Carcinogenesis of Vulvar Lesions: Morphology and Immunohistochemistry Evaluation. 14 Human papillomavirus infected cells exhibit low

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1 ORIGINAL RESEARCH ARTICLE: CERVIX AND HPV Carcinogenesis of Vulvar Lesions: Morphology and Immunohistochemistry Evaluation Raquel Camara Rivero, MD, 1 Deborah Garcia, 2 Luciano Serpa Hammes, PhD, 3 Marcelle Reesink Cerski, PhD, 4 Valentino Magno, PhD, 5 Lúcia Maria Kliemann, PhD, 4 and Edison Capp, PhD 5 Objectives: The aim of the study was to assess the 2 pathways of vulvar carcinogenesis and correlate immunohistochemical expression of p53 with histopathological findings. Materials and Methods: This cross-sectional study included 76 cases. Patients were classified according to the 2004 International Society for the Study of Vulvovaginal Disease Terminology, followed by a review of clinical records and immunohistochemical staining for p53. Results: Fifteen cases were in the human papillomavirus (HPV)-associated pathway (12 cases of usual vulvar intraepithelial neoplasia [VIN] and 3 of warty squamous cell carcinoma [SCC]), and 13 cases were in the HPVindependent pathway (5 cases of differentiated VIN and 8 of keratinizing SCC). Significant differences in p53 expression were observed between the 2 pathways of carcinogenesis: in the lesions related to the HPVindependent pathway, the percentage of p53-positive cells was greater (>25%, p <.001), and the staining pattern was basal (extending into the middle layer) in differentiated VIN and diffuse or infiltrative in warty SCC (p < 0.001). In the lesions HPV-associated pathway, p53 staining was less extensive ( 10% of cells, p < 0.001) and followed basal pattern in usual VIN, whereas warty SCCs were negative for p53 (p <0.001). Conclusions: Unique patterns of histological appearance and p53 expression can separate vulvar lesions into 2 distinct pathways of carcinogenesis. We propose that p53 immunohistochemistry may be performed simultaneously with histopathological examination in all cases of VIN and vulvar SCC, because it would aid in definition of the pathway of carcinogenesis and thus enable better clinical follow-up of patients with these conditions. Key Words: squamous cell carcinoma of the vulva, vulvar intraepithelial neoplasia, human papillomavirus, p53, vulvar carcinogenesis (J Low Genit Tract Dis 2016;21: 00 00) Squamous cell carcinoma (SCC) of the vulva is a rare disease, accounting for approximately 3% to 5% of all malignant tumors of the female genital tract and for 90% of all primary vulvar neoplasms. 1,2 Vulvar carcinogenesis is not as directly associated with human papillomavirus (HPV) as cervical carcinogenesis, which is approximately 100% HPV mediated. 3 Approximately 40% of vulvar SCCs are HPV associated, 4,5 and vulvar intraepithelial neoplasia (VIN) is found adjacent to SCCs in 50% to 70% of cases. 6,7 1 Graduate Program in Medicine: Medical Sciences, Universidade Federal do Rio Grande do Sul, School of Medicine, Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil; 2 Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil; 3 Hospital Moinhos de Vento, Porto Alegre, Rio Grande do Sul, Brazil; and Departments of 4 Pathology and 5 Obstetrics and Gynecology, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil Reprint requests to: Edison Capp, PhD, Departamento de Ginecologia e Obstetrícia de Ginecologia e Obstetrícia Faculdade de Medicina, Rua Ramiro Barcelos, 2400/4 andar, Porto Alegre, RS , Brazil. edcapp@ufrgs.br The authors have declared they have no conflicts of interest. Supported by the Fundo de Incentivo à Pesquisa. A scholarship by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) was awarded to Edison Capp. This study was approved by the Ethics Committee from Grupo de Pesquisa e Pós-Graduação, Hospital de Clínicas de Porto Alegre (# ). 2016, American Society for Colposcopy and Cervical Pathology DOI: /LGT Strong evidence suggests that 2 distinct etiopathogenetic pathways exist for the development of VIN and vulvar SCC, 1 HPV independent and 1 HPV related, 8 10 with different clinical, pathological, and epidemiological features. 6 The HPV-independent pathway is found in older women and leads to keratinizing SCC, often with lichen sclerosus and differentiated VIN. The HPV-associated pathway occurs in younger women, leads to basaloid or warty SCC, and is associated with usual VIN. 9,11 The genetic abnormality most commonly associated with cancer is a mutation in the TP53 gene, which encodes the tumor suppressor protein p p53 is responsible for recognizing DNA damage and halting the cell cycle at phase G1 or G2 to enable repair, maintaining the genomic integrity. In response to stress, p53 becomes activated, which triggers phenotypic changes ranging from DNA repair through senescence to apoptosis. 13 Inactivation of p53 is an important cell event in the process of carcinogenesis. 14 Human papillomavirus infected cells exhibit low levels of p53 protein. 15 Complete p53 negativity may be the result of true null mutations or of rapid degradation of HPV E6 protein bound p53 12 ; therefore, p53 expression is low or absent in the HPV-associated carcinogenesis pathway. Another mechanism involved in carcinogenesis is a p53 mutation with overexpression of mutant polypeptides, jeopardizing activity and inactivating the wild-type gene. 16 Mutations often have oncogenic potential and an extended half-life, leading to accumulation of p53 in tumor cells. 17 In the HPV-independent pathway, p53 expression is greater because of intracellular buildup of the mutated protein. 8 Because of the difference of p53 expression, this biomarker can be used for confirmation of histological diagnosis. 8 The objective of the present study is to assess the 2 pathways of vulvar carcinogenesis by assessing the possible relation of the immunohistochemical (IHC) expression of p53 with histopathological findings and clinical data. MATERIALS AND METHODS A cross-sectional study was performed. Surgical specimens sent for histopathological examination at the Hospital de Clínicas de Porto Alegre (HCPA) Pathology Service between January 2007 and December 2011 were selected. This study was submitted and approved by the ethics committee from Grupo de Pesquisa e Pós- Graduação do Hospital de Clínicas de Porto Alegre (# ). Specimens were reviewed by a pathologist blinded to the original diagnosis and classified in accordance with the International Society for the Study of Vulvovaginal Disease (ISSVD) Terminology as follows: differentiated VIN, keratinizing SCC or basaloid SCC (group 1, not associated with HPV pathway), high-grade squamous intraepithelial lesion and warty SCC (group 2, associated with HPV pathway). 18 Immunohistochemical staining for p53 was performed on all selected specimens and classified as none (intensity = 0), weak (intensity = 1), medium (intensity = 2), and strong (intensity = 3). Cases of VIN and carcinoma were separated into 2 groups by histopathological criteria: HPVassociated pathway (characterized by koilocytosis and warty carcinoma respectively) and HPV-independent pathway (differentiated VIN, keratinizing SCC, or basaloid SCC). 4,6,8,19 21 Journal of Lower Genital Tract Disease Volume 21, Number 1, January

2 Rivero et al. Journal of Lower Genital Tract Disease Volume 21, Number 1, January 2017 The mouse anti-p53 monoclonal antibody (BP clone) from Biogenex (Freemont, Canada) was used for manual IHC processing at the HCPA Research Center Laboratory. New sections were obtained from the archived paraffin blocks, deparaffinized, hydrated in a graded ethanol series, and placed in phosphatebuffered saline solution. The antibody was diluted to a concentration of 1:50 and used after microwaving with sodium citrate buffer (ph 6.0; Merck, Germany) for 20 minutes of blockade of endogenous peroxidase activity with 10% hydrogen peroxide for 10 minutes. The specimens were stained with the ABC or avidin-biotin complex method and counterstained with Harris hematoxylin. Samples of p53-positive infiltrative ductal carcinoma of the breast were used as a positive control. p53 IHC findings were interpreted blindly to histopathological diagnosis, as proposed by Hantschmann et al. 22 The following parameters were employed for assessment of p53 staining: intensity, percentage of stained cells, and pattern of expression. The percentage of positive cells was estimated by counting 200 epithelial cells and classified into subgroups of 0%, 1% to 10%, 11% to 25%, 26% to 50%, and more than 50%. The pattern of p53 staining was characterized as diffuse, basal, or infiltrative, again as proposed by Hantschmann et al. 22 Data were analyzed in Statistical Package for the Social Sciences 20 (IBM Corporation, Armonk, NY). The distribution of diagnoses and agreement between original and revised diagnoses were expressed as descriptive statistics (percentages and means). The Kruskal-Wallis test was used to assess p53 staining intensity and percentage of p53-positive cells. Between-group comparisons were then carried out by means of the Bonferroni-Dunn method. A Z test with Bonferroni correction was used for multiple comparisons. For comparative statistical analysis of the 2 pathways of vulvar carcinogenesis, the t test was used for analysis of patient age, whereas Fisher exact test was used for comparison of single versus multiple lesions. Fisher test was also used for analysis of p53 staining patterns, and the Mann-Whitney U test for assessment of percent staining intensity. The significance level was set at a p value of less than.05. RESULTS A total of 162 samples of vulvar lesions, including all pathological diagnosis, were recovered for the period , using the computerized system in the Department of Pathology. All samples were re-evaluated and 76 cases were diagnosed with benign lesions without any association with the vulvar carcinogenesis pathway nevi and epidermal cysts (26 cases), condylomata acuminata (15 cases), lichen sclerosus (9 cases), usual VIN (12 cases), differentiated VIN (5 cases), and SCC (11 cases). From these, 2 samples were diagnosed with VIN and SCC. These 76 samples were divided into 2 groups: (1) HPV-associated carcinogenic pathway, including usual VIN lesions and warty carcinoma, and (2) via carcinogenic pathway not associated with HPV, where cases diagnosed with differentiated VIN and carcinomas were grouped into epidermoid basaloid and keratinizing (see Table 1). TABLE 1. Distribution of the Number of Lesions by Pathway of Carcinogenesis HPV-associated HPV independent Single lesion 9 (60) 9 (69).341 a Multiple lesions 5 (33) 1 (8) No data on no. lesions 1 (7) 3 (23) a Fisher exact test. p In all cases diagnosed with benign lesions without any association with the pathways of carcinogenesis and vulvar lichen sclerosus, p53 expression remained wild pattern, that is, weak staining, expression less than 10%, and basal pattern. In this study, specimens with condyloma acuminata and vulvar low-grade squamous intraepithelial lesion had a 100% basal pattern of p53 expression, 93% had 10% p53- positive cells or less, and 53% had grade 2 staining intensity. This is consistent with what would be expected of an HPVassociated lesion. 22 Twenty-eight cases were from lesions of carcinogenic pathways. In the HPV-associated pathway, 12 usual VINs and 3 verrucous carcinomas were identified. In the pathway not associated with HPV, 5 differentiated VINs and 8 keratinizing epidermoid carcinomas were diagnosed. In the HPV-associated pathway, the mean (SD) patient age was 44 (16) years versus 66 (16) years in the HPV-independent pathway (p <.05). There were no differences between HPVassociated pathway (single lesion, 9 [60%]; multiple lesions, 5 [33%]; no data on number of lesions, 1 [7%]; and HPVindependent pathway, 9 [69%], 1 [8%], 3 [23%], respectively; Fisher exact test, p =0.341). In the 12 samples with the usual VIN diagnose, the mean (SD) patient age was 45 (17) years. Sixty-seven percent (n =8)had single lesions, 25% (n = 3) had multiple lesions, and 8% (n = 1) had no available data on number of lesions. Staining for p53 was restricted to the basal layer in 80 % of these cases. Staining intensity is showed in Figures 1A and B). Five cases of differentiated VIN were diagnosed after review of specimens (see Figures 1C, D). Mean (SD) patient age was 57 (22) years. After IHC assessment, 100% of cases had p53 staining extending suprabasally, strong staining intensity, and a high percentage of positive cells (26% 50%). Eleven cases of SCC were diagnosed; 8 were classified as keratinizing SCC (see Figures 2A, B). Mean (SD) patient age was 75 (5) years. All were positive for p53 with maximum staining intensity and at least 10% positive cells; in 75% (n =6)of cases, more than 26% of cells were positive. The staining pattern was never basal, and the diffuse or infiltrating patterns were found in 75% of cases. Three cases of warty (condylomatous) SCC were diagnosed (see Figures 2C, D). Mean (SD) patient age was 44 (20) years. Two specimens (n = 2) were negative for p53, and the positive specimen had a small percentage of stained cells (<10%). By stratifying cases of VIN and SCC into the HPVassociated and HPV-independent pathways, there were 15 cases in the former and 13 cases in the latter. Assessment of p53 expression in the HPV-associated and HPV-independent pathways showed a significant difference in staining intensity; 100% of cases in the HPV-independent pathway had a staining intensity of 3 (p =.001). Differences were also observed in percentage of p53-positive cells, with more than 26% positivity in the HPV-independent pathway and low percentages (0% 10%) related to the HPV-associated pathway (p <.001). A basal pattern of staining was significantly associated with the HPV pathway, whereas the suprabasal and diffuse staining patterns were significantly associated with the HPV-independent pathway (p <.001; Table 2). p53 presented a pattern correspondent to basal or negative in 93.3% of cases in the HPV-associated pathway. On the other hand, in the cases not associated with HPV pathway, no case showed this pattern of staining. All different VINs showed basal staining extension to the lower third of the epithelium. The squamous keratinizing carcinomas showed pattern of infiltrative or diffuse staining, whereas condylomatous epidermoid carcinomas were negative for p , American Society for Colposcopy and Cervical Pathology

3 Journal of Lower Genital Tract Disease Volume 21, Number 1, January 2017 Carcinogenesis of Vulvar Lesions FIGURE 1. Usual VIN. A, Dysplasia, characterized by failure of maturation and atypia in two thirds of the epithelium. H&E stain, 100 original magnification. B, Basal staining. p53 stain, 100 original magnification. Differentiated VIN. C, Atypia, spongiosis, and fusion of rete ridges. H&E stain, 100 original magnification. D, Immunohistochemical staining showing suprabasal extension. p53 stain, 100 original magnification. In the group not associated with the HPV pathway, 100% of the cases presented p53 expression in more than 10% of the cells, and in 23% expression, there was more than 50%. Differently, in the HPV-associated pathway, no sample was positive for p53 in more than 50 % of the cells and 80% presented expression in less than 10% of the cells. Regarding intensity, 66.7% of cases of condylomatous epidermoid carcinoma were negative for p53, and the remaining FIGURE 2. Keratinizing SCC. A, Carcinomatous nests invading the stroma. H&E stain, 100 original magnification. B, Diffuse p53 expression in the invasive nests. p53 stain, 100 original magnification. Warty SCC. C, Koilocytotic atypias in a background of invasive disease. H&E stain, 400 original magnification. D, Absence of p53 expression. p53 stain, 100 original magnification. 2016, American Society for Colposcopy and Cervical Pathology 3

4 Rivero et al. Journal of Lower Genital Tract Disease Volume 21, Number 1, January 2017 (33.3%) presented intensity 2. No cases of condylomatous carcinoma presented intensity 3. In the usual VIN cases, intensity 2 was presented in 58.3%. In the pathway not associated with HPV, 100% of differentiated VIN and keratinizing SCC showed staining intensity 3 for p53. All controls showed a pattern of basal color and low intensity, probably corresponding to the wildtype p53. DISCUSSION This cross-sectional study of the HPV-associated pathways or those not associated with vulvar carcinogenesis and the IHC expression of p53 observed that expression of p53 was consistent with that expected for normal skin as reported in the literature, 22 with weak, a basal pattern, staining of the cells. This staining corresponds to the wild-type p53 protein present in normal epithelium, predominantly in the mitotically active basal layer. 22 This finding strengthens the hypothesis that p53 mutations play an important role in vulvar carcinogenesis and suggests that the presence of such mutation may be associated with a risk of developing vulvar cancer. 22 All cases of SCC associated with differentiated VIN were of the keratinizing type, as expected, because VIN is considered a precursor of this specific subtype of vulvar SCC. 20 Differentiated VIN accounted for 6% of all cases in our sample, which confirms the rarity of this diagnosis. As well as posing a diagnostic challenge on histopathological examination, when left untreated, the rate of progression of a differentiated VIN to invasive SCC can be as high as 80% 23 (see Figure 3). In cases associated with keratinizing SCC, concomitant occurrence of differentiated VIN and lichen sclerosus has also been observed. 6,8,24 A recent review 25 assessed 60 cases diagnosed with lichen sclerosus progressing to SCC (minimum 10-year follow-up), 42% (25/60) of which were reclassified as differentiated VIN. The absence of cases of lichen sclerosus reclassified as differentiated VIN in the present sample may be due to the small sample size. Differentiated VIN was associated with suprabasal extension and a high percentage of p53 positivity, whereas usual VIN was associated with a TABLE 2. Distribution of p53 Expression Patterns by Pathway of Carcinogenesis p53 HPV-associated HPV-independent Percentage 0 2 (13.3) 0 (0) <.001 a (66.7) 0 (0) (6.7) 2 (15.4) (13.3) 8 (61.5) >50 0 (0) 3 (23.1) Intensity 0 2 (13.3) 0 (0).001 a 1 1 (6.7) 0 (0) 2 8 (53.3) 0 (0) 3 4 (26.7) 13 (100) Pattern Basal 12 (80) 0 (0) <.001 b Basal/ext 0 (0) 7 (53.8) Infiltrative 0 (0) 3 (23.1) Diffuse 2 (13.3) 3 (23.1) Absent 0 (0) 0 (0) Basal/ext indicates basal pattern of expression extending into the middle layer. a Mann-Whitney U test. b Fisher exact test. p FIGURE 3. Lower power (10) image of the HPV-associated SCC on H&E demonstrating the invasive pattern. basal staining pattern and a low percentage of positive cells (p <.05, Fischer exact test), which is in agreement with the literature. 6 p16 immunostaining was not investigated in our study. We had many difficulties to standardize the technique, and we did not obtain consistent positive results even in the controls used. A direct link or exclusive overexpression of p16 and carcinogenesis has not been demonstrated and suggests that other mechanisms may play a role in p16 expression. 26 In a recent study, just 18 of 103 HPV DNA-negative vulvar SCC were positive for p A divergence between histology and HPV or p16 detection has also been described. 28,29 CONCLUSIONS Comparison of diagnoses of VIN and vulvar SCC shows that these conditions can be separated by histopathologic findings, p53 IHC, and by significantly different distributions of cases by p53 into 2 pathways of carcinogenesis. The use of p53 staining simultaneously with histological diagnosis for all cases of VIN and vulvar SCC could aid the stratification of cases according to the carcinogenesis pathway present. This characterization of premalignant and malignant vulvar lesions is possible and could improve clinical follow-up and a better understanding of vulvar carcinogenesis. Likewise, IHC assessment of p53 expression may be an alternative when the differential diagnosis on hematoxylin and eosin (H&E) stain includes differentiated VIN in the setting of lichen sclerosus or HPV-independent SCC. REFERENCES 1. Bodelon C, Madeleine MM, Voigt LF, et al. Is the incidence of invasive vulvar cancer increasing in the United States? Cancer Causes Control 2009;20: Baandrup L, Varbo A, Munk C, et al. In situ and invasive squamous cell carcinoma of the vulva in Denmark a nationwide population-based study. Gynecol Oncol 2011;122: van de Nieuwenhof HP, van Kempen LC, de Hullu JA, et al. The etiologic role of HPV in vulvar squamous cell carcinoma fine tuned. Cancer Epidemiol Biomarkers Prev 2009;18: De Vuyst H, Clifford GM, Nascimento MC, et al. Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis. Int J Cancer 2009; 124: Scurry J, Campion M, Scurry B, et al. 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