Differentiated vulvar intraepithelial neoplasia long-term follow up and prognostic factors: An analysis of a large historical cohort.

INTRODUCTION: Differentiated vulvar intraepithelial neoplasia (dVIN) is a high-risk preinvasive vulvar lesion and precursor of human papillomavirus-independent vulvar squamous cell carcinoma (VSCC). Due to its rarity, literature data on its malignant potential are scant. The aim of the study is to assess the risk of developing VSCC in patients surgically treated for dVIN not associated with VSCC (solitary dVIN) and the risk of VSCC recurrence in patients treated for dVIN associated with VSCC (dVIN-VSCC) at first diagnosis. MATERIAL AND METHODS: A historical cohort study was performed in a northern Italy referral center for vulvar neoplasms. All consecutive women surgically treated for histologically confirmed dVIN from 1994 to 2021 were collected. Primary outcome was cancer risk or recurrent cancer risk, secondary outcomes were risk factors associated with VSCC development or recurrence. Kaplan-Meier method and log-rank test were used to estimate cancer risk or recurrent cancer risk differences and uni- and multivariate Cox regression analyses to identify risk factors associated with VSCC development in solitary dVIN and recurrence of dVIN-VSCC. RESULTS: Seventy-six patients with dVIN at preoperative biopsy were included: at excisional specimens 44 were solitary dVIN and 32 were dVIN-VSCC. The absolute risk of VSCC development after solitary dVIN treatment was 43.2% with median time to to VSCC diagnosis of 25.4 months (range 3.5-128.0 months). VSCC recurrence absolute risk in treated dVIN-VSCC patients was 31.3% with median time to VSCC recurrence of 52.9 months (range 6.5-94.8 months). At uni- and multivariate regression analyses, only compliant topical ultrapotent corticosteroid treatment after solitary dVIN excision showed an ability to prevent VSCC development. No protective effect by corticosteroid treatment was shown for VSCC recurrence in dVIN-VSCC patients. Smoking was associated with higher cancer recurrence risk in dVIN-VSCC patients on both uni- and multivariate regression analyses. CONCLUSIONS: Patients with dVIN have a high risk of developing both primary and recurring VSCC. Early recognition, long-term follow up, and compliant ultrapotent topical corticosteroid treatment are recommended.

Prevaccine Human Papillomavirus Status in Invasive and Intraepithelial Lesions of the Vulva in New Zealand Women.

OBJECTIVE: The human papillomavirus (HPV) vaccine, introduced in New Zealand (NZ) in 2008, is predicted to substantially lower the incidence of HPV-associated precancers and cancers. The aim of this study is to estimate the proportion of vulvar intraepithelial neoplasia (VIN) lesions and invasive vulvar squamous cell carcinomas (SCCV) attributable to HPV in NZ women treated by the Auckland Regional Gynecological Oncology Service, covering an estimated 50% of the NZ population. MATERIALS AND METHODS: Polymerase chain reaction and reverse hybridization were used to analyze retrospective histologically proven SCCV from 1990 to 2007 and VIN lesions from 2000 to 2007 for HPV content and genotype in a collaborative study with the Catalan Institute of Oncology. Immunohistochemistry for p16INK4a was performed on SCCV, which were attributed to HPV if both tested positive. RESULTS: Polymerase chain reaction testing for HPV content and genotype was performed on 66 VIN lesions (all high-grade squamous intraepithelial lesions) and 189 SCCV. In addition, p16 immunohistochemistry was performed on 168 of the 189 SCCV (88.9%) tested for HPV-DNA. Overall, 61 SCCV cases (36.3%) were attributed to HPV (HPV+/p16+), and 89 SCCV cases (53%) were considered to have developed independently of HPV (HPV-/p16-). Known high-risk HPV genotypes were present in 96.8% of HPV-DNA-positive vulvar high-grade squamous intraepithelial lesions and 98.4% of HPV-attributable SCCV. Human papillomavirus 16 represented the most common genotype in both. CONCLUSIONS: Overall, the HPV vaccine is likely to substantially alter the profile of SCCV in our region. The results provide a baseline assessment of the HPV status of vulvar neoplasia before the introduction of the HPV vaccine.

HPV-independent and HPV-associated vulvar squamous cell carcinoma: two different cancers.

OBJECTIVES: We report the disease-specific survival of patients with human papillomavirus (HPV)-associated and HPV-independent vulvar squamous cell carcinomas and determine whether differences exist and are independent of stage and age at diagnosis. METHODS: This was a retrospective cohort study with case note and pathology slide review of 265 consecutive women with vulvar squamous cell carcinoma. These patients were treated over a 15 year period (2001-2016) at a centralized cancer center covering half the population of New Zealand. The women's cancers were categorized dependent on their adjacent pathology, immunohistochemistry and HPV status following expert slide review. Disease-specific survival was calculated using Kaplan-Meier univariable and Cox proportional hazard (adjusting for stage, age, and HPV dependence) multivariable methods. RESULTS: The survival analysis included 236 women with follow-up to 96 months; 124 of them were HPV-associated, 95 HPV-independent, and 17 were unclassifiable. Of the 236 women, 146 were stage 1 (92 HPV-associated, 49 HPV-independent, 5 unclassifiable), 13 stage II (7 HPV-associated, 6 HPV-independent), 62 stage III (20 HPV-associated, 34 HPV-independent, 8 unclassifiable) and 15 stage IV (5 HPV-associated, 6 HPV-independent, 4 unclassifiable). HPV-independent vulvar squamous cell carcinomas had significantly worse survival than HPV-associated vulvar squamous cell carcinomas independent of stage and age at diagnosis (HR 3.6 (95% confidence interval (CI): 1.6 to 8.2)). Tumors that were unclassifiable by HPV type also had significantly worse survival than HPV-associated tumors independent of stage and age at diagnosis (HR 6.2 (95% CI: 2.4 to 16.0)). CONCLUSIONS: HPV-independent vulvar squamous cell carcinomas present more frequently in older women than HPV-associated tumors. However, the poorer prognosis is independent of age and stage, with worse outcomes even in early stage disease.

Differences Between Squamous Intraepithelial Lesions of the Vulva and the Cervix.

The Lower Anogenital Squamous Terminology project and subsequent publication have grouped preinvasive human papillomavirus-associated squamous intraepithelial lesions of the lower genital tract and adjacent skin as a single entity. We are concerned that as a result of this grouping, some of the clinically relevant differences may not be taken into consideration. We describe differences between high-grade squamous intraepithelial lesion of the vulva and cervix (vulvar intraepithelial neoplasia and cervical intraepithelial neoplasia), in embryology (arising from ectoderm vs mesoderm), clinical presentations (symptoms or signs due to many vulvar lesions vs abnormal cytology), examination techniques and diagnosis (clinical examination of potentially widely involved areas vs colposcopy of the transformation zone), natural history, management, and follow-up requirements (long-term clinical assessment vs cytology and human papillomavirus testing). We believe that failure to understand these important differences will lead to errors in management.

Trends in HPV-dependent and HPV-independent vulvar cancers: The changing face of vulvar squamous cell carcinoma.

OBJECTIVE: To investigate the incidence and survival of Vulvar Squamous Cell Carcinoma (VSCC) by etiology over a 27 year period. METHOD: Retrospective case-note and pathology slide review of 390 consecutive VSCC, treated at a Centralized Cancer Centre covering half New Zealand's population, 1990-2016. Incidence was calculated in 5-6 year cohorts and correlated with precursor of the VSCC, age and stage. RESULTS: Age-standardized incidence of all VSCC did not change significantly, however age standardized incidence of HPV-dependent VSCC increased significantly, from 0.55/100,000 (95% CI 0.38-0.72) in 1991-2000 to 0.83/100,000 (95% CI 0.68-0.97) in 2001-2016, with a significant decrease in the incidence of HPV-independent VSCC, from 0.76/100,000 (95% CI 0.58-0.95) to 0.54/100,000 (95%CI 0.43-0.65). HPV-dependent VSCC in women ≥50 years increased significantly from 0.75/100,000 (95% CI 0.45-1.17) to 1.43/100,000 (95% CI 1.14-1.77), with no significant change seen in younger women. HPV-independent VSCC in women ≥50 years has decreased significantly from 2.53/100,000 (95% CI 1.95-3.23) to 1.62/100,000 (95% CI 1.31-1.98) with no change in younger women. The proportion of HPV-dependent VSCC has increased from 25% to 50%. Age standardized death rate from VSCC has not changed significantly from 0.22/100,000 (95% CI 0.10-0.34) in 2001-5 to 0.27/100,000 (95% CI 0.15-0.40) in 2011-16. Five year survival for HPV-dependent VSCC was 93% and 68% for HPV-independent VSCC (p < .0001). CONCLUSIONS: HPV-dependent VSCC incidence has increased significantly and now accounts for half of VSCC, with a significant rise in women over 50. HPV-dependent and independent VSCC have different prognoses and should be registered and investigated separately.

Outcomes for women without conventional treatment for stage 1A (microinvasive) carcinoma of the cervix.

BACKGROUND: An unethical clinical study that entailed withholding treatment from women diagnosed with cervical intraepithelial neoplasia 3 (CIN3) was conducted at National Women's Hospital, Auckland, New Zealand. Women with microinvasive carcinoma of the cervix also had treatment withheld. AIMS: To describe the management and outcomes for women with microinvasive carcinoma for many of whom conventional treatment was withheld. MATERIALS AND METHODS: Retrospective cohort study of women with a diagnosis of stage 1A cervical carcinoma at National Women's Hospital. Medical records, cytology and histopathology were reviewed and data linked with cancer and death registries up to December 2000. RESULTS: Between 1955 and 1976, 62 women were initially diagnosed with stage 1A cervical cancer and 20 were diagnosed during follow up (to 1995). Sixty of the 82 women had initial management characterised as 'probably non-curative'; 20 of these received only a small diagnostic excision. Women in the latter group were more likely to: (i) subsequently have positive cytology (P < 0.0005), (ii) have untreated positive cytology (P = 0.02), and (iii) undergo multiple biopsies after initial management (P = 0.001). Of the women who received only a small diagnostic excision, eight of 20 developed invasive carcinoma of the cervix (≥ stage 1B) or vaginal vault, compared to two of 22 women who received initial treatment characterised as 'probably curative'. CONCLUSIONS: Women with microinvasive carcinoma were included in a natural history study of CIN3; they underwent numerous procedures designed to observe rather than treat their condition, and had a substantial risk of invasive cancer.

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells.

The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin) were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.IMPORTANCE Vesiviruses, such as San Miguel sea lion virus and feline calicivirus, are typically associated with infection in animal hosts. Following the accidental infection of a laboratory worker with San Miguel sea lion virus, a related virus was isolated in cell culture and named Hom-1. In this study, we found that Hom-1 could be propagated in a number of human cell lines, making it the first calicivirus to replicate efficiently in cultured human cells. Screening of a library of human cell surface membrane proteins showed that the virus could utilize human junctional adhesion molecule 1 as a receptor to enter cells and initiate replication. The Hom-1 virus presents a new system for the study of calicivirus biology and species specificity.

Doris Gordon: foundation of a legacy.

Doris Gordon Memorial Oration delivered to RANZCOG Annual Meeting, Wellington, 2 October, 2015.

The Natural History of Vulvar Intraepithelial Neoplasia, Differentiated Type: Evidence for Progression and Diagnostic Challenges.

Squamous cell carcinoma of the vulva (SCCV) develops through either human papillomavirus (HPV)-dependent or HPV-independent pathways. Approximately 60% of SCCV arise independently of HPV, commonly in a background of an inflammatory dermatosis, particularly lichen sclerosus. The likely direct precursor to most of these lesions is vulvar intraepithelial neoplasia (VIN), differentiated type (dVIN), although the evidence is largely circumstantial. There are few reports of progression to carcinoma, and the natural history of this pathway is not well understood. Nevertheless, dVIN is widely regarded as a potentially aggressive lesion. We identified dVIN adjacent to SCCV in 97 of 212 women (45.8%). Twenty-four of the 97 women (24.7%) had biopsies performed at least 6 mo before presentation with SCCV; slides for 47 biopsies from 21 women were available for review. dVIN was identified in 18 biopsies from 8 women (38.1%), which in 14 biopsies had been previously unrecognized. The subsequent cancer developed in the same region as the previous biopsy showing dVIN in 6 of the 8 women. The median interval between biopsy and invasive cancer was 43.5 mo (range, 8-102 mo). dVIN-associated SCCV was strongly associated with both lichen sclerosus, and HPV-negative status compared with usual type VIN (relative risk=38.35 (9.755-150.8) and 0.06485 (0.02764-0.1522), respectively). This study adds to the evidence linking dVIN with SCCV, and indicates that both clinical and histologic underrecognition contribute to the apparent rarity of dVIN as a solitary diagnosis. The morphologic spectrum of dVIN is likely to be wider than commonly appreciated; however, histologically defining the lower threshold is difficult and controversial.

A case series of young women with spontaneous regression of vulval intraepithelial neoplasia: Demographics and associated HPV genotypes.

OBJECTIVE: To review the clinical features, demography and human papillomavirus (HPV) genotype in a group of young women with usual type vulval intraepithelial neoplasia (VIN) whose lesions regressed spontaneously. MATERIALS AND METHODS: A retrospective case note review was made of the records of women with a diagnosis of usual type VIN whose lesions resolved spontaneously. The clinical features, demography, associated conditions, time to regression and follow-up data were extracted. Stored paraffin-embedded biopsy tissue was tested for the presence and genotype of HPV. RESULTS: Fifty-four women were identified. The median age at diagnosis was 19 years. Forty-four women (81%) were of non-European ethnicity. The median time to regression was 9 months. In 44 (81%) cases, the lesion was an incidental finding during clinical examination. The majority of lesions were multifocal and pigmented (44 (81%) and 48 (89%), respectively). HPV was detected in 40 (87%) of the 46 available biopsy samples, and HPV genotype 16 was identified in 33 (82.5%). Recurrences of usual type VIN occurred in three women, and these all resolved spontaneously. CONCLUSION: Women diagnosed with usual type VIN which resolves spontaneously are very young, mainly non-European, and usually present with multiple, asymptomatic pigmented lesions. HPV genotypes and their frequencies are similar to those detected in older women with usual type VIN. This clinically defined group of women may be managed by observation alone if follow-up is assured.

Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic.

The two currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines.

Currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4(+) T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20 µg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50 µg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4(+) T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or preferably in bivalent formulation to provide antigenic coverage of most of the G types of global importance.

Spindle cell carcinoma of the vulva: a series of 4 cases and review of the literature.

Spindle cell or sarcomatoid squamous cell carcinoma is an uncommon variant of squamous cell carcinoma of the vulva (SCCV) with only 12 well-documented cases reported to date. Morphologically tumors may be biphasic or monophasic, and uncommonly include heterologous elements. Only 1 reported vulval tumor has previously been investigated for human papillomavirus DNA content. We describe 4 women with spindle cell (sarcomatoid) SCCV, 3 of which occurred de novo and 1 followed radiotherapy for previous SCCV. The tumors in all 4 women arose in a background of lichen sclerosus, and 3 were associated with vulval intraepithelial neoplasia differentiated (simplex) type. All tumors were negative for human papillomavirus DNA on polymerase chain reaction analysis. One case is the second reported with malignant heterologous elements described in the vulva. These tumors seem to be more aggressive than conventional SCCV.

Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates.

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines.

Aggressive angiomyxoma [corrected] of the female genital tract and pelvis--clinicopathologic features with immunohistochemical analysis.

Aggressive angiomyxoma (AA) is a benign, slow-growing tumor that characteristically occurs in women of reproductive age. Local recurrence is cited in 30% to 40% of cases. Wide local excision is the treatment of choice. However, recent reports suggest a role for hormone manipulation in the management of these tumors. The morphology and immunophenotype of AA overlap with that of other, mainly benign vulvovaginal mesenchymal tumors. Diagnosis rests primarily on hematoxylin and eosin staining features, and distinction is important in determining appropriate treatment and follow-up. Rearrangement of HMGA2 has been shown in AA, and reports suggest that HMGA2 immunohistochemistry may have a role in the routine diagnosis of AA, its distinction from mimics, and in the evaluation of margins. Furthermore, CDK4 immunopositivity has been described in AA. We describe a series of 9 cases of AA with typical histology and long-term follow-up, and evaluate the role of HMGA2, CDK4, estrogen, and progesterone immunohistochemistry. One of 9 women (11%) experienced recurrence, with the second at 17 years, which is the longest recorded in the English literature. HMGA2 immunohistochemistry was positive in 37.5% of cases, consistent with the reported frequency of HMGA2 gene rearrangement, and negative in all benign mimics. CDK4 immunoreactivity was weak, diagnostically not helpful, and of uncertain significance. Immunohistochemistry for estrogen and progesterone were positive in 87.5% of AAs, and were widely positive in control groups.