Gynecologic Oncology

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1 Gynecologic Oncology 123 (2011) Contents lists available at ScienceDirect Gynecologic Oncology journal homepage: Residual anogenital lichen sclerosus after cancer surgery has a high risk for recurrence: A clinicopathological study of 75 women Sigrid Regauer Institute of Pathology, Working groups of Dermatopathology and Gynaecopathology, Referenzzentrum anogenitale Erkrankungen, Medical University Graz, Auenbruggerplatz 25, A-8036 Graz, Austria article info abstract Article history: Received 8 June 2011 Accepted 7 July 2011 Available online 29 July 2011 Keywords: Vulvar carcinoma Dermatoses Vulvar intraepithelial neoplasia Precursor lesions Lichen sclerosus-associated squamous cell carcinoma Objectives. Despite the strong association of lichen sclerosus (LS) and vulvar squamous cell carcinoma (SCC), the role of LS as precancerous lesion is unclear and the risk for recurrent SCC in residual LS after surgery for a LS-associated SCC is unknown. Methods. Recurrences in residual vulvar LS after complete resection of a LS-associated SCC were analyzed in 75 women. Primary SCC, recurrences and 19 biopsies obtained 1 6 months before recurrent SCC were evaluated histologically, and for presence of HPV and monoclonally rearranged T-cell receptor gamma locus (mtrg@). Results. 40/75 patients (53%; primary SCC 25pT2, 9pT1b, 6pT1a) had no recurrence for 64 months (range months), but 35/75 women (47%; primary SCC 1pT3, 18pT2, 13pT1b, 3pT1a) developed recurrences after 42 months (range months). Twenty-five women had 1 recurrence: 13SCC within 18 months, 1SCC after 26 months, 10SCC and 1 differentiated vulvar intraepithelial neoplasia (d-vin) after 74 months (range months). Ten patients suffered multiple recurrences: 3 women had 2 recurrent d-vin, 7 patients had multiple successive de-novo SCC with lymphocytes with mtrg@ in 6 patients. Wider resections correlated with no/late recurrences. Nineteen HPV-negative biopsies before diagnosis of recurrent SCC revealed 4 classical d-vin and 15 verrucous, atrophic or flat intraepithelial proliferations different from d-vin. Conclusion. With a 50% recurrence rate after cancer surgery, residual anogenital LS has a high risk for de-novo cancer. Extent of resection of LS-affected skin and activity of residual LS with lymphocytes with mtrg@ are important criteria for recurrences, which develop rapidly through a variety of HPV-negative intraepithelial lesions Elsevier Inc. All rights reserved. Introduction Only about 50% of vulvar squamous cell carcinomas (SCC) are induced by Human Papilloma Virus (HPV) [1]. HPV-negative SCCs arise often in advanced anogenital lichen sclerosus (LS), a chronic lymphocytemediated skin disease. The density of the predominant T-lymphocytic tissue inflammatory infiltrate correlates with disease activity. Tissue destruction and scarring of LS are the result of lymphokine and cytokine secretion. The accumulation of lymphocytes is believed to be the result of an exuberant local immune reaction, which is evidenced by up to 20% of T-lymphocytic infiltrate in biopsies of LS and LS-associated SCC sharing a monoclonally rearranged T-cell receptor gamma locus (mtrg@) [2 4]. The etiologic agent(s) responsible for influx of lymphocytes is still unknown. Furthermore, it is unresolved, whether LS is a precancerous condition as the exact mechanisms of LS-associated carcinogenesis is Abbreviations: d-vin, differentiated vulvar intraepithelial neoplasia; HPV, Human Papilloma Virus; LS, lichen sclerosus; SCC, Squamous cell carcinoma; VIN, vulvar intraepithelial neoplasia. Fax: address: sigrid.regauer@medunigraz.at. elusive [5]. In contrast to the basaloid HPV-induced vulvar intraepithelial neoplasia (VIN) with a slow progression to invasive carcinoma, LSassociated HPV-negative SCC has a suggested origin from welldifferentiated precursor lesions called differentiated VIN (d-vin) [6 8] with a rapid progression to invasive SCC, often within less than 6 months. d-vin was originally defined as differentiated simplex in-situ SCC [9], and is found in 25% of resection specimens of LS-associated SCC. Diagnostic features of d-vin are reported as elongated and bridging epithelial rete ridges with atypical basal keratinocytes and increased mitotic activity, although basaloid variants of d-vin [10] and other precursors different from d-vin [11] have been described. Total deep simple and radical vulvectomies for vulvar SCC have been abandoned during the past 10 years in favor of less radical, organ sparing surgical approaches. According to German and Austrian guidelines for therapy of vulvar cancer, the presently accepted standard is complete resection of a vulvar SCC with margins of 1 cm. This approach does not remove the entire LS-affected skin/mucosa and patients are discharged into oncological follow-up with more or less extensive residual anogenital LS. Assuming that LS is a risk factor for SCC, which develops via d-vin, there should be a high prevalence of de-novo SCC and d-vin in the residual LS of these patients. This paper evaluates the rate of de-novo recurrences in residual anogenital LS of 75 patients, who had an in-sano /$ see front matter 2011 Elsevier Inc. All rights reserved. doi: /j.ygyno

2 290 S. Regauer / Gynecologic Oncology 123 (2011) resection of a prior LS-associated SCC, as well as the histology of biopsies obtained shortly before diagnosis of recurrent invasive SCC. Material and methods During the past 25 years, 97 patients were diagnosed with a primary HPV-negative LS-associated vulvar SCC at the Institute of Pathology at the Medical University Graz, Austria. Twenty-two patients were excluded from analysis: 6 patients with positive resection margins of the primary LS-associated SCC, 2 women around 40 years with radiation therapy, 6 women who were lost to follow up and 8 women who died of extensive local recurrent disease at multiple sites without further intervention within 6 12 months. Seventy five patients with in-sano resections (R0=clear margins) of the primary SCC and the 1st, 2nd and 3rd recurrence were evaluated for de-novo vulvar/anogenital cancer, defined as a SCC arisingn3 months after definitive surgery. Resections were divided into s, deep hemivulvectomies, deep total simple vulvectomies and deep total radical vulvectomies with en bloc resection of inguinal lymph nodes. None of the presented patients were treated with superficial skinning vulvectomy. Lymph node metastases to the groins were not evaluated. Oncological follow-up of patients was every 3 months during the first year, and between 6 and 12 months for the following years. Endpoints were recurrence, last clinical follow-up or death of patients. SCC were classified according the 7th edition of TNM classification [12]. Nineteen of 75 patients were presented in a previous study [3]. HPV genotyping results of primary SCC were available from previous studies [3] and from analysis of formalin fixed and paraffin embedded tumor tissue (INNO-LiPA HPV GENOTYPING EXTRA; Innogenetics Diagnostic, Germany). Nineteen biopsies of suspect lesions 1 6 months before diagnosis and all subsequent surgical s were re-evaluated histologically. They were HPV-genotyped, analyzed immunohistochemically with monoclonal antibody to p53 (DAKO Denmark) and with monoclonal antibody to p16 INK4a (mtm laboratories, Heidelberg, Germany, CINtec) for over expression of p16 INK4a as surrogate marker for transforming infection with HPV-high-risk-genotypes. Primary and recurrent SCC of 33 patients were analyzed for rearrangements of the TRG@. Formalin-fixed, paraffin-embedded tissue blocks of primary SCC and recurrences of 14 patients were analyzed with PCR using the TCRG Gene Clonality Assay kit (InVivoScribe Technologies, San Diego, CA) based on the BIOMED-2 studies. Genomic DNA was used as a template for PCR with consensus primers for the variable (V1-11) and the joining regions of TRG@. PCR products were separated by capillary electrophoresis using an automated sequencing system, ABI310 (Applied Biosystems Invitrogen, Foster City, CA), and analyzed using Genescan software (Applied Biosystems Invitrogen) [13]. The remaining 19 patients were previously analyzed [3], and lymphocytes with mtrg@ were demonstrated with a different method in primary and recurrent SCC of 11 women. Results A total of 75 patients (mean age 70 years, age range years) with a surgically resected primary HPV-negative and LS-associated SCC (9pT1a, 22 pt1b, 43 pt2 and 1pT3 SCC) were evaluated for de-novo SCC arising within the residual anogenital LS. Thirty-eight patients had total deep simple and radical vulvectomies with subtotal removal of LS, and 37 women had deep hemivulvectomies and smaller s of their SCC, leaving larger areas of LS behind. All patients were discharged into oncological follow-up with more or less extensive residual LS, but did not receive local treatment of LS with high dose cortisone. Forty of the 75 patients (53%) with a mean age at diagnosis of SCC of 73 years (range years) were without vulvar/anogenital recurrent SCC during a mean follow-up of 64 months (range months). Of these 40 women, 6 patients had a pt1a SCC, 9 patients a pt1b SCC and 25 patients a pt2 SCC (see Table 1). Twenty-six of these 40 patients had either a deep hemi-vulvectomy or deep total simple or radical vulvectomy. Only 14 women had an. Two women with a pt2 SCC were without local vulvar recurrence when they died of metastatic disease to the lung after 12 and 42 months. A total of 35/75 women (47%; mean age 68 years, age range years) developed recurrences: 25/75 women (33%) had 1 recurrence only and 10/75 (13%) women suffered from multiple recurrences. The 1st recurrence was diagnosed after a mean follow-up of 42 months (range months; for details see Table 1). Patients with one recurrence (see Table 2) Twenty-five of 75 women (33%; mean age at diagnosis of primary SCC 68.5 years, range years) had 1 recurrence only. One patient developed a d-vin (Figs. 1A and B) and 24 women had 1 solitary recurrent invasive SCC (Figs. 1C and D; 2A and B) after a mean followup of 40 months (range months). In 13/25 (54%) patients, recurrent de-novo SCC were observed within the first 18 months and in 1 patient after 26 months. In the remaining 11 patients (15% of all patients, 31% of patients with recurrences), the d-vin and the keratinized invasive SCC developed after a mean latency of 74 months (range months). Ten patients with long latency had either deep hemi-vulvectomies or deep total simple/radical vulvectomies for their primary SCC. Only one patient had an for the primary cancer. Peritumoral d-vin with elongated rete ridges was identified adjacent to the recurrent invasive SCC in 8/24 patients (Figs. 1C and D). Five of the women with 1 recurrent SCC were among the previously reported patients, 3 of whom had lymphocytes with mtrg@ in the primary and recurrent SCC (after 12, 60 and 150 months). Analysis of tissue blocks of primary and recurrent SCC of 7 additional patients with dense lymphocytic infiltrates, including the two youngest women (age 21 and 25 years; Figs. 1C and D), revealed lymphocytes with mtrg@. Patients with multiple recurrences (see Table 3) Multiple successive recurrences were observed in 10/75 women (13%; 10/35 women with recurrences=29%) with a mean age of 67 years (range years) at diagnosis of primary SCC. Three patients presented with 2d-VINs each, all of which were excised locally. All d-vin were massively hyperkeratotic HPV-negative lesions of 1 2 cm diameter. They revealed the typical elongation of epidermal papillae with nuclear atypia, mitoses and a focally dense lymphocytic infiltrate. Seven women developed multiple recurrent invasive SCC. Table 1 75 patients evaluated for recurrences in residual vulvar LS after complete surgical removal of a prior LS-assoc. SCC. Classification of 1 SCC TNM classification 2009 pt1a pt1b pt2 pt3 Total Total number of patients No local Recurrence 6 9 c 25 a,b,c 0 40 Local recurrence in residual vulvar LS 3 (33%) 13 (59%) 18 (42%) 1 (100%) 35 1 recurrent d-vin recurrent invasive SCC recurrent d-vin recurrent invasive SCC recurrent invasive SCC recurrent invasive SCC a b c 2 patients without local recurrence died of lung metastases after 12 resp 42 months. 2 patients without local recurrence died during therapy after 3 resp. 6 months. 4 patients without local recurrence died of unrelated causes: 3 pts with pt1b SCC at 36, 37 and 42 months; 1 pt with pt2 SCC at 36 months.

3 S. Regauer / Gynecologic Oncology 123 (2011) Fig. 1. A) Recurrent d-vin in residual LS with a diameter of 1.2 cm excised 96 months after vulvectomy for a pt2 LS-associated SCC features massive hyperkeratosis and hypergranulosis, elongated rete ridges and a dense sub epithelial inflammatory infiltrate with. B) Inconspicuous nuclear atypia in basal keratinocytes and premature squamatization (arrow heads) of suprabasal keratinocytes. Identification of 3 mitoses (arrows) and atypia required examination at high power. C) HE stain of a recurrent, well differentiated keratinizing SCC 20 months after initial of a LS-associated SCC in a 25-year-old woman. Note peritumoral d-vin with elongated branching rete ridges. The inflammatory infiltrate contained T lymphocytes with mtrg@. D) d-vin with nuclear atypia, hyperchromasia and mitotic activity of basal keratinocytes in the elongated epidermal rete ridges and nuclear p53 staining in atypical basal keratinocytes. E) Excisional biopsy 8 weeks before diagnosis of a recurrent SCC in advanced LS features an exophytic squamous proliferation with massive hyperkeratosis, hypergranulosis, irregular elongation of rete ridges with basement membrane thickening, submucosal sclerosis and focal lymphocyte aggregates. This lesion was signed as verruca vulgaris based on HE histology. F) Only high power examination identified dyskeratotic basal keratinocytes (arrow) and individual mitoses (not shown). Note the histological features of LS with massive basement membrane thickening and sclerosis of the submucosa and entrapped sclerotic blood vessels. The 1st, 2nd and most 3rd recurrent invasive SCC were removed with clear margins. Primary and recurrent SCC of 4 women (pts.# 6 8 and 10, Table 3) were shown previously to contain lymphocytes with mtrg@. Analysis of tissue blocks of pt.#4 revealed lymphocytes with mtrg@ in the primary SCC and the second recurrence, and for pt.# 9 in the primary SCC, the first and second recurrence. No mtrg@ was detected in primary and recurrent SCC of pt.#5. Histology of lesions biopsied 1 6 months prior to diagnosis of recurrent SCC Biopsies of 19 clinically suspect lesions (Figs. 2A, C, D) were obtained 1 6 months prior to resection of the recurrent SCC. All biopsies were HPV-negative on genotyping, lacked p16 INK4a -overexpression and revealed a variety of intraepithelial lesions. Only 4/19 biopsies revealed the typical elongated epithelial rete ridges with premature squamatization, p53-positive atypical basal keratinocytes and increased mitotic activity as described for d-vin. The remaining lesions were not classifiable as d-vin, as they represented hyperkeratotic exophytic verrucous or flat squamous lesions. Prominent exophytic epithelial acanthosis with massive hyperkeratosis and hypergranulosis reminiscent of verruca vulgaris with focal clusters of atypical dyskeratotic basal keratinocytes were observed in 4/15 patients (Figs. 1E and F). In 5/19 biopsies with epithelial acanthosis and massive hyperkeratosis (Fig. 2C), microscopically small areas of cytological atypia and mitotic activity in basal and suprabasal keratinocytes in plump flattened rete ridges (Fig. 2D) were detected only after step sectioning of the biopsy. Two biopsies of atrophic flat LS showed basal cell proliferations with mitotic activity (Figs. 2E and F). One biopsy featured massive hyperkeratosis, irregular epithelial acanthosis with a microscopic focus

4 292 S. Regauer / Gynecologic Oncology 123 (2011) Fig. 2. A D: a now 77-year-old patient with a right deep hemi-vulvectomy for a pt2 SCC at age 70 years and one recurrence at age 75 years: A). Scheduled oncological control visit 54 months after right hemivulvectomy: the left vulva reveals several hyperkeratotic plaques ( and ) surrounding an central area of flat modified mucosa with a slightly irregular surface. B). Scheduled oncological control visit 6 months later reveals an ulcerated SCC in the previously flat center surrounded by hyperkeratotic plaques ( and ). C). Biopsy of the hyperkeratotic area in Fig. 2A revealed an exophytic epithelial acanthosis with striking hyperkeratosis and hypergranulosis. Rete ridges are plump and flattened. Only high power examination of the basal keratinocyte compartment (arrow head) reveals D). several mitoses (arrows). At the time of biopsy, the lesion was signed out as low-grade VIN, no high-grade VIN. E). Biopsy obtained 3 months prior to diagnosis of an extensive recurrent SCC with budding of basal keratinocytes with nuclear atypia and mitotic activity (arrow) F). Atrophic LS with a microscopically small epithelial bud with one mitosis (arrow) and nuclear atypia. G). 3 mm shave biopsy with hyperkeratosis, irregular epithelial acanthosis with partially flattened rete ridges and H). a microscopically small subepithelial area of pallor/edema ( ) with hyperchromatic and pleomorphic nuclei, prominent nucleoli and one mitosis (arrow) in atypical basal keratinocytes.

5 S. Regauer / Gynecologic Oncology 123 (2011) Table 2 Time pattern of solitary recurrent d-vin and SCC in residual vulvar LS of 25 women after surgery for LS-assoc. SCC. Primary SCC TNM classification 2009 pt1a pt1b pt2 pt3 Total Local recurrence Within b6 months 1 SCC 1 SCC 2 Within 6 12 months 2 SCC 4 SCC 6 Within months 1 SCC 4 SCC 5 Within months 0 Within months 1 SCC 1 Within months 0 Within months 2 SCC 2 Within months 1 SCC 1 After N72 months 3 SCC 3 SCC 1d-VIN 1 SCC 8 of sub epithelial edema, basal cell atypia and a single mitosis, recognizable at high power examination only (Figs. 2G and H). The remaining 3 biopsies showed intraepithelial edema in addition to cytological atypia. At the time of biopsy, 17/19 lesions were signed out as benign, with diagnosis of LS, hypertrophic or atrophic LS, hyperkeratosis, verruca vulgaris or condyloma accuminatum based on assessment of HE stained sections. Discussion Almost 50% of women with residual LS after cancer surgery for a LSassociated SCC developed a recurrence, with 90% being invasive SCC and 10% intraepithelial lesions of d-vin. The majority of recurrent SCC was observed within months after surgery. A minority of patients had a long latency between the primary SCC and 1st recurrence. These patients and the majority of patients without recurrence had radical wide s or deep hemi-/total vulvectomies, suggesting that women with LS-associated SCC may benefit from wider s with (sub) total removal of LS-affected skin. A recent clinical study from The Netherlands [14] also correlated extent of resection with recurrence rates in vulvar SCC, although only 35% patients in this study were reported to have LS. Detection of intraepithelial precursor lesions prior to diagnosis of recurrent invasive SCC was rare. Most patients returned with invasive SCC despite the short clinical follow-up intervals of 6 12 months, supporting the notion of a short intraepithelial phase of precursor lesions for LS-associated SCC. In this study, only 20% of recurrent SCC was preceded by the classical d-vin with elongated rete ridges. The majority of intraepithelial precursor lesions were flat or verrucous, hyperkeratotic, squamous proliferations with small foci of significant cytological atypia and increased mitotic activity in the basal epithelial compartment. At the time of biopsy, they were signed out as benign without further therapeutic intervention. A Dutch group reported similar observations in a retrospective analysis of LS patients, who proceeded to cancer. In other words, d-vin and atypical intraepithelial lesions were not recognized as premalignant at time biopsy [15]. In retrospect, the lesions presented in this paper need to be interpreted as true precursor lesions or variants of d-vin, a view shared by Scurry and colleagues [6]. Other colleagues referred to similar lesions as atypical LS or squamous hyperplasia with atypia or atypical hyperplastic dystrophy [5,16,17], which highlights the histopathological difficulties in separating hyperplastic LS and lichen simplex chronicus superimposed on LS from true intraepithelial precursor lesions [18,19]. Independent of epithelial thickness, all biopsies with increased mitotic activity and dyskeratoses in addition to the cytological atypia should be interpreted as true precursor lesion in the setting of a prior LS-associated SCC. In daily surgical pathology practice, many of these flat and atrophic, but also exophytic HPV-negative intraepithelial precursor lesions will go undiagnosed, since detection of the microscopically small changes requires step sectioning. Furthermore, they lack strictly defined morphologic features, as they are not addressed in the existing classification systems of WHO [20] and Table 3 10 patients with multiple recurrences in residual vulvar LS after prior cancer surgery. 1st rec d-vin 2nd rec d-vin 1st rec SCC 2nd rec SCC 3rd rec SCC 4th rec SCC mtrg@ Survival treatment of 1 SCC Age at 1 SCC TNM classification 2009 Pt.# 1; 67 years; pt1b Excision 14 months; 8 months, Neg Alive; no rec. for 10 months; Pt.# 2; 69 years; pt2 Simple vulvectomy 96 months: 24 months; Neg Alive; no rec. for 20 months; Pt.# 3; 77 years; pt1a Excision 14 months; 8 months; Neg Alive; no rec. for 12 months; pos Alive; no rec. for 30 months; pt1a; 24 months; hemi-vulvectomy Pt.# 4; 62 years; pt2 Excision pt1b; 36 months; post. exenteration Neg Alive, no rec. for 48 months pt1b; 42 months; Pt.# 5; 50 years; pt2 Radical vulvectomy pt1b; 156 months; multifocal SCC; 10 months; pt1b; 7 months; Pt.# 6; 75 years; pt1b Excision pt1a; 29 months; pt1b; 45 months; post. exenteration pt1b; 33 months; post. exenteration pt1b; 11 months; vulvectomy pt2; 12 months; hemi-vulvectomy Pt.# 7; 60 years; pt2 Excision pt1b; 13 months; Mutifocal SCC; 10 months; radiation Pt.# 8; 77 years; pt1b Excision pt1b; 14 months; Pos Alive; no recur for 5 months pt1b; 20 months; pt1b, 20 months; pt1b; 17 months; Pt.# 9; 63 years; pt1b Excision pt1b; 81 months; Multifocal SCC; 3 months; radiation Multifocal SCC; 7 months; pt1b; 8 months; Pt.#10; 66 years; pt2 Simple vulvectomy pt1b; 19 months;

6 294 S. Regauer / Gynecologic Oncology 123 (2011) ISSVD [21], and the term d-vin is too narrowly defined to incorporate the wide spectrum of basaloid, verrucous, atrophic and flat precursor lesions [10,11]. The first step/ initial hit in development of HPV-negative SCC is still unclear, but mutations in the p53 gene have been implicated [16,22]. In primary and recurrent HPV-negative SCC, particularly in patients with multiple recurrent SCC, T-lymphocytes with mtrg@ were identified. The biological implications of these observations are still unclear, but it appears, that these patients are at risk to experience an aggressive clinical course. Ineffective local immune surveillance may allow immunological escape of early invasive malignant tumor cells with subsequent rapid invasion and dissemination. This interpretation is supported by the demonstration of intraparenchymal single nucleate tumor cells capable of proliferation in sentinel lymph nodes removed for vulvar LS-associated SCC [23]. Development of SCC in LS appears more dependent on stage and duration of LS as well as activity of LS, rather than age of patients. Invasive SCC and adjacent LS in the anogenital region often are densely infiltrated by lymphocytes with mtrg@ [3,24]. The accumulation of lymphocytes with mtrg@ occurs independent of age of patients. The youngest vulvar cancer patients with lymphocytes with mtrg@ were 21 and 24 years old, and lymphocytes with mtrg@ canalsobedetectedinlsofpediatricpatients[4]. Theseobservations implicate that LS therapy should aim at reduction of lymphocytes (=remission of the disease) to avoid progression to advanced irreversible disease stages of LS, and possibly development of SCC. Treatment with topical corticosteroids and immune modulators [25,26] can effectively reduce lymphocytic infiltrates and cytokine secretions responsible for disease progression. This study, however, cannot answer the question, if local therapy for LS could have prevented development of primary or recurrent cancer, or reduced the recurrence rate. Over 90% of the presented patients were not in regular gynecological control at the time of diagnosis of their primary vulvar SCC, and they had no prior treatment for the LS. Furthermore, the protocol of oncological follow-up did not include topical treatment for residual LS. This may be partly attributed to post operative wound healing problems, which precluded the option of topical high dose corticosteroid treatment. Immune modulators have only recently emerged as treatment options, and their application is presently off-label use for anogenital LS. In conclusion, residual anogenital LS after resection of a prior LSassociated SCC with clear margins has a high risk for development of denovo cancer. This observation raises the question, if residual LS after prior cancer surgery for LS-associated SCC needs to be considered a risk factor for recurrent cancer or as a precancerous condition. Extent of resection of LS-affected skin and disease activity in residual LS along with accumulation of lymphocytes with mtrg@ appear to be important criteria for recurrence. A wide variation of exophytic, verrucous, flat and atrophic HPV-negative squamous intraepithelial lesions/precursor lesions different from d-vin, which are not addressed in the present classification systems of VIN, were identified preceding recurrent invasive SCC. Conflict of interest statement None. Acknowledgment Part of this work was supported by the Austrian Cancer Aid/Styria, Project number 04/2001. 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