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Diagnosis of lymph node metastases in pelvic lymph node dissection (PLND) is important for staging and treatment. Standard practice is to submit visible or palpable lymph nodes for histology. We assessed the added value of embedding all residual fatty tissue.Patients (n = 85) who underwent PLND for cervical (n = 50) or bladder cancer (n = 35) between 2017 and 2019 were included. Study approval was obtained (MEC-2022-0156, 18.03.2022, retrospectively registered).The median lymph node yield with conventional pathological dissection was 21 nodes (Interquartile range (IQR) 18–28). This led to discovery of positive lymph nodes in 17 (20%) patients. Extended pathological assessment found 7 (IQR 3-12) additional nodes, but did not result in identification of more node metastases.Histopathological analysis of residual fatty tissue harvested at PLND resulted in an increased lymph node yield, but not in the detection of additional lymph node metastases.

Supplementary Figure S3. Growth analysis of endometrial cancer cells in the presence/absence of Cisplatin and IL6 pathway inhibitors.

Supplementary Table S2. Summary of serial transplantation assays of primary endometrial cancer cells.

Online Supplementary Materials and Methods

Legends to Supplementary Figures

Objective: In the Netherlands a nationwide guideline was introduced in 2016, which recommended routine Lynch syndrome screening (LSS) for all women with endometrial cancer (EC)

BackgroundSerous endometrial intra-epithelial carcinoma is described as a malignant, superficial spreading lesion with risk of extra-uterine spread at time of diagnosis, and poor outcome.ObjectiveTo evaluate the surgical management of patients with serous endometrial intra-epithelial carcinoma and its impact on oncologic outcomes and complications.MethodsThis Dutch observational retrospective cohort study evaluated all patients diagnosed with pure serous endometrial intra-epithelial carcinoma in the Netherlands, between January 2012 and July 2020. The pathological examination was reviewed by two pathologists with expertise in gynecological oncology. Clinical data were obtained when the diagnosis was confirmed. Primary outcome is progression-free survival, secondary outcomes are duration of follow-up, adverse events related to surgery, and overall survival.ResultsA total of 23 patients from 13 medical centers were included, of whom 15 (65.2%) presented with post-menopausal blood loss. In 17 patients (73.9%) the intra-epithelial lesion was present in an endometrial polyp. All patients underwent hysterectomy, of whom 12 patients (52.2%) were surgically staged. None of the staged patients showed extra-uterine disease. Two patients received adjuvant brachytherapy. There were no recurrences of disease (median follow-up duration of 35.6 months (range 1.0–108.6) and no disease-related deaths in this cohort.ConclusionIn patients with serous endometrial intra-epithelial carcinoma, median progression-free survival reached nearly 3 years and no recurrences have been reported. Our results do not endorse World Health Organization 2014 advice to treat serous endometrial intra-epithelial carcinoma as high-grade, high-risk endometrial carcinoma. Full surgical staging might possibly lead to overtreatment.

The most important prognostic factor for the survival of advanced-stage epithelial ovarian cancer (EOC) is the completeness of cytoreductive surgery (CRS). Therefore, an intraoperative technique to detect microscopic tumors would be of great value. The aim of this pilot study is to assess the feasibility of near-infrared hyperspectral imaging (HSI) for EOC detection in ex vivo tissue samples. Images were collected during CRS in 11 patients in the wavelength range of 665–975 nm, and processed by calibration, normalization, and noise filtering. A linear support vector machine (SVM) was employed to classify healthy and tumorous tissue (defined as >50% tumor cells). Classifier performance was evaluated using leave-one-out cross-validation. Images of 26 tissue samples from 10 patients were included, containing 26,446 data points that were matched to histopathology. Tumorous tissue could be classified with an area under the curve of 0.83, a sensitivity of 0.81, a specificity of 0.70, and Matthew’s correlation coefficient of 0.41. This study paves the way to in vivo and intraoperative use of HSI during CRS. Hyperspectral imaging can scan a whole tissue surface in a fast and non-contact way. Our pilot study demonstrates that HSI and SVM learning can be used to discriminate EOC from surrounding tissue.

To determine the impact of pathology review on the management of patients with cervical carcinoma, 264 reports of pathology review from 230 patients referred to Erasmus MC (2010–2012) were studied retrospectively. Discrepancies between pathologic diagnoses were classified as ‘major’ if they led to changes in treatment, and as ‘minor’ where there was no change. Patient and tumor characteristics were analyzed to identify the factors influencing these discrepancies. Fifty-eight (25.2%) discrepancies were identified; 28 (12.2%) were major, these resulted frequently from missing essential information, or discordant assessment of tumor invasion. Pathology review prevented under-treatment of 3.5%, over-treatment of 1.3%, treatment for incorrect malignancy of 1.3%, and enabled definitive treatment of 6.1% of patients. This highlights the importance of pathology review for appropriate management. Major discrepancies were rare (1%) for patients with macroscopic tumor and histologic diagnosis of squamous cell carcinoma (n = 100). For these patients, yield of pathology review may be limited.

Summary Genital lichen sclerosus (LS), a chronic noninfectious dermatosis, is not rare in pediatric dermatology. The histopathological diagnosis in children and adults in both genital and nongenital LS is considered to be the same and encompasses a broad range of possible characteristics. Clinical manifestations and treatment options of genital LS in children are different depending on gender. The vast majority of boys are treated with circumcision, making for a larger amount of information on the histopathology of genital LS in boys, whereas substantial information on the histopathology of juvenile vulvar LS is lacking. In girls, vulvar LS almost always persists beyond puberty and, therefore, presents a particular challenge to clinicians and cause for concern for the patient. Vulvar LS in childhood and adolescence (juveniles) is underreported, and there are uncertainties with regard to the long-term course of the disease when it occurs at an age when the vulva is still developing. The present study investigates biopsies of 100 juvenile cases of vulvar LS and analyzes the presence or absence of the most salient histopathological characteristics of LS that are described in the literature. We found that the range of histopathological characteristics known for adult LS are also present in juvenile vulvar LS, even at very young ages, including histopathological features associated with autoimmune disease, in support of the idea of a similar pathogenesis.

Purpose: Previously, we developed breast cancer BRCA1-like and BRCA2-like copy-number profile shrunken centroid classifiers predictive for mutation status and response to therapy, targeting homologous recombination deficiency (HRD). Therefore, we investigated BRCA1- and BRCA2-like classification in ovarian cancer, aiming to acquire classifiers with similar properties as those in breast cancer. Experimental Design: We analyzed DNA copy-number profiles of germline BRCA1- and BRCA2-mutant ovarian cancers and control tumors and observed that existing breast cancer classifiers did not sufficiently predict mutation status. Hence, we trained new shrunken centroid classifiers on this set and validated them in the independent The Cancer Genome Atlas dataset. Subsequently, we assessed BRCA1/2-like classification and obtained germline and tumor mutation and methylation status of cancer predisposition genes, among them several involved in HR repair, of 300 ovarian cancer samples derived from the consecutive cohort trial AGO-TR1 (NCT02222883). Results: The detection rate of the BRCA1-like classifier for BRCA1 mutations and promoter hypermethylation was 95.6%. The BRCA2-like classifier performed less accurately, likely due to a smaller training set. Furthermore, three quarters of the BRCA1/2-like tumors could be explained by (epi)genetic alterations in BRCA1/2, germline RAD51C mutations and alterations in other genes involved in HR. Around half of the non–BRCA-mutated ovarian cancer cases displayed a BRCA-like phenotype. Conclusions: The newly trained classifiers detected most BRCA-mutated and methylated cancers and all tumors harboring a RAD51C germline mutations. Beyond that, we found an additional substantial proportion of ovarian cancers to be BRCA-like.

Simple Summary Vulvar squamous cell carcinoma (VSCC) is the most common form of vulvar malignancy, and its incidence has increased in recent years. For better diagnosis and prognostication, and to expand available treatment options, molecular characterization of VSCC is crucial. We sought to identify aberrations in DNA methylation in VSCC, as this has been implicated in the development of several cancers. To this end, we performed genome-wide methylation sequencing on a set of VSCC and normal vulvar tissue using the Infinium MethylationEPIC BeadChip array. We detected 199 genes to be differentially methylated in VSCC compared to normal vulvar tissue. Of these, 194 genes were hyper-methylated, which leads to a loss of function of the genes. As most of these genes are involved in transcription regulator activity, our results suggest that disruption of this process plays an important role in VSCC development. Abstract DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC (n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies (n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ±0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

Seborrheic keratoses (SKs) are benign lesions of uncertain etiology, which can develop in both genital and extra-genital locations. For genital SKs, there has been conjecture about the pathogenic role of human papillomavirus (HPV), in view of the frequent association of this virus with genital lesions. In light of the potential consequences on patient management, we investigated the relationship between HPV and SKs of the female genital tract (FGT). For this, we evaluated the current evidence on this relationship by performing an in-depth review of the literature. Furthermore, to add to the evidence on this association, we investigated the presence of HPV in a series of vulvar SKs (n=15), using a novel multimodal approach. This involved whole tissue section-polymerase chain reaction (WTS-PCR) using SPF10-DEIA-LipA25 for HPV detection and genotyping. In addition, immunohistochemistry (IHC) was performed with cellular biomarkers p16 and MIB-1, and viral biomarker E4, to augment HPV-testing. Finally, laser-capture microdissection-PCR (LCM-PCR) was performed to locate HPV to specific lesional cells, and to rule out incidental detection of resident HPV with WTS-PCR. Our findings from the literature review, as well as, the case-series are presented.

Objectives Carriers of BRCA1 and BRCA2 mutations are at increased risk of high grade serous carcinoma and are therefore offered risk-reducing salpingo-oophorectomy (RRSO) by 40–45 years. Most of these carcinomas are believed to arise in the fallopian tube from serous tubal intraepithelial carcinoma (STIC). We conducted a retrospective study on the prevalence of high grade serous carcinoma and STIC in BRCA1/2 carriers presenting for RRSO, and their follow-up. Methods Consecutive BRCA1/2 carriers presenting for an RRSO at Erasmus MC (2000–2016) were studied. SEE-FIM pathology protocol was followed from 2010 onwards. For the cases with carcinoma and/or STIC, the histology was reviewed and immunohistochemistry (p53 & MIB-1) was performed. Next Generation Targeted Sequencing (NGTS) for TP53 mutation was used to establish clonality in 2 cases. Results Of the 527 included patients, 68% were BRCA1, 31.6% were BRCA2, and 0.4% carried both mutations. The prevalence of high grade serous carcinoma was 2.3% (12/527); 59% of these were of tubal origin. High grade serous carcinoma was more common in patients operated on after the recommended age (p = 0.03). Isolated STIC was present in 0.8% (4/527). Two BRCA1 carriers with isolated STIC at RRSO developed peritoneal serous carcinoma >7 years later. Identical TP53 mutations in the peritoneal serous carcinoma and the preceding STIC established their clonal origin. Conclusions High grade serous carcinoma is more common in BRCA1/2 carriers presenting for RRSO after the recommended age, and is more often of tubal origin. Longer follow up of patients with STIC at RRSO should be considered.

Aims Many glandular lesions can mimic prostate cancer microscopically, including atrophic glands, adenosis and prostatic intraepithelial neoplasia. While the characteristic histopathological and immunohistochemical features of these lesions have been well established, little is known about their three-dimensional architecture. Our objective was to evaluate the three-dimensional organisation of common prostate epithelial lesions. Methods and results 500 μm-thick punches (n = 42) were taken from radical prostatectomy specimens, and stained with antibodies targeting keratin 8-18 and keratin 5 for identification of luminal and basal cells, respectively. Tissue samples were optically cleared in benzyl alcohol:benzyl benzoate and imaged using a confocal laser scanning microscope. The three-dimensional architecture of peripheral and transition zone glands was acinar, composed of interconnecting and blind-ending saccular tubules. In simple atrophy, partial atrophy and post-atrophic hyperplasia, the acinar structure was attenuated with branching blind-ending tubules from parental tubular structures. Three-dimensional imaging revealed a novel variant of prostate atrophy characterised by large Golgi-like atrophic spaces parallel to the prostate surface, which were represented by thin, elongated tubular structures on haematoxylin and eosin (H&E) slides. Conversely, adenosis lacked acinar organisation, so that it closely mimicked low-grade prostate cancer. High-grade prostatic intraepithelial neoplasia displayed prominent papillary intraluminal protrusions but retained an acinar organisation, whereas intraductal carcinoma predominantly consisted of cribriform proliferations with either spheroid, ellipsoid or complex interconnecting lumens. Conclusions While various prostate epithelial lesions might mimic malignancy on H&E slides, their three-dimensional architecture is acinar and clearly different from the tubular structure of prostate cancer, with adenosis as an exception.

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery. The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed. The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

Previously, we developed breast cancerThe detection rate of theThe newly trained classifiers detected most

Histological diagnosis of differentiated vulvar intraepithelial neoplasia (dVIN), the precursor of human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC), can be challenging, as features of dVIN may mimic those of non-dysplastic dermatoses. To aid the diagnosis, p53-immunohistochemistry (IHC) is commonly used, and mutant expression patterns are used to support a histological diagnosis of dVIN. However, a proportion of dVIN can show wild-type p53-expression, which is characteristic of non-dysplastic dermatoses. Furthermore, recent research has identified a novel precursor of HPV-independent VSCC—the p53-wild-type differentiated exophytic vulvar intraepithelial lesion (de-VIL). Currently, there are no established diagnostic IHC-markers for p53-wild-type dVIN or de-VIL. We evaluated IHC-markers, cytokeratin 17 (CK17), and SRY-box 2 (SOX2), as diagnostic adjuncts for dVIN. For this, IHC-expression of CK17, SOX2, and p53 was studied in dVIN (n = 56), de-VIL (n = 8), and non-dysplastic vulvar tissues (n = 46). For CK17 and SOX2, the percentage of cells showing expression, and the intensity and distribution of expression were recorded. We also performed next generation targeted sequencing (NGTS) on a subset of dVIN (n = 8) and de-VIL (n = 8). With p53-IHC, 74% of dVIN showed mutant patterns and 26% showed wild-type expression. Median percentage of cells expressing CK17 or SOX2 was significantly higher in dVIN (p53-mutant or p53-wild-type) and de-VIL than in non-dysplastic tissues (p &lt, 0.01). Diffuse, moderate-to-strong, full epithelial expression of CK17 or SOX2 was highly specific for dVIN and de-VIL. With NGTS, TP53 mutations were detected in both dVIN and de-VIL. We infer that immunohistochemical markers CK17 and SOX2, when used along with p53, may help support the histological diagnosis of dVIN.

Vulvar squamous cell carcinoma (VSCC) comprises two distinct etiopathological subtypes: i) Human papilloma virus (HPV)-related VSCC, which arises via the precursor high grade squamous intraepithelial lesion (HSIL); and ii) HPV-independent VSCC, which arises via precursor, differentiated vulvar intraepithelial neoplasia (dVIN), driven by TP53 mutations. However, the mechanism of carcinogen- esis of VSCC is poorly understood. The current study aimed to gain insight into VSCC carcinogenesis by identifying differentially expressed genes (DEGs) for each VSCC subtype. The expression of certain DEGs was then further assessed by performing immunohistochemistry (IHC) on whole tissue sections of VSCC and its precursors. Statistical analysis of microarrays was performed on two independent gene expres- sion datasets (GSE38228 and a study from Erasmus MC) on VSCC and normal vulva. DEGs were identified that were similarly (up/down) regulated with statistical significance in both datasets. For HPV-related VSCCs, this constituted 88 DEGs, and for HPV-independent VSCCs, this comprised 46 DEGs. IHC was performed on VSCC (n=11), dVIN (n=6), HSIL (n=6) and normal vulvar tissue (n=7) with i) signal trans- ducer and activator of transcription 1 (STAT1; an upregulated DEGs); ii) nuclear factor IB (NFIB; a downregulated DEG); iii) p16 (to determine the HPV status of tissues); and iv) p53 (to confirm the histological diagnoses). Strong and diffuse NFIB expression was observed in the basal and para-basal layers of normal vulvar tissue, whereas NFIB expression was minimal or completely negative in dVIN and in both subtypes of VSCC. In contrast, no discernable difference was observed in STAT1 expression among normal vulvar tissue, dVIN, HSIL or VSCC. By leveraging bioinformatics, the current study identified DEGs that can facilitate research into VSCC carcinogenesis. The results suggested that NFIB is down- regulated in VSCC and its relevance as a diagnostic/prognostic biomarker deserves further exploration.

Differentiated vulvar intraepithelial neoplasia (dVIN) is a premalignant lesion that is known to progress rapidly to invasive carcinoma. Accurate histological diagnosis is therefore crucial to allow appropriate treatment. To identify reliable diagnostic features, we evaluated the inter-observer agreement in the histological assessment of dVIN, among a bi-national, multi-institutional group of pathologists. Two investigators from Erasmus MC selected 36 hematoxylin-eosin-stained glass slides of dVIN and no-dysplasia, and prepared a list of 15 histological features of dVIN. Nine participating pathologists (i) diagnosed each slide as dVIN or no-dysplasia, (ii) indicated which features they used for the diagnosis, and (iii) rated these features in terms of their diagnostic usefulness. Diagnoses rendered by > 50% participants were taken as the consensus (gold standard). p53-immunohistochemistry (IHC) was performed for all cases, and the expression patterns were correlated with the consensus diagnoses. Kappa (ĸ)-statistics were computed to measure inter-observer agreements, and concordance of the p53-IHC patterns with the consensus diagnoses. For the diagnosis of dVIN, overall agreement was moderate (ĸ = 0.42), and pair-wise agreements ranged from slight (ĸ = 0.10) to substantial (ĸ = 0.73). Based on the levels of agreement and ratings of usefulness, the most helpful diagnostic features were parakeratosis, cobblestone appearance, chromatin abnormality, angulated nuclei, atypia discernable under × 100, and altered cellular alignment. p53-IHC patterns showed substantial concordance (ĸ = 0.67) with the consensus diagnoses. Histological interpretation of dVIN remains challenging with suboptimal inter-observer agreement. We identified the histological features that may facilitate the diagnosis of dVIN. For cases with a histological suspicion of dVIN, consensus-based pathological evaluation may improve the reliability of the diagnosis.

Objective. There is great need for better risk stratification in vulvar squamous cell carcinoma (VSCC). Our aim was to define the prognostic significance of stratifying VSCC based on p16 and p53 immunohistochemistry (IHC) as surrogate markers for HPV and TP53 mutations.Methods. A large retrospective cohort of surgically treated women with primary VSCC was used. VSCC were classified into three subtypes: HPV-positive (HPVpos), HPV-negative/p53 mutant (HPVneg/p53mut), and HPVnegative/p53 wildtype (HPVneg/p53wt). Overall survival (OS), relative survival (RS), and recurrence-free period (RFP) were depicted using the Kaplan-Meier method and survival curves for relative survival; associations were studied using univariable and multivariable Cox proportional hazard models.Results. Of the 413 VSCCs, 75 (18%) were HPVpos, 63 (15%) HPVneg/p53wt, and 275 (66%) HPVneg/p53mut VSCC. Patients with HPVneg/p53mut VSCC had worse OS and RS (HR 3.43, 95%CI 1.80-6.53, and relative excess risk (RER) of 4.02; 95%CI 1.48-10.90, respectively, and worse RFP (HR 3.76, 95%CI 2.02-7.00). HPVpos VSCC patients showed most favorable outcomes. In univariate analysis, the molecular subtype of VSCC was a prognostic marker for OS, RS and RFP (p = 0.003, p = 0.009, p Conclusions. Stratification of VSCC by p16and p53-IHC has potential to be used routinely in diagnostic pathology. It results in the identification of three clinically distinct subtypes and may be used to guide treatment and follow-up, and in stratifying patients in future clinical trials. (C) 2020 Elsevier Inc. All rights reserved.

Adenoid cystic carcinoma (ACC) of the Bartholin gland is a rare gynaecological entity. Despite its slow growth and inconspicuous presentation, vulvar ACC has a propensity for perineural invasion and is therefore associated with high local recurrence rates. We report a case of vulvar ACC in a 61-year-old woman with a prolonged swelling of the Bartholin gland. This patient presented with pulmonary metastases at the moment of histological diagnosis. The vulvar and the pulmonary lesions showed identical histology. Despite a history of human papilloma virus (HPV)-related usual type vulvar intra-epithelial neoplasia and cervical squamous cell carcinoma, the vulvar ACC was negative for both p16 immunohistochemistry and HPV-DNA. We conclude that HPV is not involved in the pathogenesis of pure ACC of the Bartholin gland. Additionally, we advocate a low threshold for performing biopsies of vulvar swellings in women aged >40 years, to rule out malignancy and to prevent diagnostic delays.

The precursor lesion of vulvar squamous cell carcinoma (VSCC), namely vulvar intraepithelial neoplasia (VIN), is classified as: human papillomavirus (HPV)-related high grade squamous intraepithelial lesion (HSIL), and HPV-independent differentiated VIN (dVIN). Traditionally, histology and immunohistochemistry (IHC) have been the basis of diagnosis and classification of VIN. HSIL shows conspicuous histological atypia, and positivity on p16-IHC, whereas dVIN shows less obvious histological atypia, and overexpression or null-pattern on p53-IHC. For both types of VIN, other diagnostic immunohistochemical markers have also been evaluated. Molecular characterization of VIN has been attempted in few recent studies, and novel genotypic subtypes of HPV-independent VSCC and VIN have been identified. This systematic review appraises the VSCC precursors identified so far, focusing on histology and biomarkers (immunohistochemical and molecular). To gain further insights into the carcinogenesis and to identify additional potential biomarkers, gene expression omnibus (GEO) datasets on VSCC were analyzed; the results are presented.

The extent of surgical treatment for vulvar lesions is predominantly guided by the histopathologic diagnosis rendered on the pre-operative biopsy. For premalignant lesions, local excisions are performed, whereas for vulvar squamous cell carcinoma (VSCC), more radical procedures are mandatory. However, even in the absence of a conclusive diagnosis of VSCC on biopsy, the surgeon may opt for a radical excision on grounds of strong clinical suspicion, with a view to avoiding repeat surgeries. We studied a retrospective, 10-year cohort of patients who underwent vulvar excisions, in the absence of a conclusive biopsy diagnosis of VSCC. We aimed to identify the factors predictive of VSCC in these patients, and assess their treatment.All patients who underwent vulvar excision (2005-2016) at Erasmus MC, without a definitive diagnosis of VSCC on the preoperative biopsy were included. Logistic regression analysis was performed to identify the factors predictive of a final diagnosis of VSCC. Surgical treatment was categorized as definitive, incomplete, or over-treatment, based on histopathology of the excision specimen and previous surgical history.In 57 % (64/113) of all included patients, the final diagnosis was VSCC. Higher patient age (p = 0.03), and suspicion of VSCC on pre-operative biopsy (p0.001) were associated with a final diagnosis of VSCC on univariate analysis. Suspicion of VSCC on biopsy was the only significant predictor (p0.001) on multivariable analysis. For patients with a suspicion of VSCC on biopsy, radical treatment was more frequently performed (p0.001), which resulted in over-treatment in only 1 case. Where the surgeon had performed a limited excision despite a suspicion of VSCC on biopsy, high patient age, co-morbidities, location of the tumor close to the anus, and history of previous vulvar surgeries were factors which influenced the decision. The treatment administered was definitive for 72 %., i.e. additional surgeries were not required; 25 % received incomplete treatment and needed additional surgeries, and 3% received over-treatment.Suspicion of VSCC on biopsy is strongly predictive of a final diagnosis of carcinoma. In our cohort, radical treatment performed on patients with clinical and histopathological suspicion of VSCC resulted in minimal over-treatment, and helped avoid second surgeries.

Differentiated vulvar intraepithelial neoplasia (dVIN) is the precursor lesion of HPV-negative vulvar squamous cell carcinoma (VSCC). The histopathological diagnosis of dVIN can be challenging, as it often resembles vulvar non-neoplastic epithelial disorders (NNED), especially lichen sclerosus (LS). We aimed to establish the most specific and reproducible histological features of dVIN and assessed cytokeratin 13 (CK13) and cytokeratin 17 (CK17) immunohistochemistry as a diagnostic aid. Consecutive cases of dVIN (n = 180) and LS (n = 105) from the period 2010 to 2013 were reviewed using a checklist of histological features. Each feature was recorded as ‘present’ or ‘absent’ and statistical comparison (dVIN vs LS) was made. Interobserver agreement between two pairs of pathologists was assessed for a subset of cases of dVIN (n = 31) and LS and other NNED (n = 23). Immunohistochemistry with CK13, CK17, MIB1 and p53 was performed on dVIN, LS, and other NNED cases. Macronucleoli, features of disturbed maturation and angulated nuclei were significantly more common in dVIN than LS (p

Background Differentiated oral intraepithelial dysplasia (DOIN) was described by Japanese pathologists in 2007 but is not recognized by the World Health Organization. As in the vulva and penis, differentiated dysplasia in the oral cavity is a diagnostic challenge. Objective We determined reliable histologic criteria for the diagnosis of DOIN and assessed the usefulness of cytokeratins (CK) 13 and 17, combined with proliferation marker KI67. The frequency of DOIN in oral squamous cell carcinoma (OSCC) was also estimated. Methods All OSCC cases from 2014 to 2017 were reviewed for the presence of dysplasia. For differentiated dysplasia, histologic features were studied in detail and diagnostic criteria were established. Interobserver agreement was measured. Immunohistochemistry with CK13 and CK17/Ki67 was performed. Results We noted DOIN in 69% of OSCC cases (143/207). The histologic changes of DOIN were conspicuous in only 27% of cases, whereas in 73% the changes were subtle. Immunohistochemistry with CK13 and CK17 correlated with the histology: loss of CK13 and expression of CK17 in dysplasia, combined with KI67 altered expression. Conclusions DOIN is more frequently associated with oral carcinoma than usual dysplasia. In most cases the histologic changes are subtle. In this study we have attempted to define the histologic criteria. Cytokeratins 13 and 17/KI67 can be useful to support the diagnosis, especially in cases with subtle histologic changes. Recognition of subtle dysplastic changes will lead to progress in knowledge and treatment. We hope that the World Health Organization will recognize DOIN in the future.

To determine the impact of pathology review on the management of patients with cervical carcinoma, 264 reports of pathology review from 230 patients referred to Erasmus MC (2010-2012) were studied retrospectively. Discrepancies between pathologic diagnoses were classified as 'major' if they led to changes in treatment, and as 'minor' where there was no change. Patient and tumor characteristics were analyzed to identify the factors influencing these discrepancies. Fifty-eight (25.2%) discrepancies were identified; 28 (12.2%) were major, these resulted frequently from missing essential information, or discordant assessment of tumor invasion. Pathology review prevented under-treatment of 3.5%, over-treatment of 1.3%, treatment for incorrect malignancy of 1.3%, and enabled definitive treatment of 6.1% of patients. This highlights the importance of pathology review for appropriate management. Major discrepancies were rare (1%) for patients with macroscopic tumor and histologic diagnosis of squamous cell carcinoma (n = 100). For these patients, yield of pathology review may be limited.

Introduction/Background To examine the accuracy of the diagnosis endometrial intraepithelial neoplasia (EIN) in an office endometrium sample (OES). Methodology Consecutive patients aged ≥40 years with any indication for OES by aspiration curettage (OES-A) or outpatient hysteroscopy (OES-H) were asked for written consent. Pathology was assessed according to the EIN system and the WHO94 classification. The pathology of any further, more extensive, specimen obtained within 12 months of OES, was regarded as the final pathological diagnosis. Statistical analysis was performed with SPSS24. Central study approval was obtained at the ErasmusMC, (MEC 2015-740), the study was registered on www.trialregister.nl (NL7608). Results From 01-2016 to 02-2019, 343 patients were included, as summarized in table 1. OES-A was the first sampling for 340 patients. Of these, 43 samples were non-diagnostic (12,4%). Benign, EIN or malignancy was the diagnosis in 258 (76,1%), 6 (1,8%) and 33 (9,7%) cases respectively (figure 1). Final pathological diagnosis showed 290 benign cases (85,5%), 3 of EIN (0,9%) and 46 malignancies (13,6%). The accuracy of diagnosing EIN in an OES-A was 85,5%, with sensitivity of 46,2% and specificity of 100%. Subsequently, 66 OES-H samples were obtained, the accuracy of diagnosing EIN in an OES-H was 91,3%. The incidence of EIN in any OES was 2,5%. In eight out of ten patients a malignancy was ultimately diagnosed. Analyses of the EIN cases show that, despite not all five EIN histopathological criteria were met, the diagnosis EIN was set. Conclusion EIN was diagnosed in specimen obtained through OES, although the diagnosis EIN was made despite not all histological criteria were being met. The EIN and WHO94 systems showed a considerable overlap. Given the low incidence of EIN, our study cohort will be extended to further define the place of the EIN system in an office sampling setting. Disclosure Nothing to disclose.

Introduction/Background Since 2016 routine Lynch syndrome screening (LSS) among women with endometrial cancer Objective Evaluate the implementation of the Dutch guideline to screen women with endometrial carcinoma under the age of 70 years for Lynch syndrome. Methodology From the pathology database women diagnosed with EC Results In 184 out of the 205 tumours (90%) LSS was performed (table 1). Either on the endometrial biopsy 24%, the hysterectomy (70%), or on both (6%). In 42 cases (22%) MMR protein expression was lost, in 25 cases due to hypermethylation of the MLH1 promotor. LS susceptibility was discussed with 11 of the 17 patients at risk of LS (65%), all were referred for genetic counselling. Three of them declined; eight underwent germline testing for LS after counselling. In one no germline mutation was detected, two were diagnosed with a PMS2 mutation, and five with a MSH6 mutation. Conclusion The implementation of the guideline regarding LSS has been successful, though referral for genetic counselling should be improved. Gynaecologist ought to be aware of the benefits and drawbacks of knowing mutational status, and may require training in discussing this with their patients. Disclosure Nothing to disclose.

Aims The depth of invasion is an important prognostic factor for patients with vulvar squamous cell carcinoma (SCC). The threshold of 1 mm distinguishes between FIGO stages IA and >= IB disease and guides the need for groin surgery. Therefore, high interobserver agreement is crucial. The conventional and the alternative method are described to measure the depth of invasion. The aims of this study were to assess interobserver agreement for classifying the depth of invasion using both methods and to identify pitfalls. Methods and results Fifty slides of vulvar SCC with a depth of invasion approximately 1 mm were selected, digitally scanned and independently assessed by 10 pathologists working in a referral or oncology centre and four pathologists in training. The depth of invasion was measured using both the conventional and alternative method in each slide and categorised into 1 mm. The percentage of agreement and Light's kappa for multi-rater agreement were calculated, and 95% confidence intervals were calculated by bootstrapping (1000 runs). The agreement using the conventional method was moderate (kappa = 0.57, 95% confidence interval = 0.45-0.68). The percentage of agreement among the participating pathologists using the conventional method was 85.0% versus 89.4% using the alternative method. Six pitfalls were identified: disagreement concerning which invasive nest is deepest, recognition of invasive growth and where it starts, curved surface, carcinoma situated on the edge of the tissue block, ulceration and different measurement methods. Conclusions Pathologists reached only moderate agreement in determining the depth of invasion in vulvar SCC, without a notable difference between the two measurement methods.

Immunohistochemistry (IHC) for DNA mismatch repair proteins MLH1, PMS2, MSH2, and MSH6 is used for microsatellite instability (MSI) screening in colorectal carcinoma (CRC) and endometrial carcinoma (EC). Loss of PMS2, with retained MLH1 staining occurs in germline mutations of PMS2 gene, and is an indication for genetic testing. We report a pitfall of immunohistochemical interpretation in an EC, initially regarded as MLH1-positive and PMS2-negative. Review of the MLH1-IHC (M1-clone) revealed a granular, dot-like, nuclear staining. On repeating the MLH1-IHC with a different clone (ES05-clone), complete negativity was noted, and on molecular testing, MLH1 promotor methylation was detected. The dot-like pattern was therefore adjudged a clone-dependent artefact. On reviewing the archived MLH1-IHC slides, we observed the same dot-like pattern in two CRCs; in both cases the M1-clone had been used. Awareness of this artefact may prevent reporting errors, and unnecessary referrals for germline mutation testing.

The Gleason score is one of the most important parameters for therapeutic decision-making in prostate cancer patients. Gleason growth patterns are defined by their histological features on 4- to 5-µm cross sections, and little is known about their three-dimensional architecture. Our objective was to characterize the three-dimensional architecture of prostate cancer growth patterns. Intact tissue punches (n = 46) of representative Gleason growth patterns from radical prostatectomy specimens were fluorescently stained with antibodies targeting Keratin 8/18 and Keratin 5 for the detection of luminal and basal epithelial cells, respectively. Punches were optically cleared in benzyl alcohol–benzyl benzoate and imaged using a confocal laser scanning microscope up to a depth of 500 µm. Gleason pattern 3, poorly formed pattern 4, and cords pattern 5 all formed a continuum of interconnecting tubules in which the diameter of the structures and the lumen size decreased with higher grades. In fused pattern 4, the interconnections between the tubules were markedly closer together. In these patterns, all tumor cells were in direct contact with the surrounding stroma. In contrast, cribriform Gleason pattern 4 and solid pattern 5 demonstrated a three-dimensional continuum of contiguous tumor cells, in which the vast majority of cells had no contact with the surrounding stroma. Transitions between cribriform pattern 4 and solid pattern 5 were seen. There was a decrease in the number and size of intercellular lumens from cribriform to solid growth pattern. Glomeruloid pattern 4 formed an intermediate structure consisting of a tubular network with intraluminal epithelial protrusions close to the tubule splitting points. In conclusion, three-dimensional microscopy revealed two major architectural subgroups of prostate cancer growth patterns: (1) a tubular interconnecting network including Gleason pattern 3, poorly formed and fused Gleason pattern 4, and cords Gleason pattern 5, and (2) serpentine contiguous epithelial proliferations including cribriform Gleason pattern 4 and solid Gleason pattern 5.

Cancer stem–like cells (CSC) may be critical to maintain the malignant behavior of solid and hematopoietic cancers. Recently, patients with endometrial cancer whose tumors expressed high levels of aldehyde dehydrogenase (ALDH), a detoxifying enzyme characteristic of many progenitor and stem cells, exhibited a relative reduction in survival compared with patients with low levels of ALDH. Given evidence of its role as a CSC marker, we hypothesized that high level of ALDH activity (ALDHhi) in a tumor might positively correlate with the presence of stem- and progenitor-like tumor cells in this disease setting. In support of this hypothesis, ALDH could be used to enrich for CSC in endometrial cancer cell lines and primary tumors, as illustrated by the increased tumor-initiating capacity of ALDHhi cells in immunodeficient mice. ALDHhi cells also exhibited greater clonogenic and organoid-forming capacity compared with ALDHlo cells. Notably, the number of ALDHhi cells in tumor cell lines and primary tumors inversely correlated with differentiation grade. Expression analysis revealed upregulation of IL6 receptor subunits and signal transducers CD126 and GP130 in ALDHhi endometrial cancer cells. Accordingly, targeted inhibition of the IL6 receptor and its downstream effectors JAK1 and STAT3 dramatically reduced tumor cell growth. Overall, our results provide a preclinical rationale to target IL6 or its effector functions as a novel therapeutic option in endometrial cancer. Cancer Res; 75(17); 3608–22. ©2015 AACR.

Oncological applications of Raman spectroscopy have been contemplated, pursued, and developed at academic level for at least 25 years. Published studies aim to detect pre-malignant lesions, detect cancer in less invasive stages, reduce the number of unnecessary biopsies and guide surgery towards the complete removal of the tumour with adequate tumour resection margins. This review summarizes actual clinical needs in oncology that can be addressed by spontaneous Raman spectroscopy and it provides an overview over the results that have been published between 2007 and 2017. An analysis is made of the current status of translation of these results into clinical practice. Despite many promising results, most of the applications addressed in scientific studies are still far from clinical adoption and commercialization. The main hurdles are identified, which need to be overcome to ensure that in the near future we will see the first Raman spectroscopy-based solutions being used in routine oncologic diagnostic and surgical procedures.

Hypothesis. Pathology slide review in vulvar cancer is only necessary in a restricted number of cases. Methods. A retrospective chart review of all cases of vulvar cancer treated in a tertiary centre between January 1, 2000, and April 1, 2006. Histopathology reports from the referring and tertiary centre were compared. Results. 121 pathology reports from 112 patients were reviewed. Of the original reports, 56% were deemed adequate, commenting on tumor type and depth of infiltration; of the reviews, 83% were adequate. Conclusion. There were no discrepancies that influenced patient management. We suggest that vulvar cancer biopsies need to be reviewed only when the tumor is less than 10 mm in linear extension, when the infiltration is 1 mm or less, when there is no residual tumor on inspection, and in any nonsquamous cancer.

Ovarian small-cell carcinoma of the hypercalcemic type is a rare and highly malignant tumor. In two thirds of the patients, the tumor is associated with asymptomatic paraneoplastic hypercalcemia. The diagnosis may be impeded; the tumor must be distinguished from other tumors with similar features.This tumor occurs predominantly in young women and is merely lethal. The 1-year survival is solely 50%, with an overall 5-year survival rate of approximately 10%. It is believed that the empirical treatment characterized by combination of radical surgery, chemotherapy, and radiotherapy results in the most favorable outcome in terms of survival. However, the outcome remains extremely poor despite this aggressive approach.Alternatively, these poor survival rates may justify a less aggressive fertility sparing approach without compromising the outcome. Such an approach is illustrated by a case report involving a patient with ovarian small-cell carcinoma of the hypercalcemic type, FIGO stage IIIC. A fertility-sparing approach was used, consisting of conservative surgery followed by induction chemotherapy, interval debulking surgery, and local radiotherapy. During follow-up of 60 months, there was no evidence of disease and the normal menstrual cycle resumed.In addition to this case report, histopathological features, different therapeutic modalities, and outcome of ovarian small-cell carcinoma of the hypercalcemic type is reviewed. This report suggests that a fertility-sparing approach may be just as feasible as the generally applied aggressive approach.

Aims Cold ischaemic and formalin fixation time (CIT and FFT) are considered to be crucial parameters for intralaboratory variation in immunohistochemistry (IHC). Here we describe a new method to optimize IHC, by using control tissue blocks with known pre-analytical history and comparing the IHC outcome with digitized reference slides. Methods and results Tissue specimens (two per tissue type) were divided into eight samples, which were subjected to different CIT and FFT. Immunohistochemistry was performed with 34 routinely used antibodies, following standard operating procedures. Relative staining intensity of four sections per slide was scored. Of the antibodies studied, seven were influenced by CIT, 13 by FFT and five by both parameters. IHC protocols were adapted until most sections on the slide showed the same intensity. Changing the antibody dilution for 10 protocols and the antigen retrieval method for six protocols improved the consistency of the IHC staining. Nine protocols could not be optimized. The optimized staining results were compared to reference slides and were found to be of adequate quality. Conclusions It was possible to optimize most IHC protocols by adapting the analytical, rather than the pre-analytical, phase. If global references can be established, this method could decrease interlaboratory variation, preceding standardization of the pre-analytical workflow.

We investigated the significance of prognostic markers-estrogen receptor, progesterone receptor, p53, MIB-1 and bcl-2 - in adenocarcinoma of the uterine cervix. In 101 patients with primary cervical adenocarcinoma, treated from 1989 to 2000, we evaluated clinical parameters in relation to these prognostic markers. Mean age of patients was 45 years. Seventy eight percent of the patients were in FIGO stage I, 16% stage II, 7% stage III and IV. estrogen receptor, progesterone receptor, p53 and bcl-2 immunoreactivity was scored as 0 (up to 5% positive cells), 1+ (5-25% of cells positive), 2+ (26-50% of cells positive), 3+ (51-75% of cells positive) or 4+ (>76% of cells positive). MIB-1 was scored in 10 categories: 0-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100. The overall survival rate was 67%. Survival was not influenced by estrogen receptor, progesterone receptor, MIB-1, or bcl-2 strongly positive staining. Only p53 showed significant influence on survival, even when adjusted for stage or tumor grade. In conclusion, it does not seems useful to determine estrogen receptor, progesterone receptor, MIB-1 or bcl-2 in cervical adenocarcinomas as an indication of prognosis: survival is not influenced by presence or absence. However, if p53 staining is strongly positive survival is significantly worse than in tumors scored as negative or weak positive.

To determine the behaviour of adenocarcinomas of the uterine cervix during the last 10 years in the southwest region of the Netherlands, and to determine prognostic factors.Three hundred and five cases of primary cervical adenocarcinomas (ACs) diagnosed between 1989 and 1999 in the region of Rotterdam, The Netherlands, were retrieved. Clinical and pathological data were reviewed and analysed.Mean age at presentation was 52 years. The mean follow-up time for surviving patients was 61 months. The overall survival was 60% at 5 years. The 5-year survival rates for stages I and II were, respectively, 79% and 37%. The 5-year survival rates for stages III and IV were less than 9%. Using univariate analysis stage, grade, age35 years and histological type were significant prognostic factors. In the group of patients who underwent surgery (n = 200), stages I-IIA, lymph node metastases, lymph-vascular-space-invasion (LVSI) and depth of stromal invasion were significant for survival. For patients with stages I and II-A disease, survival was significantly better where the primary treatment was surgical as opposed to primary radiotherapy (P = 0.002). Using multivariate analysis, only stage, grade and lymph node metastases remained significant independent predictors for survival.This report about cervical adenocarcinoma in the southwest region of The Netherlands shows similar results for survival to previous reports. Longest survival was for patients with early stage disease, younger patients and after primary surgery. We found FIGO stage, grade and lymph node metastases of significant prognostic value for survival in cervical adenocarcinoma.