Case report: Primary vulvar adenocarcinoma of mammary gland type-its genetic characteristics by focused next-generation sequencing.

Mammary-like vulvar adenocarcinoma (MLVA) is an exceedingly rare subtype of vulvar adenocarcinoma that shares features with mammary gland tissue. Due to its rarity and lack of consensus, MLVA presents diagnostic challenges to pathologists. We present the case of a 59-year-old female with an ulcerated mass on the right side of the external genitalia, diagnosed as MLVA. Comprehensive immunohistochemistry (IHC) and gene sequencing studies were performed to characterize the tumor. IHC analysis revealed triple expression of hormonal receptors (estrogen receptor, progesterone receptor, and HER2), supporting the mammary gland origin of the tumor. Gene sequencing identified unique genetic mutations associated with the expression of hormonal markers. One fusion gene (ERBB2-NAGLU) has not been reported in any tumors, and other mutations with unique mutation types have not been previously reported in MLVA. Our findings shed light on the molecular characteristics of MLV and may help improve the diagnosis and treatment of this rare type of vulvar adenocarcinoma.

Primary vulvar adenocarcinoma of intestinal type: Report of two cases showing molecular similarity with colorectal adenocarcinoma.

Primary vulvar adenocarcinoma is a particularly rare tumor with poorly understood histogenesis and unclear clinical characteristics and prognosis. Vulvar adenocarcinoma of intestinal type (VAIt) is a very uncommon subtype of primary vulvar adenocarcinoma and only 27 cases have been described in the literature in the past. Of these cases, two have been described as human papillomavirus (HPV)-associated VAIt. The current report presents two additional cases of primary VAIt showing variants in the KRAS, TP53, and DPYD genes and no evidence of HPV DNA by real-time polymerase chain reaction (RT-PCR). Next-generation sequencing (NGS) revealed TP53 pathogenic variants in both cases, but only one case had aberrant p53 protein immunohistochemical characteristics. KRAS and DPYD mutations were identified separately in the two cases. Due to their capacity to imitate the spread of more prevalent gastrointestinal carcinomas, these tumors may present diagnostic issues. Additional cases can contribute to a better understanding of the pathophysiology and prognosis of VAIt.

Genetics and beyond: Precision Medicine Real-World Data for Patients with Cervical, Vaginal or Vulvar Cancer in a Tertiary Cancer Center.

Advances in molecular tumor diagnostics have transformed cancer care. However, it remains unclear whether precision oncology has the same impact and transformative nature across all malignancies. We conducted a retrospective analysis of patients with human papillomavirus (HPV)-related gynecologic malignancies who underwent comprehensive molecular profiling and subsequent discussion at the interdisciplinary Molecular Tumor Board (MTB) of the University Hospital, LMU Munich, between 11/2017 and 06/2022. We identified a total cohort of 31 patients diagnosed with cervical (CC), vaginal or vulvar cancer. Twenty-two patients (fraction: 0.71) harbored at least one mutation. Fifteen patients (0.48) had an actionable mutation and fourteen (0.45) received a recommendation for a targeted treatment within the MTB. One CC patient received a biomarker-guided treatment recommended by the MTB and achieved stable disease on the mTOR inhibitor temsirolimus for eight months. Factors leading to non-adherence to MTB recommendations in other patient cases included informed patient refusal, rapid deterioration, stable disease, or use of alternative targeted but biomarker-agnostic treatments such as antibody-drug conjugates or checkpoint inhibitors. Despite a remarkable rate of actionable mutations in HPV-related gynecologic malignancies at our institution, immediate implementation of biomarker-guided targeted treatment recommendations remained low, and access to targeted treatment options after MTB discussion remained a major challenge.

NOTCH1 and PIK3CA mutation are related to HPV-associated vulvar squamous cell carcinoma.

NOTCH1 and PIK3CA are members of important cell signalling pathways that are deregulated in squamous cell carcinomas of various organs. Vulvar squamous cell carcinomas (vulvSCC) are classically divided into two pathways, HPV-associated or HPV-independent, but the effect of NOTCH1 and PIK3CA mutations in both groups is unclear. We analysed two different cohorts of vulvSCC using Hybrid Capture-based Comprehensive Genomic Profiling and identified NOTCH1 and PIK3CA mutations in 35% and 31% of 48 primary vulvSCC. In this first cohort, PIK3CA and NOTCH1 mutations were significantly correlated with HPV infection (p < 0.01). Furthermore, mutations in both genes were associated with an advanced tumor stage and poorly differentiated status (p < 0.05). PIK3CA and NOTCH1 mutations were also associated with shorter patient survival which did not reach significance. In the second cohort of 735 advanced vulvSCC from metastatic site biopsies or from sites of unresectable loco-regional disease, NOTCH1 and PIK3CA mutations were reported in 14% and 20.3%, respectively. 4 of 48 (8%) and 22 of 735 vulvSCC (3.0%) featured genomic alterations (short variants and/or copy number changes and/or rearrangements) in both NOTCH1 and PIK3CA. NOTCH1 mutations were mostly located in the extracellular EGF-like domains, were inactivating and indicated that NOTCH1 functions predominantly as a tumor suppressor gene in vulvSCC. In contrast, PIK3CA mutations favored hotspot codons 1624 and 1633 of the gene, indicating that PIK3CA acts as an oncogene in vulvar carcinogenesis. In conclusion, NOTCH1 and PIK3CA mutations are detectable in a substantial proportion of vulvSCC and are related to HPV infection and more aggressive tumor behaviour.

The Spectrum of HPV-independent Penile Intraepithelial Neoplasia: A Proposal for Subclassification.

Compared with vulva, precursor lesions of human papillomavirus (HPV)-independent invasive squamous cell carcinoma (SCC) of the penis are insufficiently characterized. We analyzed the histologic and immunohistochemical characteristics of 70 peritumoral precursor lesions and correlated them with the histology and mutational profile of the adjacent HPV-negative invasive penile SCC. Atypical basal keratinocyte proliferation with variously elongated epithelial rete with premature squamatiziation, but regular superficial cornification, termed differentiated penile intraepithelial neoplasia (d-PeIN), were identified adjacent to 42/70 (60%) SCC (36/42 keratinizing ( P <0.001); 3 papillary, and 1 each verrucous, clear cell, sarcomatoid SCC). d-PeIN were associated with chronic inflammatory dermatoses (32/42; P <0.001), p53 overexpression (26/42; P <0.001), and hotspot mutations in TP53 (32/42; P <0.001), CDKN2A (26/42; P <0.001) or both (21/42; P =0.003) in the adjacent SCC. Cytoplasmic p16 ink4a overexpression in 5/42 d-PeIN correlated with CDKN2A missense mutations in the adjacent SCC. In all, 21/70 (30%) cornified verrucous or glycogenated verruciform precursors with minimal atypia and wild-type p53 (18/21; P <0.001) occurred adjacent to verrucous or papillary SCC (17/21; P <0.001) and keratinizing (4/21) SCC, which harbored mutations in HRAS and/or PIK3CA (12/21; P <0.004). Undifferentiated p16 ink4a -negative full-thickness precursors were identified in 7/70 (10%) SCC. Four histologically different HPV-independent penile precursor lesions can be assigned to 2 major genetic/biological pathways with characteristic highly differentiated precursors requiring different clinical management decisions. These include d-PeIN in chronic inflammatory dermatoses, with p53 overexpression and TP53/CDKN2A mutations, and the p53 wild-type verrucous and verruciform precursors unassociated with dermatoses, but with mutations in oncogenes PIK3CA and HRAS .

TP53 Mutation-driven Stratified Mucin-producing Carcinoma Coexisting With Squamous Cell Carcinoma of the Vulva: A Case Study.

Associated with high-risk human papillomavirus infection, invasive stratified mucin-producing carcinoma is a recently characterized adenocarcinoma of the cervix. It often occurs in association with adjacent stratified mucin-producing intraepithelial lesion. Differentiated vulvar intraepithelial neoplasia and related invasive squamous cell carcinoma often arise in background vulvar lichen sclerosus with TP53 mutation as the underlying molecular signature. We present a unique case of vulvar invasive stratified mucin-producing carcinoma-like component coexisting with invasive squamous cell carcinoma in a 64-year-old woman. Both neoplastic components were proven TP53 -driven processes arising in the background of differentiated vulvar intraepithelial neoplasia and lichen sclerosus. The invasive stratified mucin-producing carcinoma-like component behaved aggressively in this case, evidenced by the presence of lymphovascular invasion and inguinal lymph node metastasis.

Clinical validation of methylation biomarkers for optimal detection of high-grade vulvar intraepithelial neoplasia.

The precursor lesions of vulvar squamous cell carcinoma (VSCC) include human papillomavirus (HPV)-associated and HPV-independent squamous neoplasia with a varying cancer risk. Our study aimed to validate the accuracy of previously identified DNA methylation markers for detection of such high-grade vulvar intraepithelial neoplasia (VIN). A large clinical series of 751 vulvar lesions, originally diagnosed as high-grade VIN, were reassessed and categorized into HPV-associated or HPV-independent vulvar disease categories. Together with 113 healthy vulvar controls, all samples were tested for 12 methylation markers with quantitative multiplex methylation-specific PCR (qMSP). Performance of individual markers and selection of an optimal marker panel for detection of high-grade VIN was determined by logistic regression analysis. SST was the best-performing individual marker (AUC 0.90), detecting 80% of high-grade VIN cases, with excellent detection of HPV-independent VIN (95%), known to have the highest cancer risk. Merely 2% of controls tested methylation positive for SST. Selection of a marker panel, including ZNF582, SST and miR124-2, resulted in a comparably high accuracy for detection of high-grade VIN (AUC 0.89). In conclusion, we clinically validated the accuracy of 12 DNA methylation markers for detection of high-grade VIN. SST, as a sole marker or in a panel, provides an optimal diagnostic tool to distinguish high-grade VIN in need of treatment, particularly HPV-independent VIN, from low-grade or reactive vulvar lesions. These findings warrant further prognostic validation of methylation biomarkers for cancer risk stratification of patients with VIN.

Mammary-like adenocarcinoma of the vulva: a rare case report with next generation sequencing.

Vulvar adenocarcinomas are rare tumors, representing approximately 5% of vulvar cancers. Mammary-like adenocarcinomas of the vulva (MLAV) are extremely rare, and their molecular features are poorly described in the scientific literature. We report a case of an 88-year-old woman affected by MLAV with comedo-like features, with a detailed description of the pathological, immunohistochemical and molecular features. Immunohistochemistry (IHC) showed strong staining for cytokeratin 7, GATA3, androgen receptor, GCFPD15, and weak staining for mammaglobin; no staining for Her-2 was found. The proliferation index (Ki-67) was 15%. Molecular testing detected a pathogenic mutation of the AKT1 gene, a likely pathogenic frameshift insertion of the JAK1 gene, and two likely pathogenic frameshift deletions of the KMT2C gene; in addition, two variants of unknown significance (VUS) involving the ARID1A and OR2T4 genes were detected. Finally, two CNVs of the BRCA1 gene were identified.

Malignant rhabdoid tumors of the vulva versus epithelioid sarcomas: a clinicopathologic, immunohistochemical, and molecular genetics study.

It has been suggested that most, if not all, extrarenal rhabdoid tumors of the vulva represent "proximal-type" epithelioid sarcomas. To better understand rhabdoid tumors of the vulva, we studied the clinicopathologic, immunohistochemical (IHC), and molecular features of 8 of these tumors and 13 extragenital epithelioid sarcomas. IHC analysis for cytokeratin AE1/AE3, EMA, S100, CD34, ERG, smooth muscle actin, desmin, and SMARCB1 (INI1) was performed. Ultrastructural study was done in one vulvar rhabdoid tumor. Next-generation sequencing of the SMARCB1 gene was performed in all cases. The 8 vulvar tumors occurred in adult women (mean age, 49 years). They were poorly differentiated neoplasms with a rhabdoid morphology. The ultrastructural study showed large amounts of intermediate filaments (10 nm). All cases had loss of expression of INI1 and were negative for CD34 and ERG. One case showed 2 SMARCB1 mutations: c.592C>T in exon 5 and c.782delG in exon 6. Follow-up revealed that 4 patients died of disease, 1 was alive with disease, and 3 were alive without evidence of disease. Epithelioid sarcomas occurred in young adults (mean age, 41 years), mostly men. Seven tumors arose in the distal extremities and the other 6 had a proximal location. They showed the characteristic "granulomatous" arrangement of the neoplastic cells. The recurrent tumors were more proximal and often showed a rhabdoid morphology. All cases had loss of expression of INI1. CD34 and ERG were expressed by 8 (62%) and 5 (38%) tumors, respectively. No SMARCB1 mutations were encountered. Follow-up revealed that 5 patients died of disease, 1 was alive with disease, and 7 were alive without evidence of disease. Based on their different morphology and biological behavior, we conclude that rhabdoid tumors of the vulva and epithelioid sarcomas are different diseases with distinct clinicopathologic features. Undifferentiated vulvar tumors with rhabdoid morphology should be classified as malignant rhabdoid tumors, rather than "proximal-type" epithelioid sarcomas.

Adenoid cystic carcinoma of Bartholin's gland, a case report with genomic data and literature review.

Adenoid cystic carcinoma of the Bartholin's gland (ACCBG) is a rare, slowly but aggressive malignancy. We reported the case of a 31-year-old woman who was treated by local excision and then hemi-vulvectomy, with positive margins and perineural invasion. Radiation therapy (RT) was then performed delivering 45Gy in 25 fractions in bilateral inguinal lymph nodes and 64.8Gy in 36 fractions on the vulvar area. After 30 months, there was no local relapse (LR) but the patient presented a histologically documented lung recurrence. Genomic profiling of the tumor showed a MYB-NFIB fusion transcript and a somatic mutation of PLCG1. A treatment by Lenvatinib was started. We conducted a literature review of 100 published cases. Patients were mainly treated by radical vulvectomy (30%), hemi-vulvectomy (17%), wide or local excision (21% and 24%, respectively) or other. Forty-four percent of patients received postoperative RT, more frequently in case of positive margin (71.9% versus 29.5%). RT may reduce the risk of LR regardless of margin status, with 15.4% vs. 41.9% of LR with or without RT, respectively, in patients with negative margins, and 13% vs. 33.3% of LR with or without RT, respectively, in patients with positive margins. The risk of relapse of any type was 40.9% in patients who received adjuvant RT vs. 48.2% in patients who did not. Median time to relapse was 24 months (range 6-156 months). The most frequent metastatic sites were lung (76.7%) and bone (26.7%). Optimal treatment for ACCBG is still not clearly defined but pooling the data from published case report help us better understand this rare disease and help in the therapeutic decision.

N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) is an uncommon gynecologic malignancy but with an increasing incidence in recent years. Etiologically, VSCC is classified into two subtypes: HPV-dependent and HPV-independent. Localized VSCC is treated surgically and/or with radiation therapy, but for advanced, metastatic or recurrent disease, therapeutic options are still limited. N6-methyladenosine (m6A) is the most prevalent post-transcriptional messenger RNA (mRNA) modification and involved in many physiological processes. The group of m6A proteins can be further divided into: 'writers' (METTL3, METTL4, METTL14, WTAP, KIAA1429), 'erasers' (FTO, ALKBH5), and 'readers' (HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1-3). Dysregulated m6A modification is implicated in carcinogenesis, progression, metastatic spread, and drug resistance across various cancer entities. Up to date, however, only little is known regarding the role of m6A in VSCC. METHODS: Here, we comprehensively investigated protein expression levels of a diverse set of m6A writers, readers and erasers by applying immunohistochemical staining in 126 patients with primary VSCC. RESULTS: In the entire study cohort, dominated by HPV-independent tumors, m6A protein expression was not associated with clinical outcome. However, we identified enhanced protein expression levels of the 'writers' METTL3, METTL14 and the 'reader' YTHDC1 as poor prognostic markers in the 23 patients with HPV-dependent VSCC. CONCLUSION: Our study suggests dysregulated m6A modification in HPV-associated VSCC.

The First Human Vulvar Intraepithelial Neoplasia Cell Line with Naturally Infected Episomal HPV18 Genome.

Persistent infection with high-risk HPV leads to cervical cancers and other anogenital cancers and head and neck carcinomas in both men and women. There is no effective drug fortreating HPV infection and HPV-associated carcinomas, largely due to a lack of models of natural HPV infection and the complexity of the HPV life cycle. There are no available cell lines from vulvar, anal, or penile lesions and cancers in the field. In this study, we established the first human cell line from vulvar intraepithelial neoplasia (VIN) with naturally infected HPV18 by conditional reprogramming (CR) method. Our data demonstrated that VIN cells possessed different biological characteristics and diploid karyotypes from HPV18-positive cancer cells (HeLa). Then, we determined that VIN cells contained episomal HPV18 using approaches including the ratio of HPV E2copy/E7copy, rolling cycle amplification, and sequencing. The VIN cells expressed squamous epithelium-specific markers that are different from HeLa cells, a cervical adenocarcinoma cell line. When cultured under 3D air-liquid interface (ALI) system, we observed the expression of both early and late differentiation markers involucrin and filaggrin. Most importantly, we were able to detect the expression of viral late gene L1 in the cornified layer of ALI 3D culture derived from VIN cells, suggesting quite different HPV genomic status from cancer cells. We also observed progeny viral particles under transmission electron microscopy (TEM) in ALI 3D cultures, confirming the episomal HPV18 genome and active viral life cycle in the new cell line. To our knowledge, this is the first human VIN cell line with naturally infected HPV18 genome and provides a valuable model for HPV biology studies, HPV-associated cancer initiation and progression, and drug-screening platforms.

Combined HPV 16 E2 and L1 methylation predict response to treatment with cidofovir and imiquimod in patients with vulval intraepithelial neoplasia.

BACKGROUND: Topical cidofovir and imiquimod can effectively treat approximately 55% of patients with vulval intraepithelial neoplasia (VIN), thus avoiding the need for surgery. Human papillomavirus (HPV) E⁢2 gene methylation predicts response to treatment but a methylation measurement is only obtainable in approximately 50% of patients. OBJECTIVE: This work aimed to determine if the applicability and predictive power of the E⁢2 methylation assay could be improved by combining it with the components of a host and viral DNA methylation panel (S5) that has been found to predict disease progression in patients with cervical intraepithelial neoplasia. METHODS: HPV E2 methylation and S5 classifier score were measured in fresh tissue samples collected pre-treatment from 132 patients with biopsy-proven VIN grade 3 who participated in a multicentre clinical trial and were randomised to treatment with cidofovir or imiquimod. RESULTS: Combining HPV16 E⁢2 and HPV16 L⁢1 methylation provides a biomarker that is both predictive of response to topical treatment and that can produce a clinically applicable result for all patients. Patients with HPV 16 L⁢1^high and HPV 16 E⁢2^high (36/132 (27.3%)) were more likely to respond to treatment with cidofovir (12/15 (80.0%)) than imiquimod (9/21 (42.9%)) (p= 0.026). Patients with HPV 16 L⁢1^low or HPV 16 E⁢2^low (including those with no HPV/unassessable methylation) were more likely to respond to imiquimod: 23/50 (46.0%) vs 31/46 (67.4%) (p= 0.035). CONCLUSIONS: Combined HPV E⁢2 and L⁢1 methylation is a potential predictive marker in treatment for all patients with VIN. These findings justify validation in a prospective trial.

Regulatory T Cells with Additional COX-2 Expression Are Independent Negative Prognosticators for Vulvar Cancer Patients.

Vulvar cancer incidence numbers have been steadily rising over the past decades. In particular, the number of young patients with vulvar cancer has recently increased. Therefore, the need to identify new prognostic factors and, in addition, therapeutic options for vulvar carcinoma is more apparent. The aim of this study was to analyze the influx of COX-2 positive tumor-infiltrating lymphocytes and monocytes and their influence on prognosis. Using subtyping by immunofluorescence, the majority of COX-2 expressing immune cells were identified as FOXP3-positive regulatory T cells. In addition, peri- and intra-tumoral macrophages in the same tumor tissue were detected simultaneously as M2-polarized macrophages. COX-2 positive immune cells were independent negative prognostic markers in long-term overall survival of patients with vulvar cancer. These results show an influence of immune cell infiltration for vulvar carcinoma patients. Immune cell infiltration and immune checkpoint expression may, therefore, become interesting targets for further research on new vulvar cancer treatment strategies.

Forty-eight-year-old female MUTYH carrier presenting with five concurrent primary cancers.

BACKGROUND: MUTYH-associated polyposis is a rare disorder resulting from mutations involved in DNA mismatch repair. This results in an increased susceptibility to colonic adenomatosis and other cancers. Studies have examined the resulting frequency of extracolonic manifestations; however, these typically occur alone, concurrently, or temporally separate from an already diagnosed colorectal cancer in individuals with a biallelic mutation. CASE: Reported here is a case of five distinct primary neoplasms presenting simultaneously in a patient monoallelic for an MYH mutation. These neoplasms included squamous cell carcinoma of the vulva, rectal adenocarcinoma, synchronous anal adenocarcinoma, papillary thyroid carcinoma, and ovarian serous psammocarcinoma. Throughout her course, she underwent multiple surgical procedures, neoadjuvant chemoradiation, with further adjuvant therapy, and treatment ongoing. Due to her unique presentation, she underwent genetic testing that demonstrated she was monoallelic for an MYH mutation. CONCLUSION: The patient had a positive response to her treatment and surgical procedures with ongoing adjuvant therapy. She will continue to undergo further genetic testing, and testing for her children is being considered. This case demonstrates a unique presentation associated with a monoallelic MYH mutation that is not described in the current literature and warrants further investigation.