Carlos Parra‐Herran

Lesions sub‐diagnostic of endometrioid intra‐epithelial neoplasia/atypical hyperplasia: value of morphology and immunohistochemistry in predicting neoplastic outcome

AimsAreas of gland crowding that do not fulfil diagnostic criteria of endometrioid intra‐epithelial neoplasia (EIN) are often encountered in endometrial biopsies. In this study, we document the prevalence of neoplastic outcome in patients with these subdiagnostic lesions (SL) and assess the utility of morphological features and a three‐marker immunohistochemistry panel (PAX2, PTEN, beta‐catenin) to predict outcome.Methods and resultsOf 430 women with SL on endometrial sampling at Brigham and Women's Hospital between 2001 and 2021 with available follow‐up biopsy, 72 (17%) had a neoplastic outcome (EIN or endometrioid carcinoma). Multilayered epithelium and mitoses in SL were statistically associated with a neoplastic outcome. Abnormal three‐marker staining was observed in 93% (53 of 57) of SL with neoplastic outcome and 60% (37 of 62) of a control group with benign outcome. Among the 72 patients with neoplastic outcome, EIN/carcinoma tissue was available in 33; of these, 30 (91%) showed abnormal staining for one or more markers. Remarkably, in 84% of these cases the EIN/carcinoma had the aberrant expression seen in the preceding SL. Based on a prevalence of 17%, the positive and negative predictive values of abnormal staining in one or more markers were 24 and 97%, respectively.ConclusionsThe presence of SL warrants clinical surveillance and repeat sampling because it is followed by endometrioid neoplasia in a significant subset of patients. Normal three‐marker staining identifies women with a very low risk of neoplastic outcome. Conversely, abnormal staining is frequent in SL with benign outcome leading to poor specificity and positive predictive value.

Editorial: Infiltrative pattern of invasion is independently associated with shorter survival and desmoplastic stroma markers FAP and THBS2 in mucinous ovarian carcinoma

Pattern A endocervical adenocarcinomas with ovarian metastasis are indolent and molecularly distinct from destructively invasive adenocarcinomas

AimsThe invasive pattern in HPV‐associated endocervical adenocarcinoma (HPVA) has prognostic value. Non‐destructive (pattern A) HPVA has excellent prognosis mirroring adenocarcinoma in‐situ (AIS). However, the rare occurrence of ovarian spread in these tumours suggests aggressiveness in a subset of patients with these otherwise indolent lesions. We hypothesise that AIS/pattern A HPVA with ovarian metastases are biologically different than metastatic destructively invasive HPVA.Methods and resultsSamples from patients with HPVA and synchronous or metachronous metastases were retrieved and reviewed to confirm diagnosis and determine the Silva pattern in the primary lesion. For each case, normal tissue, cervical tumour and at least one metastasis underwent comprehensive sequencing using a 447‐gene panel. Pathogenic single‐nucleotide variants and segmental copy‐number alterations (CNA), tumour mutational burden and molecular signatures were evaluated and compared between primary and metastases and among invasive pattern categories. We identified 13 patients: four had AIS/pattern A primaries, while nine had pattern B/C tumours. All AIS/pattern A lesions had metastasis only to ovary; 50% of patients with ovarian involvement, regardless of invasive pattern, also had involvement of the endometrium and/or fallopian tube mucosa by HPVA. In the ovary, AIS/pattern A HPVA showed deceptive well‐differentiated glands, often with adenofibroma‐like appearance. Conversely, pattern C HPVAs consistently showed overt infiltrative features in the ovary. Sequencing confirmed the genetic relationship between primary and metastatic tumours in each case. PIK3CA alterations were identified in three of four AIS/pattern A HPVAs and three of eight pattern B/C tumours with sequenced metastases. Pattern C tumours showed a notably higher number of CNA in primary tumours compared to pattern A/B tumours. Only one metastatic AIS/pattern A HPVA had a novel pathogenic variant compared to the primary. Conversely, five of eight pattern B/C tumours with sequenced metastases developed novel pathogenic variants in the metastasis not seen in the primary. All four AIS/pattern A patients were alive and free of disease at 31, 47, 58 and 212 months after initial diagnosis. Conversely, cancer‐related death was documented in five of nine pattern B/C patients with follow‐up at 7, 20, 20, 43 and 87 months.ConclusionMorphologically and genomically, AIS/pattern A HPVA with secondary ovarian involvement appears distinct from destructively invasive tumours. In at least a subset of these cases, ovarian spread appears to occur via trans‐Mullerian superficial extension, different from the stromal and lymphatic vascular spread typical of more aggressive tumours (pattern C). These differences may explain the indolent outcome observed in the rare subset of patients with AIS/pattern A HPVA and ovarian metastasis. Our data underscore the potential for conservative surgical management approaches to pattern A HPVA.

Reproducibility of scoring criteria for HER2 immunohistochemistry in endometrial serous carcinoma: a multi-institutional interobserver agreement study

Targeted anti-human epidermal growth factor receptor 2 (HER2) therapy has recently been proven to improve progression-free and overall survival of patients with advanced stage or recurrent endometrial serous carcinoma. To date, no specific pathology HER2 testing or scoring guidelines exist for endometrial cancer. However, based on evidence from the recent successful clinical trial and comprehensive pre-trial pathologic studies, a new set of HER2 scoring criteria have been proposed for endometrial serous carcinoma-distinct from the existing breast and gastric cancer-specific criteria. We present the first study assessing interobserver agreement of HER2 scores using the proposed serous endometrial cancer-specific scoring system. A digitally scanned set of 40 HER2-immunostained slides of endometrial serous carcinoma were sent to seven gynecologic pathologists, who independently assigned HER2 scores for each slide following a brief tutorial. Follow-up fluorescent in situ hybridization (FISH) for HER2 gene amplification was performed on cases with interobserver disagreement when a 2+ HER2 score was assigned by at least one observer. Complete agreement of HER2 scores among all 7 observers was achieved on 15 cases, and all but one case had an agreement by at least 4 observers. The overall agreement was 72.3% (kappa 0.60), 77.5% (kappa 0.65), and 83.3% (kappa 0.65), using four (0 to 3+ ), three (0/1+ , 2+ , 3+ ), or two (0/1+ , 2/3+ ) HER2 scoring categories, respectively. Based on the combination of HER2 immunostaining scores and FISH, the interobserver disagreement may have potentially resulted in a clinically significant difference in HER2 status only in three tumors. We conclude, that the proposed serous endometrial cancer-specific HER2 scoring criteria are reproducible among gynecologic pathologists with moderate to substantial interobserver agreement rates comparable to those of previously reported in breast and gastric carcinomas. Our findings significantly strengthen the foundation for establishing endometrial cancer-specific HER2 scoring guidelines in the future.

Assessing para‐aortic nodal status in high‐grade endometrial cancer patients with negative pelvic sentinel lymph node biopsy

Abstract Objective To determine the accuracy of pelvic sentinel lymph node biopsy (SLN) in detecting positive para‐aortic (PA) lymph nodes in high‐grade uterine cancer, and to determine the recurrence rate in patients with high‐grade uterine cancers who did not receive adjuvant chemotherapy based on negative pelvic SLNs. Methods This was a retrospective cohort study of patients with newly diagnosed, high‐grade endometrial cancer who underwent surgery, including pelvic SLNs with or without PA node dissection, at a tertiary care institution between 2015 and 2020. Baseline demographics, surgical management, pathology data, and outcomes were analyzed using descriptive statistics, and survival analysis. Results Postoperative histology of the 110 patients meeting inclusion criteria was 45.5% grade 3 endometrioid, 36.4% serous, 10.9% clear cell, and 7.3% carcinosarcoma. On final pathology, 63.7% were stage 1, and 23.6% were stage 3C with positive nodes. A total of 63 patients (57.3%) had a PA lymph node dissection (56 bilateral, 7 unilateral) in addition to the pelvic SLN. Among this group, 5.8% (95% confidence interval 1.2%–16.0%) had a positive PA node despite a negative pelvic SLN. Among those with a negative pelvic SLN and no adjuvant chemotherapy ( n  = 75), the rate of distant recurrence was 14.7%, and 3‐year recurrence‐free survival was 71.9%. Conclusion The rate of isolated PA node metastasis in high‐grade endometrial cancers despite a negative pelvic SLN may be significantly higher than the accepted rate of isolated PA node metastasis in low‐grade endometrial cancer. This supports adjuvant treatment decisions continuing to incorporate primary tumor pathology and molecular classification.

Genomic profiling of dedifferentiated endometrial carcinomas arising in the background of high‐grade carcinoma: a targeted next‐generation sequencing study

AimsOur understanding of dedifferentiated endometrial carcinoma (DEC), a rare and aggressive malignancy, mainly reflects undifferentiated carcinomas (UC) arising in the setting of low‐grade endometrial cancer (DEC‐LG). However, cases of UC arising in the setting of high‐grade EC (DEC‐HG) have been noted in the literature. Our knowledge of the genomics of DEC‐HG is limited. To characterise the molecular landscape of DEC‐HC, targeted genomic sequencing and immunohistochemical analysis was carried out on seven DEC‐HG and four DEC‐LG.Methods and resultsDEC‐HG and DEC‐LG, including undifferentiated and differentiated components, both showed a similar frequency and spectrum of mutations. ARID1A mutations were identified in 6/7 (86%) DEC‐HG and 4/4 (100%) DEC‐LG, while SMARCA4 mutations were present in 4/7 (57%) DEC‐HG and in 1/4 (25%) DEC‐LG. Concurrent SMARCA4/BRG1 protein loss by immunohistochemistry was observed in 3/4 and 1/1 SMARCA4 mutated DEC‐HG and DEC‐LG, respectively. Neither genomic alterations nor protein loss in SMARCB1/INI1 were observed in any of our cases. TP53 mutations were detected in 4/7 (57%) DEC‐HG and in 2/4 (50%) DEC‐LG, while mutation‐pattern p53 immunohistochemistry expression was observed in 2/7 (29%) DEC‐HG and none of the DEC‐LG. MLH1 mutations were observed in 1/7 (14%) DEC‐HG and 1/4 (25%) DEC‐LG. MSH2 and MSH6 mutations were each detected in 1/7 (14%) DEC‐HG, but neither was associated with corresponding loss of protein expression.ConclusionThe findings support expanding the definition of DEC to include DEC‐HG, a previously under‐recognised phenomenon with genomic similarities to DEC‐LG.

Localized Endometrial Proliferations of Pregnancy are Clonal Glandular Outgrowths Characterized by PTEN Loss and PIK3CA Pathogenic Variants

Gestational endometrium can demonstrate a spectrum of atypical but benign changes. One such lesion is localized endometrial proliferation of pregnancy (LEPP), first described in a series of 11 cases. To understand its biological and clinical importance, we explore the pathologic, immunophenotypic, and molecular features of this entity. Nine cases of LEPP identified in 15 years were retrieved from departmental archives and reviewed. Immunohistochemistry and next-generation sequencing using a comprehensive 446-gene panel were performed when the material was available. Eight cases were identified in curettage specimens performed after first-trimester pregnancy loss, and 1 in the basal plate of a mature placenta. The mean patient age was 35 (range 27-41) years. The mean lesion size was 6.3 (range 2-12) mm. Architectural patterns, often coexisting in the same case, included cribriform (n = 7), solid (n = 5), villoglandular (n = 2), papillary (n = 2), and micropapillary (n = 1). Cytologic atypia was mild in 7 cases and moderate in 2. Mitotic activity was low (up to 3 per 2.4 mm

Variability in endometrial carcinoma pathology practice: opportunities for improvement with molecular classification

We assessed the landscape of diagnostic pathology practice and how molecular classification could potentially impact management of patients with endometrial cancer by collecting patient samples, clinicopathologic data, and patient outcomes from EC patients diagnosed in 2016 at 10 Canadian tertiary cancer centers and 19 community hospitals. ProMisE molecular subtype (POLEmut, MMRd, p53abn, No Specific Molecular Profile (NSMP)) was assigned retrospectively. 1357 patients were fully evaluable including 85 POLEmut (6.3%), 380 MMRd (28.0%), 643 NSMP (47.4%), and 249 p53abn ECs (18.3%). Immunohistochemistry (IHC) for MMR proteins was undertaken at the time of primary diagnosis in 2016 in only 42% of the cohort (570/1357; range 3.5-95.4%/center). p53 IHC had only been performed in 21.1% of the cohort (286/1357; range 10.1-41.9%/center). Thus, based on the retrospective molecular subtype assignment, 54.7% (208/380) of MMRd EC had not been tested with MMR IHC (or MSI) and 48.2% (120/249) of p53abn ECs were not tested with p53 IHC in 2016. Molecular subtype diversity within histotypes was profound; most serous carcinomas were p53abn (91.4%), but only 129/249 (51.8%) p53abn EC were serous. Low-grade (Gr1-2) endometrioid carcinomas were mostly NSMP (589/954, 61.7%) but included all molecular subtypes, including p53abn (19/954, 2.0%). Molecular subtype was significantly associated with clinical outcomes (p < 0.001) even in patients with stage I disease (OS p = 0.006, DSS p < 0.001, PFS p < 0.001). Assessment of national pathologic practice in 2016 shows highly variable use of MMR and p53 IHC and demonstrates significant opportunities to improve and standardize biomarker reporting. Inconsistent, non-reflexive IHC resulted in missed opportunities for Hereditary Cancer Program referral and Lynch Syndrome diagnosis, and missed potential therapeutic implications (e.g., chemotherapy in p53abn EC, immune blockade for MMRd EC). Routine integration of molecular subtyping into practice can improve the consistency of EC pathology assessment and classification.

Claudin-18 and Mutation Surrogate Immunohistochemistry in Gastric-type Endocervical Lesions and their Differential Diagnoses

Gastric-type endocervical adenocarcinomas (GAS) are aggressive HPV-independent neoplasms with molecular alterations in TP53, STK11, CDKN2A, and SMAD4. Claudin-18 (CLDN18) has emerged as a useful marker to distinguish GAS from HPV-associated neoplasia. Its role in separating GAS from benign proliferations and exuberant endocervical glands is unknown. We studied the utility of immunohistochemistry for CLDN18, progesterone receptor (PR), and mutation surrogate stains (P53, STK11/LKB1, MTAP, SMAD4/DPC4) in 46 GAS, 12 benign gastric-type endocervical lesions, 54 benign Mullerian endocervical populations, and 11 HPV-associated endocervical adenocarcinomas. PD-L1 and HER2 immunostains were evaluated in GAS. Gastric-type lesions were more often positive for CLDN18 (100% benign, 78% GAS, most often well to moderately differentiated) compared to benign Mullerian endocervical specimens (all negative) and HPV-associated neoplasia (18%, always focal). Conversely, PR was negative in all gastric-type lesions and positive in 92% of benign Mullerian endocervical populations. GAS revealed aberrant/mutant expression of P53 in 35%, STK11/LKB1 in 25%, MTAP in 23%, and SMAD4/DPC4 in 9% of cases. Abnormal staining in at least one of these 4 mutation surrogate markers was present in 63% of GAS. HER2 score of 3+ was seen in 25% of GAS, and PD-L1 was positive in 37% based on a combined positive score. CLDN18 is a sensitive and highly specific marker of gastric-type benign and malignant endocervical lesions. Once a gastric-type phenotype is confirmed, mutation surrogate immunostains can be used to support a diagnosis of GAS. PD-L1 and HER2 expression is seen in a subset of GAS offering therapeutic options for this aggressive tumor.

Atypical Endometriosis: Comprehensive Characterization of Clinicopathologic, Immunohistochemical, and Molecular Features

Atypical endometriosis (A-EMS), defined by cytologic atypia and/or crowded glands resembling endometrial intraepithelial neoplasia, remains poorly understood. We aimed to refine the morphologic, immunohistochemical, and molecular features of A-EMS in an institutional series. Cases were identified through a structured search and reviewed by 2 pathologists. Immunohistochemistry and comprehensive sequencing using a panel 447-gene coverage were performed in suitable cases. A-EMS with synchronous and/or subsequent EMS-related neoplasia were compared with those without. Of 4598 EMS cases over an 11-yr period, 36 A-EMS were identified. The mean age at presentation was 46 (range 26–68) yr. Locations included the ovary (24, 66%), tubo-ovary (6, 17%), fallopian tube (3, 8%), and peritoneum (3, 8%). The mean size was 6.5 (range 0.5–40) mm. Cytologic atypia was mild in 4 (11%), moderate in 21 (58%), and severe in 11 (31%). Most lesions were partially or completely flat (28, 78%); of these, 66% showed hobnail nuclei. Crowded/cribriform and micropapillary/papillary patterns were seen in 11 (31%) and 16 (44%) A-EMS, respectively. Immunohistochemistry, performed in 33 A-EMS, showed wildtype p53 (100%) retained PMS2/MSH6 (100%), and positive estrogen receptor (97%, mean 65% cells), progesterone receptor (76%, mean 30% cells), and Napsin A (39%). Ki67 labelling was &lt;1% to 10% (median 5%). Nine (25%) patients presented with concurrent or subsequent ipsilateral endometrioid, seromucinous, or clear cell neoplasia (4 borderline tumors and 4 carcinomas). The only A-EMS feature statistically more frequent in this subset was crowded/glands (6/9 vs. 2/27 A-EMS without, P=0.001 Fisher exact test). Sequencing showed pathogenic variants in 5 of 6 cases analyzed, involving ATM, BRCA2, KRAS, AKT, CTNNB1, PTEN, and ARID1A among other genes. In 2 cases, synchronous neoplasia showed an accumulation of additional variants. A-EMS is characterized by cytologic atypia and crowded architecture but low proliferation index, positive estrogen receptor, and normal p53 and MMR, which can be helpful in the distinction from malignancy. The prevalence of synchronous/subsequent tubo-ovarian neoplasia in our series was 25%, significantly higher than the reported 1% in conventional EMS. Moreover, A-EMS harbors genomic alterations seen in EMS-related tumors and shares pathogenic variants with synchronous ipsilateral neoplasia. Therefore, it is important to report A-EMS as currently defined and describe its architectural features, especially gland crowding as this appears to increase the risk of EMS-related epithelial neoplasia. Napsin-A is often positive in A-EMS and should be interpreted with caution.

Histological grading of ovarian mucinous carcinoma – an outcome‐based analysis of traditional and novel systems

AimsGrading of primary ovarian mucinous carcinoma (OMC) is inconsistent among practices. The International Collaboration on Cancer Reporting recommends grading OMC using the International Federation of Gynecology and Obstetrics (FIGO) system for endometrial endometrioid carcinoma, when needed. The growth pattern (expansile versus infiltrative), a known prognostic variable in OMC, is not considered in any grading system. We herein analysed the prognostic value of various grading methods in a well‐annotated cohort of OMC.Methods and resultsInstitutional OMCs underwent review and grading by the Silverberg and FIGO schemes and a novel system, growth‐based grading (GBG), defined as G1 (expansile growth or infiltrative invasion in ≤10%) and G2 (infiltrative growth &gt;10% of tumour). Of 46 OMCs included, 80% were FIGO stage I, 11% stage II and 9% stage III. On follow‐up (mean = 52 months, range = 1–190), five patients (11%) had adverse events (three recurrences and four deaths). On univariate analysis, stage (P = 0.01, Cox proportional analysis), Silverberg grade (P = 0.01), GBG grade (P = 0.001) and percentage of infiltrative growth (P &lt; 0.001), but not FIGO grade, correlated with disease‐free survival. Log‐rank analysis showed increased survival in patients with Silverberg grade 1 versus 2 (P &lt; 0.001) and those with GBG G1 versus G2 (P &lt; 0.001). None of the parameters evaluated was significant on multivariate analysis (restricted due to the low number of adverse events).ConclusionsSilverberg and the new GBG system appear to be prognostically significant in OMC. Pattern‐based grading allows for a binary stratification into low‐ and high‐grade categories, which may be more appropriate for patient risk stratification. Despite current practices and recommendations to utilise FIGO grading in OMC, our study shows no prognostic significance of this system and we advise against its use.

Ovarian mucinous and seromucinous neoplasms: problematic aspects and modern diagnostic approach

The morphological spectrum of primary ovarian mucinous and seromucinous tumours is broad, and presents an array of diagnostic challenges, many unique to these tumour types. This reflects the heterogeneous nature of these lesions, their varied histogenesis and evolving classification systems over recent decades, with further modification to the seromucinous category incorporated in the recently published 5th edition of the World Health Organisation (WHO) Classification of female genital tumours. In this review we provide an update on the classification of these neoplasms and discuss their histogenesis and diverse morphology, focusing on areas which are diagnostically problematic. We also cover tumour grading, differential diagnosis, immunohistochemistry, the recent elucidation of the molecular underpinnings of ovarian mucinous neoplasia and discuss the gross and intra‐operative handling of these tumours. A number of diagnostic issues remain unresolved, highlighting the importance of further research on this front, as well as a multidisciplinary approach in the care of patients with ovarian mucinous and seromucinous neoplasia.

Ovarian microcystic stromal tumour: from morphological observations to syndromic associations

Microcystic stromal tumour (MST) is a rare, usually benign, ovarian neoplasm characterized morphologically in its classic form by a distinctive triad of features comprising microcysts, solid cellular regions and fibrous stroma. Variant morphology also occurs, including the presence of nests, tubules, cords and signet ring cells. Immunohistochemically, this neoplasm is characterized by diffuse nuclear expression of β‐catenin, cyclin D1, Wilms' tumour 1 (WT1) and steroidogenic factor 1 (SF1), as well as diffuse staining with forkhead box ligand 2 (FoxL2) and CD10. Inhibin and calretinin are typically negative. At the genomic level, these neoplasms harbour mutually exclusive mutations in CTNNB1 or APC genes, with the former being significantly more common. This molecular characteristic raises possible links to other rare ovarian lesions, including solid pseudopapillary tumour, signet‐ring stromal tumour and Sertoli cell tumour. Rarely, MST is an extracolonic manifestation of familial adenomatous polyposis (FAP) and serves as a sentinel event that could trigger the identification of the syndrome. Herein, we review the published literature on ovarian MST and provide practical advice for pathologists reporting these rare neoplasms.

Verruciform Acanthotic Vulvar Intraepithelial Neoplasia Harbors Recurrent Genomic Alterations Found in HPV-independent Squamous Cell Carcinoma

The term verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN) was coined to describe HPV-independent p53-wildtype lesions with characteristic clinicopathologic characteristics and association with vulvar squamous cell carcinoma (vSCC). We aimed to expand on the molecular landscape of vaVIN using comprehensive sequencing and copy number variation profiling. vaVIN diagnosis in institutional cases was confirmed by a second review, plus negative p16 and wildtype p53 by immunohistochemistry. Multigene next-generation sequencing and shallow-whole genome sequencing were used to survey for single-nucleotide variants (SNV), copy number alterations, and structural variants. Targeted TERT promoter sequencing was also carried out. Nineteen patients with vaVIN were included; 4 had concurrent vSCC. The median patient age was 74 (range 56–90) years. Genomic aberrations were noted in 18 cases (95%) as follows: PIK3CA in 10 (53%), CDKN2A in 7 (37%), HRAS in 6 (32%), FAT1 and NOTCH1-2 in 5 each (26%), TSC2 in 2 (11%), and PTEN, ARID2, and KRAS in 1 (5%) each. TERT promoter variants were detected in 11 of 13 cases successfully tested (85%). Five vaVINs harbored a TP53 variant but showed wild-type p53 immunohistochemical expression. In one of these, the concurrent carcinoma showed abnormal p53 and biallelic TP53 mutations. Out of 15 patients with follow-up (mean: 20, range: 2–50 mo), vaVIN persistence/recurrence was seen in 8 (53%), and subsequent vSCC in 2 (13%). At the last encounter, 3 (20%) patients had persistent disease and 1 (7%) died of vSCC. vaVIN is characterized by a wider molecular spectrum, beyond known alterations in PIK3CA, HRAS, and ARID2, to include TERT promoter, CDKN2A, FAT1, and NOTCH1-2, which are characteristic of HPV-independent vSCC. vaVIN can occur with concurrent or subsequent carcinoma, sometimes with fatal outcomes. These findings support the concept of vaVIN as a neoplastic process within the family of HPV-independent vulvar neoplasia.

Diagnosis of verruciform acanthotic vulvar intra‐epithelial neoplasia (vaVIN) using CK17, SOX2 and GATA3 immunohistochemistry

AimsVerruciform acanthotic vulvar intra‐epithelial neoplasia (vaVIN) is an HPV‐independent, p53 wild‐type lesion with distinct morphology and documented risk of recurrence and cancer progression. vaVIN is rare, and prospective distinction from non‐neoplastic hyperplastic lesions can be difficult. CK17, SOX2 and GATA3 immunohistochemistry has emerging value in the diagnosis of HPV‐independent lesions, particularly differentiated VIN. We aimed to test the combined value of these markers in the diagnosis of vaVIN versus its non‐neoplastic differentials in the vulva.Methods and resultsCK17, SOX2 and GATA3 immunohistochemistry was evaluated on 16 vaVINs and 34 mimickers (verruciform xanthoma, lichen simplex chronicus, lichen sclerosus, psoriasis, pseudo‐epitheliomatous hyperplasia). CK17 was scored as 3+ = full‐thickness, 2+ = partial‐thickness, 1+ = patchy, 0 = absent; SOX2 as 3+ = strong staining ≥ 10% cells, 2+ = moderate, 1 + =weak, 0 = staining in &lt; 10% cells; and GATA3 as pattern 0 = loss in &lt; 25% basal cells, 1 = loss in 25–75% basal cells, 2 = loss in &gt; 75% basal cells. For analysis, results were recorded as positive (CK17 = 3+, SOX2 = 3+, GATA3 = patterns 1/2) or negative (CK17 = 2+/1+/0, SOX2 = 2+/1+/0, GATA3 = pattern 0). CK17, SOX2 and GATA3 positivity was documented in 81, 75 and 58% vaVINs, respectively, versus 32, 17 and 22% of non‐neoplastic mimickers, respectively; ≥ 2 marker positivity conferred 83 sensitivity, 88 specificity and 86% accuracy in vaVIN diagnosis. Compared to vaVIN, SOX2 and GATA3 were differentially expressed in lichen sclerosus, lichen simplex chronicus and pseudo‐epitheliomatous hyperplasia, whereas CK17 was differentially expressed in verruciform xanthoma and adjacent normal mucosa.ConclusionsCK17, SOX2 and GATA3 can be useful in the diagnosis of vaVIN and its distinction from hyperplastic non‐neoplastic vulvar lesions. Although CK17 has higher sensitivity, SOX2 and GATA3 are more specific, and the combination of all markers shows optimal diagnostic accuracy.

Reply to Comment on HPV-independent, p53-Wild-type Vulvar Intraepithelial Neoplasia: A Review of Nomenclature and the Journey to Characterize Acanthotic Precursor Lesions of the Vulva

HPV-independent, p53-wild-type vulvar intraepithelial neoplasia: a review of nomenclature and the journey to characterize verruciform and acanthotic precursor lesions of the vulva

Vulvar squamous cell carcinomas and their precursors are currently classified by the World Health Organization based on their association with high-risk human papillomavirus (HPV). HPV independent lesions often harbor driver alterations in TP53, usually seen in the setting of chronic vulvar inflammation. However, a group of pre-invasive vulvar squamous lesions is independent from both HPV and mutant TP53. The lesions described within this category feature marked acanthosis, verruciform growth and altered squamous maturation, and over the last two decades several studies have added to their characterization. They have a documented association with verrucous carcinoma and conventional squamous cell carcinoma of the vulva, suggesting a precursor role. They also harbor recurrent genomic alterations in several oncogenes, mainly PIK3CA and HRAS, indicating a neoplastic nature. In this review, we provide a historical perspective and a comprehensive description of these lesions. We also offer an appraisal of the terminology used over the years, going from Vulvar Acanthosis with Altered Differentiation and Verruciform Lichen Simplex Chronicus to Differentiated Exophytic Vulvar Intraepithelial Lesion and Vulvar Aberrant Maturation, the latter term having been recently proposed by the International Society for the Study of Vulvovaginal Diseases. In line with the recognition of these lesions by the 2020 World Health Organization Classification of Tumours as a neoplastic precursor, we herein propose the term HPV-independent, p53-wild-type verruciform acanthotic Vulvar Intraepithelial Neoplasia (HPVi(p53wt) vaVIN), which better conveys not only the pathology but also the neoplastic nature and the biologic risk inherent to these uncommon and challenging lesions. We outline strict morphologic and immunohistochemical criteria for its diagnosis and distinction from mimickers. Immunohistochemistry for p16 and p53 should be performed routinely in the diagnostic work-up of these lesions, and the morphologic alternative term vaVIN should be reserved for instances in which p16/HPV/p53 status is unknown. We also discuss management considerations and the need to further explore precursors within and beyond the spectrum of verruciform acanthotic vulvar intraepithelial neoplasia.

Differentiated exophytic vulvar intraepithelial lesion: Clinicopathologic and molecular analysis documenting its relationship with verrucous carcinoma of the vulva

Verruciform proliferations of the vulva unrelated to HPV infection are rare. The term differentiated exophytic vulvar intraepithelial lesion (DEVIL) was recently proposed for these lesions, which harbor recurrent PIK3CA mutations. It is still unclear whether DEVIL is related to verrucous carcinoma, a neoplasm characterized by persistence and local recurrence but nil risk of distant spread. Specimens identified using the words "verruciform" and "verrucous" were reviewed. Diagnosis of DEVIL required verruciform acanthosis, hyper and/or parakeratosis, hypogranulosis, cytoplasmic pallor, and bland nuclei. Verrucous carcinoma required, in addition, discontinuous, bulbous, puzzle-like nests in the stroma. A targeted next-generation sequencing using a custom 11-gene panel was performed. Eighteen specimens corresponding to ten patients with DEVIL and/or verrucous carcinoma were included. Median age at presentation was 66 years for DEVIL and 70 years for verrucous carcinoma. A similar spectrum of prevalent mutations was found in both lesions involving HRAS, PIK3CA, and BRAF. DEVIL preceded verrucous carcinoma and/or was diagnosed concurrently or in subsequent follow-up in five patients. In four of these, the same mutation was identified in DEVIL and synchronous or metachronous carcinoma. All cases showed wild-type 53 staining and lacked pathogenic TP53 mutations. DEVIL is a rare form of squamous proliferation characterized by prevalent PIK3CA and HRAS mutations. Its temporal relationship with verrucous carcinoma and their shared mutational profile in some patients suggest that DEVIL is a precursor of verrucous carcinoma. Moreover, given their morphologic and molecular overlap and the nil risk of verrucous carcinoma for distant spread, it is conceivable that DEVIL and verrucous carcinoma represent a spectrum of the same entity.

Clinical correlation of lymphovascular invasion and Silva pattern of invasion in early-stage endocervical adenocarcinoma: proposed binary Silva classification system

Silva invasion pattern can help predict lymph node metastasis risk in endocervical adenocarcinoma. We analysed Silva pattern of invasion and lymphovascular invasion to determine associations with clinical outcomes in stage IA and IB1 endocervical adenocarcinomas. International Federation of Gynecology and Obstetrics (FIGO; 2019 classification) stage IA-IB1 endocervical adenocarcinomas from 15 international institutions were examined for Silva pattern, presence of lymphovascular invasion, and other prognostic parameters. Lymph node metastasis status, local/distant recurrences, and survival data were compared using appropriate statistical tests. Of 399 tumours, 152 (38.1%) were stage IA [IA1, 77 (19.3%); IA2, 75 (18.8%)] and 247 (61.9%) were stage IB1. On multivariate analysis, lymphovascular invasion (p=0.008) and Silva pattern (p<0.001) were significant factors when comparing stage IA versus IB1 endocervical adenocarcinomas. Overall survival was significantly associated with lymph node metastasis (p=0.028); recurrence-free survival was significantly associated with lymphovascular invasion (p=0.002) and stage (1B1 versus 1A) (p=0.002). Five and 10 year overall survival and recurrence-free survival rates were similar among Silva pattern A cases and Silva pattern B cases without lymphovascular invasion (p=0.165 and p=0.171, respectively). Silva pattern and lymphovascular invasion are important prognostic factors in stage IA1-IB1 endocervical adenocarcinomas and can supplement 2019 International Federation of Gynecology and Obstetrics staging. Our binary Silva classification system groups patients into low risk (patterns A and B without lymphovascular invasion) and high risk (pattern B with lymphovascular invasion and pattern C) categories.