receiving at least 1 dose of HPV vaccine increased from approximately 30% in 2011 to 76% in 2022.²² Given its rarity, there are no currently accepted screening methods for scrotal cancer at the population level, so primary prevention with HPV vaccine is particularly important.

Although the International Agency for Research on Cancer does not currently consider scrotal SCC an HPV-associated cancer,⁶ experts have confirmed that it plays a role,² but larger studies are needed to replicate findings. Currently, there is no systematic effort to routinely determine the HPV types in HPV-associated cancer cases in the United States. In this study, CDC used a novel methodology to obtain HPV-associated cancer tissue, leveraging partnerships with population-based cancer registries to obtain cancer tissue for HPV typing. A major limitation of our study was the small number of scrotal cancer cases from whom we were able to obtain tissue. Although our ability to obtain more samples and additional prior years of data was limited in the current study, this framework could be applied to future studies. In addition, we were limited to sociodemographic data available in the cancer registry, which does not include information on other relevant factors, including occupational exposures, sexual history, or immunocompromising conditions, such as HIV infection. There may be future opportunities to improve the data through linkages, such as links to immunization registries. In addition, we focused on only 2 markers most related to HPV-associated cancers, HPV DNA, and p16 overexpression. Other studies have used additional markers to help determine true cancer etiology, which would be helpful in a future iteration of this study. These markers include mRNA, which can confirm HPV is present and the demonstration of viral integration.

In our case series of 5 scrotal SCCs, 3 were HPV-positive, providing further evidence supporting a potential causal link between HPV and this rare cancer. With the reduction in environmental exposures that have traditionally caused SCC, HPV infection may become a more important etiologic factor in scrotal SCC. Currently, scrotal SCC is not captured in HPV-associated cancer surveillance summaries, given its low frequency and the evolving but limited data to support a causal role of HPV. Additional studies with larger sample sizes will be crucial to elucidate the role that HPV plays in the development of scrotal cancer. Continued monitoring of scrotal cancer incidence rates will be important in evaluating the potential effects of the HPV vaccine on this cancer.