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Role of human papillomavirus infection in the etiology of vulvar cancer in Italian women

Role of human papillomavirus infection in the etiology of vulvar cancer in Italian women Background: Vulvar squamous cell carcinoma (VSCC) is a rare malignancy of the female genital tract. We aimed to determine the mucosal high-risk human papillomavirus (HPV)-attributable fraction of VSCCs from Italian women using multiple markers of viral infections. Methods: VSCCs and 8 metastatic lymph node samples from 107 Italian women were analyzed by a highly type- specific multiplex genotyping assay for the presence of DNA from 119 different HPVs. Tissues were further analyzed INK4a for HPV RNA and for upregulation of the cellular protein p16 . Results: The rate of mucosal HPV-related tumors defined by viral DNA and RNA positivity was low (7.8%). HPV16 INK4a was the most prevalent, followed by 53, 56, and 58. Only five (4.9%) p16 -positive tumors were also positive for both viral DNA and RNA. One (14.3%) metastatic lymph node sample was positive for all three markers. DNA of cutaneous HPVs was detected in only two VSCCs, i.e. genus beta types 5 and 110. Conclusion: A small proportion of Italian VSCCs is putatively HPV-related, i.e. positive for both viral DNA and RNA of the same type, thus reinforcing the importance of HPV vaccination. Moreover, this study suggests that a direct role of HPV from genus beta and gamma in vulvar carcinogenesis is unlikely. Keywords: Cancer, Human papillomavirus, Lymph node metastatic tissues, Vulvar squamous cell carcinoma, Multiple markers of viral infections Introduction chemotherapy/radiotherapy [5], with negative psychosex- Vulvar squamous cell carcinoma (VSCC) is a rare tumor ual consequences [1, 6]. VSCC includes two distinct types, of the female genital tract, accounting for about 5% of all with apparently different etiology: (i) VSCC associated gynecological malignancies [1, 2]. In North America, the with infection with mucosal high-risk (HR) human papil- incidence of VSCC has increased by 0.6% annually in the lomaviruses (HPVs), and (ii) HPV-independent VSCC [7, past decade [1, 3, 4]. Moreover, tumor nodal recurrence is 8]. HPV-related VSCC is more common in younger typically fatal [1]. The primary treatment approach for women and is always preceded by a pre-malignant lesion, VSCC is surgery, complemented in selected cases by i.e. vulvar high-grade squamous intraepithelial lesion (VHSIL) [9, 10]. HPV-independent VSCC occurs mainly in older women [1, 11] and arises from a chronic inflam- * Correspondence: gheitt@iarc.fr; icb@iarc.fr International Agency for Research on Cancer, Lyon, France matory dermatosis, referred to as lichen sclerosus [12, 13], Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 2 of 8 that can evolve into differentiated vulvar intraepithelial types in the anogenital tract [24, 25]. In addition, several neoplasia (VIN), a precursor lesion of VSCC [14]. investigations have highlighted the transforming proper- Based on a pooled prevalence rate of HPV DNA, 34% ties of genus beta and gamma HPV types in various ex- of vulvar cancers have been reported to be attributable perimental models [26–28]. to HPV [15]. In this study, we aimed to determine the HPV- Although detection of HR-HPV DNA is a valid testing attributable proportion of VSCC by analyzing HPV approach for the identification of pre-malignant and ma- DNA and RNA status, and by determining p16 expres- lignant cervical lesions, its use for other anatomical sites, sion level, in a large retrospective cohort of VSCC cases including the vulva, is insufficient proof for viral causal- from Italy. In addition, we examined the possible role of ity and can lead to misclassification of the lesion [16, genus beta and/or gamma HPV types in VSCC. 17]. Therefore, the use of additional markers for active HR-HPV infections, such as overexpression of the hu- Materials and methods INK4a man cyclin-dependent kinase-4 inhibitor p16 (p16) Sample collection or/and detection of viral RNA, may enable more precise The Ethics Committee of the Hospital of Turin approved estimation of the proportion of VSCC that may be at- this study to be in compliance with the Declaration of tributable to HPV, as previously shown in head and neck Helsinki (reference number 1005). All patients had signed cancer [16]. informed consent at the time of hospital admission. The As recently suggested, a useful protocol for the identi- inclusion criteria were: invasive vulvar carcinoma (i.e. an fication of HPV-related VSCC is the simultaneous detec- invasive depth > 1 mm), squamous cell histological type, tion of HPV DNA and RNA [16, 17]. and no prior surgical treatment for vulvar cancer or any The evaluation of p16 protein expression by immuno- other malignancy (apart from biopsy). The exclusion histochemistry (IHC) enables discrimination between criteria were: primary site other than vulva, recurrent tu- HPV-independent and HPV-related cancers at anatom- mors, HIV- or hepatitis C virus (HCV)-positive patients, ical sites outside the cervix, including the vulva [17–21]. and non-consenting patients. However, because there are a limited number of studies Formalin-fixed, paraffin-embedded (FFPE) VSCC samples evaluating p16 expression in VSCC, the actual reliability obtained from 107 women (mean [±standard deviation (SD)] of p16 protein expression as a single marker of onco- age, 73 [11.8] years) collected during 2013–2016 were genic HPV infection for this tumor entity is not entirely retrieved from the archive of the Department of Obstetrics known [17, 18, 22, 23]. Detection of both HPV DNA and Gynecology, University of Turin, Sant’Anna Hospital, and p16 overexpression to classify the two different Turin, Italy. According to the International Federation of VSCC subtypes has also been proposed, but for VSCC Gynecology and Obstetrics (FIGO) classification of vulvar this algorithm has not yet been fully validated [17, 18, cancer [29], the VSCCs were in the following stages: IB 21]. However, the detection of viral transcripts as a (54.2%); II (0.9%); IIIA (19.6%); IIIB (11.2%); IIIC (4.7%); and marker of biological HPV activity in the lesion tissue, in IVA (9.3%). The mean tumor diameter was 29 mm (SD, 15.8 addition to HPV DNA and p16 overexpression, may sig- mm), and 42.1, 45.8, and 12.1% of tumors showed depth of nificantly improve the classification of truly HPV- invasion of < 5 mm, 5–12mm,and>12mm,respectively.Ar- positive VSCC. chived FFPE metastatic lymph node samples (MTS) were also A multicenter study using HPV DNA and p16 positiv- available from 8 women with VSCC tissue (mean [±SD] age, ity as markers has shown that approximately 25% of vul- 69 [16.5] years). var cancers worldwide may be associated with HPV [18]. This study, which included tumor specimens collected Preparation of paraffin sections and tumor evaluation from cancer cases in 39 countries on five continents, Ten sections of 10 μm from each FFPE tissue block were highlighted some variability in HPV positivity in VSCC processed at the Department of Pathology, University of in the different geographical regions. However, some Turin, Italy. Sections S1 and S10 were used for geographical variations in HPV-positive VSCC may be hematoxylin and eosin histology, and S2 and S9 were due simply to the analysis of a relatively low number of used for p16 IHC. In addition, S3–S5 and S6–S8 were specimens in specific countries. Thus, additional studies collected for DNA and RNA extraction, respectively. To are required in individual countries, where only limited avoid cross-contamination between different samples, information on the association between HR-HPV and each time a new case was sectioned, the microtome was VSCC is available. extensively washed with DNA Away (Dutscher, Regarding HR-HPV-negative VSCC, the involvement Brumath, France) and a new microtome blade was used. of other infectious agents, including the cutaneous genus Empty paraffin blocks were cut after every 10 cancer beta and gamma HPV types, has been poorly investi- specimens and blindly analyzed. Tumor size/status was gated. Studies have reported the presence of these HPV documented by routine pathologic evaluation, in Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 3 of 8 accordance with the World Health Organization (WHO) the specific HR- and pHR-HPV types(s) detected. In classification [30]. addition, tissues that were DNA-positive for LR-HPV6 or 11 were analyzed for the presence of unspliced E6 mRNA Nucleic acid extraction of HPV6 and 11, respectively. RNA detection from FFPE Total DNA was prepared by incubating overnight at 37 °C sections was performed as previously described [32]. three internal FFPE sections (S3–S5) from each VSCC/ Briefly, 1 μl of extracted RNA was subjected to a one-step MTS tissue in 250 μl of digestion buffer (10 mM Tris/HCl reverse transcription PCR protocol with the QuantiTect at pH 7.4, 0.5 mg/ml proteinase K, and 0.4% Tween 20). Virus Kit (Qiagen, Hilden, Germany) using HPV type- Then, to inactivate the proteinase K and to separate paraf- specific primers to amplify 65–75 bp cDNA sequences fin from the aqueous phase, samples were incubated at across the E6*I splice sites [32]. The biotinylated strands 95 °C for 10 min, centrifuged, and chilled on ice [31]. The of the amplicons were detected by hybridization with aqueous phase was transferred to a new tube. type- and splice site-specific oligonucleotide probes Total RNA was prepared from tissue sections S6–S8 coupled to fluorescence-labelled Luminex beads (Luminex using the PureLink FFPE Total RNA Isolation Kit (Invitro- Corp., Austin, TX, USA). The E6*I mRNA assays are gen, Carlsbad, CA, USA). DNase I (RNase-Free DNase available for 20 HR- or pHR-HPV types for which exist- Set, Qiagen, Hilden, Germany) treatment was carried out ence of splice sites was demonstrated [32]. For detection on the RNA purifying columns during sample processing of unspliced LR-HPV6 and 11 mRNA, primers designed as previously described [32]. Extracted RNA was eluted in to amplify a 77 bp amplicon of the full-length HPV6 or 11 50 μl of RNase-free water and stored at − 80 °C until use. E6 gene, and an oligonucleotide probe for detection of full-length HPV11 E6, were applied. HPV DNA detection and genotyping Samples that were negative in duplicate assays for HPV DNA was detected by E7 type-specific multiplex HPV E6*I or E6 and for UbC mRNA were considered genotyping (E7-MPG), which combines multiplex poly- RNA-invalid. merase chain reaction (PCR) and hybridization to type- specific oligonucleotide probes on fluorescent beads p16 immunohistochemistry (Luminex Corp., Austin, TX, USA) [33, 34]. E7-MPG Expression of p16 was evaluated manually by IHC in uses HPV type-specific primers targeting the E7 region FFPE sections using the CINtec p16 Histology Kit of 12 HR-HPVs (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, (Roche mtm laboratories AG, Mannheim, Germany) ac- 56, 58, 59), 7 possible/probable HR (pHR)-HPVs cording to the manufacturer’s instructions and as previ- (HPV26, 53, 66, 68, 70, 73, 82), and two low-risk (LR) ously described [42]. Expression of p16 was evaluated by HPVs (HPV6 and 11). Forty-six genus beta HPVs IHC in all VSCC and MTS cases. Continuous, diffuse (HPV5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, nuclear and cytoplasmic staining in 70% or more of the 37, 38, 47, 49, 75, 76, 80, 92, 93, 96, 98, 99, 100, 104, tumor cells was considered p16-positive, and focal stain- 105, 107, 110, 111, 113, 115, 118, 120, 122, 124, 143, ing or no staining was considered p16-negative. All 145, 150, 151, 152, 159, 174) and 52 genus gamma HPVs slides were evaluated three times by three different eval- (HPV4, 48, 50, 60, 65, 88, 95, 101, 103, 108, 109, 112, uators (SSP, RW, RR), who were blinded to the clinical 116, 119, 121, 123, 126, 127, 128, 129, 130, 131, 132, and epidemiological data. The final classification of the 133, 134, 148, 149, 156, 161, 162, 163, 164, 165, 166, staining was based on the majority consensus. 167, 168, 169, 170, 171, 172, 173, 175, 178, 179, 180, 184, 197, 199, 200, 201, 202, SD2) [35–41] were detected using the same methodology. Results The sensitivity has been evaluated using serial dilu- HPV infection markers in VSCCs tions of DNA from HPV types. This multiplex PCR Of 107 VSCC and 8 MTS cases initially selected, five protocol is highly sensitive, with the ability to detect VSCC cases and one MTS case were excluded due to in- only 10 copies of the viral genome [34, 40]. valid RNA (n =3) and p16 (n = 2) data, or to the absence of cancer tissue in the block (n= 1). Thus, a total of 102 HPV mRNA analysis VSCC and 7 MTS tissues were included in the analysis. All HPV DNA-positive VSCC/MTS tissues, including The 102 VSCC cases had a mean age of 70 years (SD, HPV16 and non-HPV16 DNA-positive samples, and a 12.8 years); FIGO stages were IB (52.9%), II (1%), IIIA group of 10 randomly selected HPV DNA-negative tissues (20.6%), IIIB (10.8%), IIIC (4.9%), and IVA (9.8%). Mean were analyzed for HPV16 E6*I mRNA and, as a control tumor diameter was 30 mm (SD, 15.7 mm) and mean for tissue and RNA quality, for human UbC mRNA. Tis- depth of stromal invasion 8 mm (SD, 6.3 mm), with 40.2, sues that were DNA-positive for HR- and pHR-HPV types 47.1 and 12.7% of tumors showing depth of invasion of < other than HPV16 were also analyzed for E6*I mRNA of 5mm, 5–12 mm, and > 12 mm, respectively. Furthermore, Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 4 of 8 MTS selected from 7 cases were analyzed (mean [±SD] positivity, the percentage of HPV-related VSCCs was age, 73.9 [13.5] years). 7.8% (8/102), i.e. HPV16 (n = 6), HPV53 (n = 1), HPV56 Table 1 and Supplementary Table S1 show the HPV (n = 1), and HPV58 (n = 1). One VSCC case simultan- DNA, RNA, and p16 detection in VSCC and MTS cases. eously tested positive for HPV DNA and RNA from Mucosal HPV DNA was detected in 17 of 102 (16.7%) HPV16 and HPV56. Of the 7 mucosal HPV DNA- VSCCs and all 7 MTS cases (100%). HPV16 was the most positive MTS cases, only one (14.3%) had E6*I mRNA of prevalent type; it was present in 10 of the 17 HPV DNA- the same type (HPV16). None of the HPV6 DNA- positive VSCCs (58.8%), followed by HPV6 (2/17, 11.8%), positive cases tested positive for full-length HPV6 E6 HPV18 (1/17, 5.9%), HPV53 (1/17, 5.9%), HPV56 (1/17, mRNA. 5.9%), HPV58 (1/17, 5.9%), and HPV82 (1/17, 5.9%). Only Overexpression of the cell-cycle inhibitor p16 is a one multiple infection was detected, containing HPV16, well-validated surrogate marker of HPV transformation 18, and 56. All MTS cases were positive for mucosal HPV in the cervix [43]. We determined the p16 IHC positivity DNA. Five MTS cases contained a single HPV type, i.e. in all 102 VSCC and 7 MTS cases (Fig. 1). Eleven VSCC HPV16 (n = 1), HPV6 (n = 3), and HPV56 (n = 1). Multiple cases were positive for p16 (11/102, 10.8%). Of these, HPV infections were detected in two MTS cases; both five were also HPV DNA- and RNA-positive (Table 1). were positive for HPV6 and HPV16 (Table 1). Matched Thus, when all three markers were considered, the per- VSCC and MTS cases of three women were positive for centage of HPV-related VSCC cases dropped from 7.8 to the same HPV DNA, i.e. HPV6 (n = 2) and HPV16 (n =1). 4.9% (5/102) (Table 1). The remaining p16-positive cases There was no concordance for the HPV types in the (n = 6) were HPV DNA-positive and HPV RNA-negative remaining MTS cases (n = 4) and the corresponding (n = 2) or HPV DNA-negative and HPV RNA-negative VSCCs (Supplementary Table S1). (n = 2), or were not tested for HPV DNA and RNA (n = To evaluate the etiological role of the mucosal HPV 2). Only one MTS case showed p16 overexpression (1/7, detected, all 17 HPV DNA-positive VSCC and all 7 MTS 14.3%), and was also positive for HPV16 DNA and RNA. cases were analyzed for the presence of HR- and pHR- The corresponding VSCC from the same case was also HPV E6*I and unspliced E6 for LR-HPV (Table 1). All positive for all three markers. 24 HPV DNA-positive VSCC and MTS cases were ubC The presence of DNA of cutaneous beta and gamma mRNA-positive. Considering both HPV DNA and RNA HPV in cytological samples of the genital tract has been Table 1 HPV DNA, RNA, and p16 positivity in 102 VSCC and 7 MTS cases VSCC (N = 102) MTS (N =7) HPV type Marker positivity Positive Positive n (%) n (%) Any HPV DNA 17 (16.7) 7 (100.0) DNA and RNA 8 (7.8) 1 (14.3) DNA, RNA, and p16 5 (4.9) 1 (14.3) HPV16 single infection DNA 9 (8.8) 1 (14.3) DNA and RNA 5 (4.9) 0 (0.0) DNA, RNA, and p16 4 (3.9) 0 (0.0) b d Mucosal HPV other than HPV16 single infections DNA 7 (6.9) 4 (57.1) DNA and RNA 2 (2.0) 0 (0.0) DNA, RNA, and p16 0 (0.0) 0 (0.0) e g Multiple infections DNA 1 (1.0) 2 (28.6) f h DNA and RNA 1 (1.0) 1 (14.3) DNA, RNA, and p16 1 (1.0) 1 (14.3) HPV Human papillomavirus, MTS Metastatic lymph node samples, VSCC Vulvar squamous cell carcinoma HPV RNA was examined in 17 VSCC and 7 MTS HPV DNA-positive cases and a randomly selected subset of HPV DNA-negative cases (n = 10). p16 expression was examined in all VSCC (n = 102) and MTS (n = 7) cases Single infections: HPV6 (n = 2), HPV18 (n = 1), HPV53 (n = 1), HPV56 (n = 1), HPV58 (n = 1), HPV82 (n =1) Single infections: HPV53 (n = 1), HPV58 (n =1) Single infections: HPV6 (n = 3), HPV56 (n =1) Coinfection: HPV16, HPV18, and HPV56 (n =1) Coinfection: HPV16 and HPV56 (n =1) Coinfection: HPV6 and HPV16 (n =2) Single infection: HPV16 (n =1) Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 5 of 8 ink4a ink4a ink4a Fig. 1 p16 immunohistochemistry staining. Negative p16 staining (a), and diffuse nuclear and cytoplasmic p16 staining (b) in VSCC cases. Magnification X10 reported [24, 37, 44]. We also determined the presence of HPV DNA-positive VSCC cases, with HPV16 RNA of DNA of 46 genus beta and 52 genus gamma HPV showing the highest prevalence. Eight of 102 VSCC types in all VSCC and MTS cases. Only two VSCC cases cases (7.8%) were likely to be HPV-driven, i.e. positive were positive for beta HPV types (i.e. HPV5 and for both HPV DNA and RNA. None of the HPV6 DNA- HPV110), and no gamma HPV types were detected in positive samples tested positive for HPV RNA. The up- the VSCC and MTS cases analyzed. regulation of p16 has been suggested as a surrogate marker for oncogenic HR-HPV infections, and p16 IHC Discussion has frequently been used not only to determine and/or Mucosal HR-HPV types have been clearly associated confirm positivity for HPV in various tumors but also to with the development of a small subset of VSCC. How- define HPV-related tumors [19]. Therefore, p16 expres- ever, the contribution of the viral infectious agents ap- sion has also been studied in VSCC before [17, 22, 48, pears to vary in different geographical regions [15]. In 49]. In the present study, this cellular marker was over- particular, limited information is available for the contri- expressed in approximately 50% of HR-HPV DNA- bution of HR-HPV to VSCC development in Italy. In positive VSCC cases (7/15). Other series of VSCC stud- this study, we analyzed a large number of VSCCs col- ies previously showed 70–80% of HPV DNA-positive lected in a hospital in northern Italy during 2013–2016. VSCC cases expressing p16 [17, 46]. However, in our In the first step, we investigated the presence of HPV study, p16 overexpression was observed in the majority DNA in VSCCs by using a sensitive HPV genotyping ap- (5/8, 62.5%) of HR-HPV DNA- and RNA-positive proach covering all oncogenic and probably/possibly VSCCs. A lower p16 positivity rate in HR-HPV DNA- oncogenic HPV types. The prevalence of HPV DNA- negative VSCCs (4/85, 4.7%) and in HR-HPV DNA- positive cases was observed to be nearly 17%. Previous positive and HPV RNA-negative VSCCs (2/7, 28.6%) was studies on VSCC reported HPV DNA frequencies ran- also detected. Overexpression of p16 in HPV DNA- ging from 29 to 45% [17, 18, 21, 45, 46]. However, the negative VSCCs may be a consequence of either a muta- overall results indicate that HPV could have a role in the tion of CDKN2A, the gene coding for p16 [50], or the development of a proportion of VSCC cases. In agree- inactivation of the retinoblastoma protein pRb (e.g. by ment with a previous study [17], we observed HPV16 to mutation), which then leads to upregulation of p16 [51]. be the most common HR-HPV type detected in VSCC. Previous reports showed higher concordance between In addition, viral DNA from HPV18, 53, 56, 58, and 82 the HR-HPV transcript expression and p16 overexpres- was also detected, with low frequency. The LR-HPV type sion, ranging from 83 to 90% [17, 21, 49]. The causes of 6 was found to be the second most common viral type this discrepancy between our results and those of previ- in this study, suggesting that it may be involved, together ous published studies are unknown. with other factors, in VSCC onset or progression [17, In this study, we were also able to retrieve MTSs for a 46]. Recently, Faber et al. (2017) reported a prevalence small number of patients with VSCC. Only one case of 6.8% (19/277) for HPV6 DNA as a single infection in showed concordance in HPV DNA/RNA positivity and VIN, and 1.1% (6/527) in VSCC [47]. p16 expression between VSCC and MTS. Because HPV transcriptional activity may reflect its The investigation of the presence of HR-HPV E6 oncogenic activity, we applied a HPV-type-specific E6*I mRNA instead of analyzing E6 oncoprotein, which may mRNA detection method, established for 20 HR- or be more informative on the oncogenic implications of pHR-HPV types and validated for use in FFPE tissues, to HR-HPVs in VSCC development, may be considered a evaluate the presence of HPV RNA in HPV DNA- limitation of this study. However, we focused our atten- positive VSCCs. We observed viral RNA in 47.1% (8/17) tion on evaluating the expression of the p16 protein, Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 6 of 8 which is considered a surrogate marker for all trans- Ethics approval and consent to participate The Ethics Committee of the Hospital of Turin approved this study to be in forming HR-HPV infections and represents a single tar- compliance with the Declaration of Helsinki (reference number 1005). All get molecule, different from the E6 proteins with type- patients had signed informed consent at the time of hospital admission. specific polypeptide sequence variability. Finally, although independent studies have reported that Consent for publication Not applicable. cutaneous beta and gamma HPV types can be detected in the genital tract, our study does not provide evidence for Competing interests their involvement in the development of VSCC. R. Ridder is an employee of Roche. All other authors report no conflict of interest. Conclusion Author details We characterized the HPV-independent and HPV- Department of Surgical Sciences, University of Turin, Turin, Italy. 2 3 International Agency for Research on Cancer, Lyon, France. Department of related VSCCs in a cohort of more than 100 VSCC cases Morphology, Surgery and Experimental Medicine; Section of Pathology, from Italy. We identified as putative HPV-related tumors Oncology and Experimental Biology; Laboratories of Cell Biology and a small proportion of VSCC cases that tested positive for Molecular Genetics, University of Ferrara, Ferrara, Italy. Infections and Cancer Epidemiology, Infections and Cancer Program, German Cancer Research both HPV DNA and RNA. Our results support the Center (DKFZ), Heidelberg, Germany. Department of Oncology, Città della causative role of HPV in vulvar carcinogenesis in a dis- Salute e della Scienza, University of Turin, Turin, Italy. Roche mtm tinct subset of cases and highlight the prevalence of laboratories, Mannheim, Germany. Ventana Medical Systems Inc., Tucson, AZ, USA. HPV16, thus reinforcing the importance of HPV vaccin- ation programs. Received: 15 January 2020 Accepted: 23 March 2020 Supplementary information References Supplementary information accompanies this paper at https://doi.org/10. 1. Clancy AA, Spaans JN, Weberpals JI. 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Role of human papillomavirus infection in the etiology of vulvar cancer in Italian women

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Abstract

Background: Vulvar squamous cell carcinoma (VSCC) is a rare malignancy of the female genital tract. We aimed to determine the mucosal high-risk human papillomavirus (HPV)-attributable fraction of VSCCs from Italian women using multiple markers of viral infections. Methods: VSCCs and 8 metastatic lymph node samples from 107 Italian women were analyzed by a highly type- specific multiplex genotyping assay for the presence of DNA from 119 different HPVs. Tissues were further analyzed INK4a for HPV RNA and for upregulation of the cellular protein p16 . Results: The rate of mucosal HPV-related tumors defined by viral DNA and RNA positivity was low (7.8%). HPV16 INK4a was the most prevalent, followed by 53, 56, and 58. Only five (4.9%) p16 -positive tumors were also positive for both viral DNA and RNA. One (14.3%) metastatic lymph node sample was positive for all three markers. DNA of cutaneous HPVs was detected in only two VSCCs, i.e. genus beta types 5 and 110. Conclusion: A small proportion of Italian VSCCs is putatively HPV-related, i.e. positive for both viral DNA and RNA of the same type, thus reinforcing the importance of HPV vaccination. Moreover, this study suggests that a direct role of HPV from genus beta and gamma in vulvar carcinogenesis is unlikely. Keywords: Cancer, Human papillomavirus, Lymph node metastatic tissues, Vulvar squamous cell carcinoma, Multiple markers of viral infections Introduction chemotherapy/radiotherapy [5], with negative psychosex- Vulvar squamous cell carcinoma (VSCC) is a rare tumor ual consequences [1, 6]. VSCC includes two distinct types, of the female genital tract, accounting for about 5% of all with apparently different etiology: (i) VSCC associated gynecological malignancies [1, 2]. In North America, the with infection with mucosal high-risk (HR) human papil- incidence of VSCC has increased by 0.6% annually in the lomaviruses (HPVs), and (ii) HPV-independent VSCC [7, past decade [1, 3, 4]. Moreover, tumor nodal recurrence is 8]. HPV-related VSCC is more common in younger typically fatal [1]. The primary treatment approach for women and is always preceded by a pre-malignant lesion, VSCC is surgery, complemented in selected cases by i.e. vulvar high-grade squamous intraepithelial lesion (VHSIL) [9, 10]. HPV-independent VSCC occurs mainly in older women [1, 11] and arises from a chronic inflam- * Correspondence: gheitt@iarc.fr; icb@iarc.fr International Agency for Research on Cancer, Lyon, France matory dermatosis, referred to as lichen sclerosus [12, 13], Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 2 of 8 that can evolve into differentiated vulvar intraepithelial types in the anogenital tract [24, 25]. In addition, several neoplasia (VIN), a precursor lesion of VSCC [14]. investigations have highlighted the transforming proper- Based on a pooled prevalence rate of HPV DNA, 34% ties of genus beta and gamma HPV types in various ex- of vulvar cancers have been reported to be attributable perimental models [26–28]. to HPV [15]. In this study, we aimed to determine the HPV- Although detection of HR-HPV DNA is a valid testing attributable proportion of VSCC by analyzing HPV approach for the identification of pre-malignant and ma- DNA and RNA status, and by determining p16 expres- lignant cervical lesions, its use for other anatomical sites, sion level, in a large retrospective cohort of VSCC cases including the vulva, is insufficient proof for viral causal- from Italy. In addition, we examined the possible role of ity and can lead to misclassification of the lesion [16, genus beta and/or gamma HPV types in VSCC. 17]. Therefore, the use of additional markers for active HR-HPV infections, such as overexpression of the hu- Materials and methods INK4a man cyclin-dependent kinase-4 inhibitor p16 (p16) Sample collection or/and detection of viral RNA, may enable more precise The Ethics Committee of the Hospital of Turin approved estimation of the proportion of VSCC that may be at- this study to be in compliance with the Declaration of tributable to HPV, as previously shown in head and neck Helsinki (reference number 1005). All patients had signed cancer [16]. informed consent at the time of hospital admission. The As recently suggested, a useful protocol for the identi- inclusion criteria were: invasive vulvar carcinoma (i.e. an fication of HPV-related VSCC is the simultaneous detec- invasive depth > 1 mm), squamous cell histological type, tion of HPV DNA and RNA [16, 17]. and no prior surgical treatment for vulvar cancer or any The evaluation of p16 protein expression by immuno- other malignancy (apart from biopsy). The exclusion histochemistry (IHC) enables discrimination between criteria were: primary site other than vulva, recurrent tu- HPV-independent and HPV-related cancers at anatom- mors, HIV- or hepatitis C virus (HCV)-positive patients, ical sites outside the cervix, including the vulva [17–21]. and non-consenting patients. However, because there are a limited number of studies Formalin-fixed, paraffin-embedded (FFPE) VSCC samples evaluating p16 expression in VSCC, the actual reliability obtained from 107 women (mean [±standard deviation (SD)] of p16 protein expression as a single marker of onco- age, 73 [11.8] years) collected during 2013–2016 were genic HPV infection for this tumor entity is not entirely retrieved from the archive of the Department of Obstetrics known [17, 18, 22, 23]. Detection of both HPV DNA and Gynecology, University of Turin, Sant’Anna Hospital, and p16 overexpression to classify the two different Turin, Italy. According to the International Federation of VSCC subtypes has also been proposed, but for VSCC Gynecology and Obstetrics (FIGO) classification of vulvar this algorithm has not yet been fully validated [17, 18, cancer [29], the VSCCs were in the following stages: IB 21]. However, the detection of viral transcripts as a (54.2%); II (0.9%); IIIA (19.6%); IIIB (11.2%); IIIC (4.7%); and marker of biological HPV activity in the lesion tissue, in IVA (9.3%). The mean tumor diameter was 29 mm (SD, 15.8 addition to HPV DNA and p16 overexpression, may sig- mm), and 42.1, 45.8, and 12.1% of tumors showed depth of nificantly improve the classification of truly HPV- invasion of < 5 mm, 5–12mm,and>12mm,respectively.Ar- positive VSCC. chived FFPE metastatic lymph node samples (MTS) were also A multicenter study using HPV DNA and p16 positiv- available from 8 women with VSCC tissue (mean [±SD] age, ity as markers has shown that approximately 25% of vul- 69 [16.5] years). var cancers worldwide may be associated with HPV [18]. This study, which included tumor specimens collected Preparation of paraffin sections and tumor evaluation from cancer cases in 39 countries on five continents, Ten sections of 10 μm from each FFPE tissue block were highlighted some variability in HPV positivity in VSCC processed at the Department of Pathology, University of in the different geographical regions. However, some Turin, Italy. Sections S1 and S10 were used for geographical variations in HPV-positive VSCC may be hematoxylin and eosin histology, and S2 and S9 were due simply to the analysis of a relatively low number of used for p16 IHC. In addition, S3–S5 and S6–S8 were specimens in specific countries. Thus, additional studies collected for DNA and RNA extraction, respectively. To are required in individual countries, where only limited avoid cross-contamination between different samples, information on the association between HR-HPV and each time a new case was sectioned, the microtome was VSCC is available. extensively washed with DNA Away (Dutscher, Regarding HR-HPV-negative VSCC, the involvement Brumath, France) and a new microtome blade was used. of other infectious agents, including the cutaneous genus Empty paraffin blocks were cut after every 10 cancer beta and gamma HPV types, has been poorly investi- specimens and blindly analyzed. Tumor size/status was gated. Studies have reported the presence of these HPV documented by routine pathologic evaluation, in Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 3 of 8 accordance with the World Health Organization (WHO) the specific HR- and pHR-HPV types(s) detected. In classification [30]. addition, tissues that were DNA-positive for LR-HPV6 or 11 were analyzed for the presence of unspliced E6 mRNA Nucleic acid extraction of HPV6 and 11, respectively. RNA detection from FFPE Total DNA was prepared by incubating overnight at 37 °C sections was performed as previously described [32]. three internal FFPE sections (S3–S5) from each VSCC/ Briefly, 1 μl of extracted RNA was subjected to a one-step MTS tissue in 250 μl of digestion buffer (10 mM Tris/HCl reverse transcription PCR protocol with the QuantiTect at pH 7.4, 0.5 mg/ml proteinase K, and 0.4% Tween 20). Virus Kit (Qiagen, Hilden, Germany) using HPV type- Then, to inactivate the proteinase K and to separate paraf- specific primers to amplify 65–75 bp cDNA sequences fin from the aqueous phase, samples were incubated at across the E6*I splice sites [32]. The biotinylated strands 95 °C for 10 min, centrifuged, and chilled on ice [31]. The of the amplicons were detected by hybridization with aqueous phase was transferred to a new tube. type- and splice site-specific oligonucleotide probes Total RNA was prepared from tissue sections S6–S8 coupled to fluorescence-labelled Luminex beads (Luminex using the PureLink FFPE Total RNA Isolation Kit (Invitro- Corp., Austin, TX, USA). The E6*I mRNA assays are gen, Carlsbad, CA, USA). DNase I (RNase-Free DNase available for 20 HR- or pHR-HPV types for which exist- Set, Qiagen, Hilden, Germany) treatment was carried out ence of splice sites was demonstrated [32]. For detection on the RNA purifying columns during sample processing of unspliced LR-HPV6 and 11 mRNA, primers designed as previously described [32]. Extracted RNA was eluted in to amplify a 77 bp amplicon of the full-length HPV6 or 11 50 μl of RNase-free water and stored at − 80 °C until use. E6 gene, and an oligonucleotide probe for detection of full-length HPV11 E6, were applied. HPV DNA detection and genotyping Samples that were negative in duplicate assays for HPV DNA was detected by E7 type-specific multiplex HPV E6*I or E6 and for UbC mRNA were considered genotyping (E7-MPG), which combines multiplex poly- RNA-invalid. merase chain reaction (PCR) and hybridization to type- specific oligonucleotide probes on fluorescent beads p16 immunohistochemistry (Luminex Corp., Austin, TX, USA) [33, 34]. E7-MPG Expression of p16 was evaluated manually by IHC in uses HPV type-specific primers targeting the E7 region FFPE sections using the CINtec p16 Histology Kit of 12 HR-HPVs (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, (Roche mtm laboratories AG, Mannheim, Germany) ac- 56, 58, 59), 7 possible/probable HR (pHR)-HPVs cording to the manufacturer’s instructions and as previ- (HPV26, 53, 66, 68, 70, 73, 82), and two low-risk (LR) ously described [42]. Expression of p16 was evaluated by HPVs (HPV6 and 11). Forty-six genus beta HPVs IHC in all VSCC and MTS cases. Continuous, diffuse (HPV5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, nuclear and cytoplasmic staining in 70% or more of the 37, 38, 47, 49, 75, 76, 80, 92, 93, 96, 98, 99, 100, 104, tumor cells was considered p16-positive, and focal stain- 105, 107, 110, 111, 113, 115, 118, 120, 122, 124, 143, ing or no staining was considered p16-negative. All 145, 150, 151, 152, 159, 174) and 52 genus gamma HPVs slides were evaluated three times by three different eval- (HPV4, 48, 50, 60, 65, 88, 95, 101, 103, 108, 109, 112, uators (SSP, RW, RR), who were blinded to the clinical 116, 119, 121, 123, 126, 127, 128, 129, 130, 131, 132, and epidemiological data. The final classification of the 133, 134, 148, 149, 156, 161, 162, 163, 164, 165, 166, staining was based on the majority consensus. 167, 168, 169, 170, 171, 172, 173, 175, 178, 179, 180, 184, 197, 199, 200, 201, 202, SD2) [35–41] were detected using the same methodology. Results The sensitivity has been evaluated using serial dilu- HPV infection markers in VSCCs tions of DNA from HPV types. This multiplex PCR Of 107 VSCC and 8 MTS cases initially selected, five protocol is highly sensitive, with the ability to detect VSCC cases and one MTS case were excluded due to in- only 10 copies of the viral genome [34, 40]. valid RNA (n =3) and p16 (n = 2) data, or to the absence of cancer tissue in the block (n= 1). Thus, a total of 102 HPV mRNA analysis VSCC and 7 MTS tissues were included in the analysis. All HPV DNA-positive VSCC/MTS tissues, including The 102 VSCC cases had a mean age of 70 years (SD, HPV16 and non-HPV16 DNA-positive samples, and a 12.8 years); FIGO stages were IB (52.9%), II (1%), IIIA group of 10 randomly selected HPV DNA-negative tissues (20.6%), IIIB (10.8%), IIIC (4.9%), and IVA (9.8%). Mean were analyzed for HPV16 E6*I mRNA and, as a control tumor diameter was 30 mm (SD, 15.7 mm) and mean for tissue and RNA quality, for human UbC mRNA. Tis- depth of stromal invasion 8 mm (SD, 6.3 mm), with 40.2, sues that were DNA-positive for HR- and pHR-HPV types 47.1 and 12.7% of tumors showing depth of invasion of < other than HPV16 were also analyzed for E6*I mRNA of 5mm, 5–12 mm, and > 12 mm, respectively. Furthermore, Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 4 of 8 MTS selected from 7 cases were analyzed (mean [±SD] positivity, the percentage of HPV-related VSCCs was age, 73.9 [13.5] years). 7.8% (8/102), i.e. HPV16 (n = 6), HPV53 (n = 1), HPV56 Table 1 and Supplementary Table S1 show the HPV (n = 1), and HPV58 (n = 1). One VSCC case simultan- DNA, RNA, and p16 detection in VSCC and MTS cases. eously tested positive for HPV DNA and RNA from Mucosal HPV DNA was detected in 17 of 102 (16.7%) HPV16 and HPV56. Of the 7 mucosal HPV DNA- VSCCs and all 7 MTS cases (100%). HPV16 was the most positive MTS cases, only one (14.3%) had E6*I mRNA of prevalent type; it was present in 10 of the 17 HPV DNA- the same type (HPV16). None of the HPV6 DNA- positive VSCCs (58.8%), followed by HPV6 (2/17, 11.8%), positive cases tested positive for full-length HPV6 E6 HPV18 (1/17, 5.9%), HPV53 (1/17, 5.9%), HPV56 (1/17, mRNA. 5.9%), HPV58 (1/17, 5.9%), and HPV82 (1/17, 5.9%). Only Overexpression of the cell-cycle inhibitor p16 is a one multiple infection was detected, containing HPV16, well-validated surrogate marker of HPV transformation 18, and 56. All MTS cases were positive for mucosal HPV in the cervix [43]. We determined the p16 IHC positivity DNA. Five MTS cases contained a single HPV type, i.e. in all 102 VSCC and 7 MTS cases (Fig. 1). Eleven VSCC HPV16 (n = 1), HPV6 (n = 3), and HPV56 (n = 1). Multiple cases were positive for p16 (11/102, 10.8%). Of these, HPV infections were detected in two MTS cases; both five were also HPV DNA- and RNA-positive (Table 1). were positive for HPV6 and HPV16 (Table 1). Matched Thus, when all three markers were considered, the per- VSCC and MTS cases of three women were positive for centage of HPV-related VSCC cases dropped from 7.8 to the same HPV DNA, i.e. HPV6 (n = 2) and HPV16 (n =1). 4.9% (5/102) (Table 1). The remaining p16-positive cases There was no concordance for the HPV types in the (n = 6) were HPV DNA-positive and HPV RNA-negative remaining MTS cases (n = 4) and the corresponding (n = 2) or HPV DNA-negative and HPV RNA-negative VSCCs (Supplementary Table S1). (n = 2), or were not tested for HPV DNA and RNA (n = To evaluate the etiological role of the mucosal HPV 2). Only one MTS case showed p16 overexpression (1/7, detected, all 17 HPV DNA-positive VSCC and all 7 MTS 14.3%), and was also positive for HPV16 DNA and RNA. cases were analyzed for the presence of HR- and pHR- The corresponding VSCC from the same case was also HPV E6*I and unspliced E6 for LR-HPV (Table 1). All positive for all three markers. 24 HPV DNA-positive VSCC and MTS cases were ubC The presence of DNA of cutaneous beta and gamma mRNA-positive. Considering both HPV DNA and RNA HPV in cytological samples of the genital tract has been Table 1 HPV DNA, RNA, and p16 positivity in 102 VSCC and 7 MTS cases VSCC (N = 102) MTS (N =7) HPV type Marker positivity Positive Positive n (%) n (%) Any HPV DNA 17 (16.7) 7 (100.0) DNA and RNA 8 (7.8) 1 (14.3) DNA, RNA, and p16 5 (4.9) 1 (14.3) HPV16 single infection DNA 9 (8.8) 1 (14.3) DNA and RNA 5 (4.9) 0 (0.0) DNA, RNA, and p16 4 (3.9) 0 (0.0) b d Mucosal HPV other than HPV16 single infections DNA 7 (6.9) 4 (57.1) DNA and RNA 2 (2.0) 0 (0.0) DNA, RNA, and p16 0 (0.0) 0 (0.0) e g Multiple infections DNA 1 (1.0) 2 (28.6) f h DNA and RNA 1 (1.0) 1 (14.3) DNA, RNA, and p16 1 (1.0) 1 (14.3) HPV Human papillomavirus, MTS Metastatic lymph node samples, VSCC Vulvar squamous cell carcinoma HPV RNA was examined in 17 VSCC and 7 MTS HPV DNA-positive cases and a randomly selected subset of HPV DNA-negative cases (n = 10). p16 expression was examined in all VSCC (n = 102) and MTS (n = 7) cases Single infections: HPV6 (n = 2), HPV18 (n = 1), HPV53 (n = 1), HPV56 (n = 1), HPV58 (n = 1), HPV82 (n =1) Single infections: HPV53 (n = 1), HPV58 (n =1) Single infections: HPV6 (n = 3), HPV56 (n =1) Coinfection: HPV16, HPV18, and HPV56 (n =1) Coinfection: HPV16 and HPV56 (n =1) Coinfection: HPV6 and HPV16 (n =2) Single infection: HPV16 (n =1) Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 5 of 8 ink4a ink4a ink4a Fig. 1 p16 immunohistochemistry staining. Negative p16 staining (a), and diffuse nuclear and cytoplasmic p16 staining (b) in VSCC cases. Magnification X10 reported [24, 37, 44]. We also determined the presence of HPV DNA-positive VSCC cases, with HPV16 RNA of DNA of 46 genus beta and 52 genus gamma HPV showing the highest prevalence. Eight of 102 VSCC types in all VSCC and MTS cases. Only two VSCC cases cases (7.8%) were likely to be HPV-driven, i.e. positive were positive for beta HPV types (i.e. HPV5 and for both HPV DNA and RNA. None of the HPV6 DNA- HPV110), and no gamma HPV types were detected in positive samples tested positive for HPV RNA. The up- the VSCC and MTS cases analyzed. regulation of p16 has been suggested as a surrogate marker for oncogenic HR-HPV infections, and p16 IHC Discussion has frequently been used not only to determine and/or Mucosal HR-HPV types have been clearly associated confirm positivity for HPV in various tumors but also to with the development of a small subset of VSCC. How- define HPV-related tumors [19]. Therefore, p16 expres- ever, the contribution of the viral infectious agents ap- sion has also been studied in VSCC before [17, 22, 48, pears to vary in different geographical regions [15]. In 49]. In the present study, this cellular marker was over- particular, limited information is available for the contri- expressed in approximately 50% of HR-HPV DNA- bution of HR-HPV to VSCC development in Italy. In positive VSCC cases (7/15). Other series of VSCC stud- this study, we analyzed a large number of VSCCs col- ies previously showed 70–80% of HPV DNA-positive lected in a hospital in northern Italy during 2013–2016. VSCC cases expressing p16 [17, 46]. However, in our In the first step, we investigated the presence of HPV study, p16 overexpression was observed in the majority DNA in VSCCs by using a sensitive HPV genotyping ap- (5/8, 62.5%) of HR-HPV DNA- and RNA-positive proach covering all oncogenic and probably/possibly VSCCs. A lower p16 positivity rate in HR-HPV DNA- oncogenic HPV types. The prevalence of HPV DNA- negative VSCCs (4/85, 4.7%) and in HR-HPV DNA- positive cases was observed to be nearly 17%. Previous positive and HPV RNA-negative VSCCs (2/7, 28.6%) was studies on VSCC reported HPV DNA frequencies ran- also detected. Overexpression of p16 in HPV DNA- ging from 29 to 45% [17, 18, 21, 45, 46]. However, the negative VSCCs may be a consequence of either a muta- overall results indicate that HPV could have a role in the tion of CDKN2A, the gene coding for p16 [50], or the development of a proportion of VSCC cases. In agree- inactivation of the retinoblastoma protein pRb (e.g. by ment with a previous study [17], we observed HPV16 to mutation), which then leads to upregulation of p16 [51]. be the most common HR-HPV type detected in VSCC. Previous reports showed higher concordance between In addition, viral DNA from HPV18, 53, 56, 58, and 82 the HR-HPV transcript expression and p16 overexpres- was also detected, with low frequency. The LR-HPV type sion, ranging from 83 to 90% [17, 21, 49]. The causes of 6 was found to be the second most common viral type this discrepancy between our results and those of previ- in this study, suggesting that it may be involved, together ous published studies are unknown. with other factors, in VSCC onset or progression [17, In this study, we were also able to retrieve MTSs for a 46]. Recently, Faber et al. (2017) reported a prevalence small number of patients with VSCC. Only one case of 6.8% (19/277) for HPV6 DNA as a single infection in showed concordance in HPV DNA/RNA positivity and VIN, and 1.1% (6/527) in VSCC [47]. p16 expression between VSCC and MTS. Because HPV transcriptional activity may reflect its The investigation of the presence of HR-HPV E6 oncogenic activity, we applied a HPV-type-specific E6*I mRNA instead of analyzing E6 oncoprotein, which may mRNA detection method, established for 20 HR- or be more informative on the oncogenic implications of pHR-HPV types and validated for use in FFPE tissues, to HR-HPVs in VSCC development, may be considered a evaluate the presence of HPV RNA in HPV DNA- limitation of this study. However, we focused our atten- positive VSCCs. We observed viral RNA in 47.1% (8/17) tion on evaluating the expression of the p16 protein, Preti et al. Infectious Agents and Cancer (2020) 15:20 Page 6 of 8 which is considered a surrogate marker for all trans- Ethics approval and consent to participate The Ethics Committee of the Hospital of Turin approved this study to be in forming HR-HPV infections and represents a single tar- compliance with the Declaration of Helsinki (reference number 1005). All get molecule, different from the E6 proteins with type- patients had signed informed consent at the time of hospital admission. specific polypeptide sequence variability. Finally, although independent studies have reported that Consent for publication Not applicable. cutaneous beta and gamma HPV types can be detected in the genital tract, our study does not provide evidence for Competing interests their involvement in the development of VSCC. R. Ridder is an employee of Roche. All other authors report no conflict of interest. Conclusion Author details We characterized the HPV-independent and HPV- Department of Surgical Sciences, University of Turin, Turin, Italy. 2 3 International Agency for Research on Cancer, Lyon, France. Department of related VSCCs in a cohort of more than 100 VSCC cases Morphology, Surgery and Experimental Medicine; Section of Pathology, from Italy. We identified as putative HPV-related tumors Oncology and Experimental Biology; Laboratories of Cell Biology and a small proportion of VSCC cases that tested positive for Molecular Genetics, University of Ferrara, Ferrara, Italy. Infections and Cancer Epidemiology, Infections and Cancer Program, German Cancer Research both HPV DNA and RNA. Our results support the Center (DKFZ), Heidelberg, Germany. Department of Oncology, Città della causative role of HPV in vulvar carcinogenesis in a dis- Salute e della Scienza, University of Turin, Turin, Italy. Roche mtm tinct subset of cases and highlight the prevalence of laboratories, Mannheim, Germany. Ventana Medical Systems Inc., Tucson, AZ, USA. HPV16, thus reinforcing the importance of HPV vaccin- ation programs. Received: 15 January 2020 Accepted: 23 March 2020 Supplementary information References Supplementary information accompanies this paper at https://doi.org/10. 1. Clancy AA, Spaans JN, Weberpals JI. 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