Abstract

Vulvar cancers are rare tumors, representing approximately 5% of gynecological malignancies. 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-years-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 found. Finally, two CNVs of the BRCA1 gene were identified.

Introduction

Vulvar cancers are rare tumors, representing 5% of all gynecologic malignancies ¹. Only 10% of vulvar cancers are adenocarcinomas, and they are often associated with human papilloma virus (HPV) infection. Adenocarcinomas of the vulva include sweat gland carcinomas, Bartholin gland carcinomas, extramammary Paget disease and mammary-like adenocarcinomas ^2,3. Mammary-like adenocarcinomas of the vulva (MLAV) are extremely rare, with only a few cases reported in the scientific literature. It has been hypothesized that they origin from ectopic breast tissue located in the vulvar area. Ectopic breast tissue in this area was first described by Kazakov et al. in 1872 ⁴, who suggested that it belongs to the embryonic milk line arising from the ectoderm and extend from the axilla to the medial aspect of the groin. This ectopic tissue undergoes spontaneous regression during embryogenesis; when regression is incomplete tissue remnants may persist and may be subsequently involved in cancerogenesis ^3,5,6.

We report herein a case of an 88-year-old woman affected by MLAV with comedo-like features of the vulva, with a detailed description of the pathological, immunohistochemical and molecular features of the tumor.

Case Report

An 88-year-old woman was referred at the Unit of Gynecology and Obstetrics of the University of Sassari for a painful vulvar lesion, which was fully excised and sent for pathological analysis. The sample was fixed in 10% formalin immediately after surgery and consisted of a 3 x 2 x 1.5 cm fragment of the right labia majora, with a prominent polypoid mass, surrounded by apparently normal mucosa. The mass itself measured 2 x 1 x 1 cm, had a grayish-white color, and a mixed solid-cystic consistency with cystic-like spaces full of dense mucous-like content.

Histological examination revealed a solid dermal neoplasia with cystic areas; the solid component consisted of atypical epithelial cells with vesicular nucleus, prominent nucleolus and large eosinophilic cytoplasm, organized in cribriform, solid or papillary patterns, often with a central area of comedo-like necrosis and sclerotic surrounding stroma (Fig. 1).

Immunohistochemistry (IHC) showed strong immunostaining for cytokeratin 7, GATA3, androgen receptor (AR), GCFPD15, and weak staining for mammaglobin (Fig. 2); no staining for Her-2 was found. The proliferation index (Ki-67) was 15%. These features led to the pathological diagnosis of a mammary-like infiltrating adenocarcinoma of the vulva with comedo-like aspects; the margins of resection were free of disease and clinicians were advised to complete the clinical staging to exclude loco-regional or distant metastases. Nevertheless, the patient refused any further clinical investigation and treatment, as well as evaluation and management by the team of oncological psychologists of our hospital.

Given the lack of data on the molecular profile of mammary-like tumors of the external genitalia, we decided to perform next generation sequencing (NGS) testing with a wide gene panel, to investigate the molecular alterations occurring in these tumors. Slides 5 μm-thick of formalin-fixed parafin-embedded (FFPE) samples with more than 70% of viable neoplastic cells were obtained and used for nucleic acid extraction. Paraffin was removed from the samples with Bio-Clear (Bio-optica, Milan, Italy) and DNA was purified using the QIAamp DNA FFPE Tissue kit (QIAGEN Inc., Valencia, CA, USA). DNA quantitation and quality assessment were carried out with Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). DNA fragmentation status was evaluated with the Agilent 2200 TapeStation system using the Genomic DNA ScreenTape assay (Agilent Technologies, Santa Clara, CA, USA) able to produce a DNA Integrity Number (DIN).

NGS experiments were carry out using the Ion Torrent S5 System. The molecular analysis was carried out with the Oncomine Comprehensive Assay Plus (OCA Plus) panel, which profiles more than 500 unique driver genes involved in tumorigenesis, for single gene and multiple gene biomarker insights, detecting all variant types including single nucleotide variations (SNV), insertions-deletions (indel), copy number variations (CNV), splice variants, tumor mutational burden (TMB), and microsatellite instability (MSI). The TMB value was calculated following a stringent protocol; briefly, after high accuracy detection, variant calling was optimized, by applying a software filter-chain to remove germline variants. The TMB parameters used were: a minimum base coverage of 60 and a minimum variant allele frequency of 0.05, including SNV and indel occurring in exonic regions only (thus, with effects on gene products). The MSI score was estimated considering a panel of 76 microsatellite loci and based for each MSI marker on the peak shift in tested samples. Libraries were generated starting from 10 ng of input genomic DNA with the Ion AmpliSeq Library Kit Plus and barcoded with Ion Xpress Barcode Adapters (Life Technologies). After dilution to a final concentration of 50 pM libraries were pooled together, placed in the Ion Chef for emulsion PCR and Chip (550) loading steps. Sequencing of libraries loaded on the Chip was done with the Ion S5TM System (Thermofisher) using the recommended reagents. Sequencing data were processed with the Ion Torrent platform-specific pipeline (Torrent Suite, V5.2.1). The Ion Reporter™ V5.12 software and Integrative Genome Viewer software () were used for variant annotation and reads visualizations, respectively.

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 found. Finally, two CNVs of the BRCA1 gene were identified; specifically, the observed deletions of exons 4 and 14 were classified as functionally deleterious losses by the Oncomine software, which compares mutation records in all available databases. Details of the molecular alterations found are depicted in Figure 3.

Discussion

Mammary-like tumors of the vulva are rare gynecological malignancies ². Their origin and pathogenesis are not clear. It is currently debated whether mammary-like glands arise from extra-truncal remnants of milk-line mammary tissue or from modified anogenital vulvar eccrine glands, with potential for undergo malignant transformation and give rise to adenocarcinomas ⁷. Diniz da Costa et al. advocated that ectopic breast tissue shows the same pathophysiological changes as normal breast tissue ⁸, and therefore, MLAV are primary vulvar cancers that biologically and pathologically resemble classic breast cancers. Indeed, the morphological patterns of MLAV include entities like ductal, lobular, mixed ductal and lobular, tubulolobular, mucinous, and others ⁴. They are generally aggressive tumors, with high local recurrence and progression rates and local lymph node metastasis in 60% of the cases described. The differential diagnosis of these cancers includes primary adenocarcinomas of the vulva, extramammary Paget disease, and metastatic disease which amounts for about 5-8% of all vulvar cancers and requires the presence of a primary breast cancer. Differential diagnosis with vulvar adenocarcinomas is based on immunostaining for CEA, CK8-18 and MUC1, while Paget disease can be ruled out based on immunostaining for PAS-diastase, CEA and GCDFP-15. Moreover, squamous cell carcinomas express CK5, and metastases from the gynaecological and gastrointestinal tract express PAX8 and CK20 respectively, which are not common features of vulvar mammary-like tumors.

IHC characterization of MLAVs is, therefore, essential for the pathological diagnosis, risk stratification and prognostication. In addition, subtyping MLAVs according to St. Gallen criteria is important for planning appropriate treatment schemes, considering the current availability of hormone and anti-HER2 therapies. The most common IHC markers used for the study of MLAVs are those used in breast cancers, namely estrogen receptors (ER), progesterone receptors (PR), AR, HER2, GATA3, CK7, CK5, CK14, and Ki67. Table I summarizes the immunostaining frequency of these and other IHC markers reported in the scientific literature.

Only few sporadic data are currently available regarding the molecular profile of MLAVs. Our NGS testing with an OCA PLUS panel containing 500 genes detected a pathogenic mutation of the AKT1 gene. AKT1 protein is part of the PTEN/PIK3CA/AKT1 signaling pathway, which is implied in the pathogenesis of numerous solid cancers, like colorectal and breast cancer. In addition, AKT1 is activated by numerous PTEN-independent substrates, playing a great variety of physiologic and pathologic roles. Intrinsic or external AKT1 constitutive activation enhances cell survival by blocking the function of pro-apoptotic proteins; in addition, AKT1 activates of the mTOR complex 1 (mTORC1), which is regulated by both nutrients and growth factor signalling and plays an essential role in the cell mass increase. In vivo studies evidenced that AKT1 governs breast cancer progression in vivo. Furthermore, genetic alterations of the PTEN/PIK3CA/AKT1 pathway have been associated with poor prognosis in patients with breast cancer and have been involved in resistance to anti-HER2 therapy. Medications against PIK3CA and mTORC have been recently introduced in clinical practice for specific subgroups of patients with breast cancer; the use of these medications in MLAVs harboring genetic alterations in the PTEN/PIK3CA/AKT1/mTORC axis is a very interesting perspective.

A likely pathogenic insertion of the JAK1 gene has also been detected; JAK proteins are part of a different signaling pathway, the JAK/STAT pathway, which participates in numerous cell responses including proliferation, differentiation, migration, apoptosis, and cell survival, depending on the signal, tissue, and cellular context. Alterations of the JAK/STAT pathway are common in breast cancer and have been associated with resistance to specific types of therapy. Also, mutations of the KMT2C gene are common in breast cancer, and a role as biomarkers of chemotherapy response has been recently reported. Germline BRCA1 and BRCA2 CNVs are associated with the pathogenesis of breast cancer, accounting for < 5% of known pathogenic variants in these genes and have been shown to be modifiers of hereditary breast cancer risk; a similar role of somatic CNVs in MLAVs, like those detected in our case in two different loci of the BRCA1 gene, need to be elucidated in the future. Globally, the genetic alterations found in our case are relatively common in breast cancer, which outlines a certain affinity between these pathologic entities.

Treatment options for MLAVs are similar to those available for other localized primary vulvar adenocarcinomas or early-stage breast cancer. Several authors reported that local resection and sentinel lymph-node excision with subsequent regional lymphadenectomy in case of sentinel lymph-node involvement, provide excellent results in early-stage disease. In advanced stages, neoadjuvant therapy and radical vulvectomy/hemivulvectomy, with or without lymphadenectomy, and/or adjuvant postoperative chemoradiation have been suggested. The role of gene-targeted or immunotherapy approaches remain to be evaluated. Interestingly, it has been shown that the treatments described above lead to better responses in MLAVs than the respective ones applied in breast cancers, despite the pathological, immunohistochemical and molecular similarities described. This suggests a different pathophysiologic background, which needs to be further investigated. Unfortunately, in our case the patient denied all the treatments proposed by the gynecologists.

In conclusion, we described inhere a rare case of MLAV with comedo-like features, mutations involving the AKT1, JAK1, and KMT2C genes, along with CNVs of the BRCA1 gene. These genetic alterations are often observed in breast cancer, but other cases with detailed genetic profiling are necessary to better understand the biologic, pathogenic and clinical relation of MLAVs with breast cancer, and to establish better approaches and strategies for their oncological management and treatment.

ACKNOWLEDGEMENTS

The Authors wish to thank Gabriela Uras and Maria Antonia Pinna for their technical support in preparing tissue slides for immunohistochemistry and molecular analyses.

CONFLICTS OF INTEREST

None of the authors has a conflict of interest.

FUNDING

Antonio Cossu received research funds by “Fondo di Ateneo per la Ricerca 2020” of the University of Sassari, Italy.

ETHICAL CONSIDERATION

The information contained in this manuscript complies with the journal’s ethical standards.

AUTHOR CONTRIBUTIONS

RL: reviewed the scientific literature, prepared illustrations and tables, and drafted parts of the manuscript; AM: made the histological diagnosis, prepared illustrations and made critical revisions in the manuscript; AC and GP: performed the molecular analysis and drafted parts of the manuscript, MP: performed excisional biopsy and drafted parts of the manuscript; PP: supervised all the phases of the study, drafted parts of the manuscript and revised the final version. All authors gave their approval for publication of the final version of the manuscript.

Figures and tables

Figure 1.Morphological features of the tumor: (a) a mixed pattern with solid and cystic areas is evident, hematoxylin and eosin (H&E), magnification 10x; (b) cystic comedo-like areas, H&E, magnification 20x; (c) the same features seen in a are seen here in higher magnification, H&E, magnification 20x; (d) in the solid areas of the tumor cells are organized mainly in cribriform patterns, H&E, 10x.

Figure 2.Main immunohistochemical features of the tumor: strong staining for androgen receptors (a), GATA 3 (b) and cytokeratin 7 (c); week staining for mammaglobin (d).

Figure 3.Details of the genetic alterations found through next generation molecular testing.

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