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OBJECTIVE To compare the diagnostic accuracy of transvaginal ultrasound (TVS) and magnetic resonance imaging (MRI) for assessing myometrial infiltration (MI) in patients with low grade endometrioid endometrial cancer. METHODS Observational prospective study performed at a single tertiary care center from 2016 to 2020, comprising 156 consecutive patients diagnosed by endometrial sampling as having an endometrioid grade 1/grade 2 endometrial cancer. TVS and MRI were performed prior to surgical staging for assessing MI, which was estimated using subjective examiner's impression and Karlsson's method for both TVS and MRI. During surgery, intraoperative assessment of MI was also performed. Definitive pathological study considered as reference standard. Diagnostic accuracy for ultrasound, MRI, and intraoperative biopsy was estimated and compared. RESULTS Sensitivity and specificity of TVS for detecting deep MI were 75 and 73.5% for subjective impression and 65 and 70% for Karlsson method, respectively (P = .54). Sensitivity and specificity of MRI for detecting deep MI were 80 and 87% for subjective impression and 70 and 71.3% for Karlsson method. MRI subjective impression showed a significant better specificity than MRI Karlsson method (P = .03). MRI showed better specificity than TVS when subjective impression was considered (P

Most succinate dehydrogenase (SDH)-deficient renal cell carcinomas (RCCs) demonstrate stereotypical morphology characterized by bland eosinophilic cells with frequent intracytoplasmic inclusions. However, variant morphologic features have been increasingly recognized. We therefore sought to investigate the incidence and characteristics of SDH-deficient RCC with variant morphologies. We studied a multi-institutional cohort of 62 new SDH-deficient RCCs from 59 patients. The median age at presentation was 39 years (range 19-80), with a slight male predominance (M:F = 1.6:1). A relevant family history was reported in 9 patients (15%). Multifocal or bilateral tumors were identified radiologically in 5 patients (8%). Typical morphology was present at least focally in 59 tumors (95%). Variant morphologies were seen in 13 (21%) and included high-grade nuclear features and various combinations of papillary, solid, and tubular architecture. Necrosis was present in 13 tumors, 7 of which showed variant morphology. All 62 tumors demonstrated loss of SDHB expression by immunohistochemistry. None showed loss of SDHA expression. Germline SDH mutations were reported in all 18 patients for whom the results of testing were known. Among patients for whom follow-up data was available, metastatic disease was reported in 9 cases, 8 of whom had necrosis and/or variant morphology in their primary tumor. Three patients died of disease. In conclusion, variant morphologies and high-grade nuclear features occur in a subset of SDH-deficient RCCs and are associated with more aggressive behavior. We therefore recommend grading all SDH-deficient RCCs and emphasize the need for a low threshold for performing SDHB immunohistochemistry in any difficult to classify renal tumor, particularly if occurring at a younger age.

Mismatch repair deficiency (MMRD) is involved in the initiation of both hereditary and sporadic tumors. MMRD has been extensively studied in colorectal cancer and endometrial cancer, but not so in other tumors, such as ovarian carcinoma. We have determined the expression of mismatch repair proteins in a large cohort of 502 early-stage epithelial ovarian carcinoma entailing all the 5 main subtypes: high-grade serous carcinoma, endometrioid ovarian carcinoma (EOC), clear cell carcinoma (CCC), mucinous carcinoma, and low-grade serous carcinoma. We studied the association of MMRD with clinicopathologic and immunohistochemical features, including tumor-infiltrating lymphocytes in EOC, the histologic type in which MMRD is most frequent. In addition, MLH1 promoter methylation status and massive parallel sequencing were used to evaluate the proportion of sporadic and Lynch syndrome-associated tumors, and the most frequently mutated genes in MMRD EOCs. MMRD occurred only in endometriosis-associated histologic types, and it was much more frequent in EOC (18%) than in CCC (2%). The most frequent immunohistochemical pattern was loss of MLH1/PMS2, and in this group, 80% of the cases were sporadic and secondary to MLH1 promoter hypermethylation. The presence of somatic mutations in mismatch repair genes was the other mechanism of MMRD in sporadic tumors. In this series, the minimum estimated frequency of Lynch syndrome was 35% and it was due to germline mutations in MLH1, MSH2, and MSH6. ARID1A, PTEN, KTM2B, and PIK3CA were the most common mutated genes in this series. Interestingly, possible actionable mutations in ERRB2 were found in 5 tumors, but no TP53 mutations were detected. MMRD was associated with younger age and increased tumor-infiltrating lymphocytes. Universal screening in EOC and mixed EOC/CCC is recommended for the high frequency of MMRD detected; however, for CCC, additional clinical and pathologic criteria should be evaluated to help select cases for analysis.

Langerhans cell histiocytosis (LCH) of the vulva is rare and even moreso in postmenopausal women. Twenty-six cases of primary vulvar LCH have been described in the current literature, and only 8 cases are in postmenopausal women. We report an additional case of primary vulvar LCH in a 59-yr-old woman with subsequent multiorgan involvement. In this article, we briefly describe the clinical presentation, histopathological findings, and immunohistochemistry results of vulvar LCH. We want to emphasize the importance of recognizing this entity in a woman with vulvar lesions both for the clinician and the pathologist.

The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix &ndash, SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell&reg, invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC, and explain why p16 overexpression fails to stop CC growth.

This paper presents a clinicopathologic and immunohistochemical report of 2 gastrointestinal-type tumors, one in the endometrium and the other in the cervix. Both showed extensive invasion into the pelvic structures with acellular mucin, identical to pseudomyxoma but in the absence of appendiceal or ovarian tumors. Case 1 was an 81-yr-old female with a Stage III endometrial gastrointestinal-type adenocarcinoma who had had an endometrial polyp with intestinal metaplasia 4 years previously. Case 2 was a 68-yr-old female with Stage IIIB endocervical gastrointestinal-type adenocarcinoma. Both were associated with a pseudomyxoma type of invasion, which in the endometrial case was transmural through the myometrium, and in the cervical case involved parametria, pelvic floor, and lymph nodes. Immunohistochemically, both tumors had a gastrointestinal phenotype coexpressing cytokeratins 7 and 20, CDX2, villin, MUC2, MUC5AC, and MUC6 and were negative for human papillomavirus, analyzed by real-time polymerase chain reaction. The first case exemplifies intestinal endometrial metaplasia as a precursor lesion of the rare gastrointestinal type of adenocarcinoma and also proves its progression into carcinoma. The second case exemplifies the highly aggressive nature of cervical invasion forming mucin lakes. Extensive pseudomyxoma in the uterus and cervix was associated with high clinical stages with marked lymphovascular invasion and lymph node metastases.

Immunohistochemistry is a basic routine in establishing the diagnosis of many tumors. However, immunomarkers are often irregularly distributed across different regions of the same tumor, alternating positive and negative areas without any apparent cause. Full identification of this type of intratumor heterogeneity is crucial for patients since the expression of many markers is linked to the prognosis and/or treatment of some tumors. We have quantified this variability testing 406 tumor samples from eight clear cell renal cell carcinomas using four epithelial-mesenchymal transition markers (vimentin, ZEB-1, β-catenin, and E-cadherin) and two different sampling protocols. Routine sampling included an amount of 59 samples (average, 7.3 samples/case) and multisite tumor sampling did a total of 347 samples (average, 43.3 samples/case). High variability of immunostaining was detected with E-cadherin and ZEB-1 in all high-grade cases. Irregular patterns of expression were detected in all tumors including all histologically homogeneous low-grade tumors. Multisite tumor sampling protocol detected a significant decreased number of E-cadherin, β-catenin and ZEB-1 positive samples in high-grade tumors. We conclude that high levels of intratumor heterogeneity characterize the immunohistochemical expression of epithelial-mesenchymal transition markers in high-grade clear cell renal cell carcinomas. Multisite tumor sampling protocol outperforms routine sampling in detecting immunohistochemical intratumor heterogeneity. Sin financiación 1.608 JCR (2018) Q3, 50/76 Pathology 0.633 SJR (2018) Q2, 1043/2844 Medicine (miscellaneous), 81/210 Pathology and Forensic Medicine No data IDR 2018 UEM

Heterogeneity is an inherent event to tumour development that is lately receiving much attention in oncologic research. The topic is being addressed primarily at the molecular level, and results are promising. However, translation to practical medicine is still pending. Our intention in this study is to approach the problem in a series of clear cell renal cell carcinomas with the tools that pathologists use in routine practice. Three randomly selected areas of 48 clear cell renal cell carcinomas prospectively collected in two different institutions were analysed for intratumour heterogeneity. The evaluated parameters were tumour size, cell type (clear vs. eosinophilic), Fuhrman’s grade and immunohistochemical expression of carbonic anhydrase IX, BRCA1-associated protein-1 (BAP-1), cyclooxygenase-2 (COX-2) and Ki67. Intratumour heterogeneity was detected in 26 cases (54 %). Cell type, grade and Ki67 index were the parameters more frequently heterogeneous amounting, respectively, 44, 42 and 38 %. Tumour size was a significantly discriminative factor to predict tumour heterogeneity, with a cut-off of 3.8 cm (p < 0.001). Aside from tumour size, the most relevant parameters related with intratumour heterogeneity were cell type (clear vs. eosinophilic), Fuhrman’s grade and Ki67 and COX-2 expression patterns. Carbonic anhydrase 9 and BAP-1 did not show statistical relevance. We conclude that heterogeneity is a common event in clear cell renal cell carcinomas that may be overlooked in cases insufficiently sampled. Tumour size appears as a reliable tool in identifying this situation since clear cell renal cell carcinomas under 3.8 cm in diameter are always homogeneous. This point may help the pathologist to make decisions in tumour sampling.

Nephrogenic adenoma is a benign condition of the urinary tract resulting from the displacement and seeding of renal tubular cells from the renal pelvis to the urethra. A retrospective series of 134 cases collected from four hospitals in three different countries was analyzed in this study. Recorded clinical data included age and sex, topography, urological antecedents, coexistent lesions, and follow-up. Cytonuclear and architectural features were reviewed, and PAX-8, p63, PSMA, S100A1, CEA, EMA, CD117, cannabinoid receptor CB1, AMACR, E-cadherin, and CD10 antibodies were included in an immunohistochemical panel. Males predominated (105 M/29 F) with an average age of 66 years (range, 14-96). Urothelial carcinoma was the most frequent clinical antecedent (43.2 %) and also the most common coexisting lesion (14 %). Tubular architecture was the most frequent pattern detected (40 %) although most cases showed a mixed pattern (45.5 %). Deep infiltrative growth into the muscularis propria occurred in two cases. EMA and PAX-8 were expressed in 100 % of nephrogenic adenomas, while E-cadherin reactivity was observed in 66.6 % of cases, cannabinoid receptor CB1 in 25 %, CD10 in 13.6 %, CD117 in 4.1 %, and AMACR in 2.7 %. For the rest of the antigens, no reactivity was found. The average time lapse between the pathological antecedent and the discovery of a nephrogenic adenoma was 32 months. We conclude that nephrogenic adenoma displays a broad spectrum of histological features that may mimic malignancy. In our experience, CB1 immunostaining adds a further argument in favor of a renal origin of this lesion. The combination of PAX-8+, p63-, and EMA + distinguishes nephrogenic adenoma from urothelial and prostate carcinoma, its most frequent malignant look-alikes.

OBJECTIVE: For patients with metastatic renal cell cancer (mRCC), treatment choice is mainly based on clinical parameters. With many treatments available and the limited response to treatment and associated toxicities, there is much interest in identifying better biomarkers for personalized treatment. EuroTARGET aims to identify and characterize host- and tumor-related biomarkers for prediction of response to tyrosine kinase inhibitor therapy in mRCC. Here, we describe the EuroTARGET mRCC patient cohort. METHODS AND MATERIALS: EuroTARGET is a European collaborative project designed as an observational study for which patients with mRCC were recruited prospectively in 62 centers. In addition, 462 patients with mRCC from previous studies were included. Detailed clinical information (baseline and follow-up) from all patients was entered in web-based case record forms. Blood was collected for germline DNA and pharmacokinetic/pharmacodynamic analyses and, where available, fresh-frozen tumor material was collected to perform tumor DNA, RNA, kinome, and methylome analyses. RESULTS: In total, 1,210 patients with mRCC were included. Of these, 920 received a tyrosine kinase inhibitor as first-line targeted treatment (sunitinib [N = 713, 78%], sorafenib [N = 41, 4%], or pazopanib [N = 166, 18%]) and had at least 6 months of outcome assessment (median follow-up 15.3 months [interquartile range: 8.5-30.2 months]). Germline DNA samples were available from 824 of these patients, fresh-frozen tumor material from 142 patients, fresh-frozen normal kidney tissue from 95 patients, and tissue microarrays created from formalin-fixed paraffin-embedded tumor material from 247 patients. Of the 920 patients, germline DNA variant chip data were successfully generated for 811 patients (Illumina HumanOmniExpress BeadChip). For 80 patients, next-generation exome sequencing of germline and tumor DNA was performed, tumor RNA sequencing was performed for 124 patients, kinome activity measured and processed for 121 patients (PamChip), and methylome data (Illumina Infinium HumanMethylation450 BeadChip) were created for 116 RCC tissues (and 23 normal kidney tissues). For 73 out of the 920 patients, all platform data types were generated. In addition, 40 patients were included in a pharmacokinetic/pharmacodynamic phase IV substudy. CONCLUSIONS: Analysis of EuroTARGET cohort data will contribute to personalization of therapy for patients with mRCC. The extensive clinical data and multiplatform EuroTARGET data will be freely available. We would like to thank all patients who participated and their treating physicians for inviting them. Sí

Clear cell renal cell carcinoma is an aggressive neoplasm related to VHL gene inactivation. The molecular events derived from this initial alteration lead to a permanent intracellular pseudo‐hypoxic status that stimulates vascular proliferation. The resulting increased intratumor angiogenesis is the target of most modern therapies. Although intratumor angiogenesis has received full attention in the last years, few studies have focused on its potential importance from a strict morphological approach. Intratumor angiogenesis has been analyzed in a retrospective series of clear cell renal cell carcinomas (n = 208) with long‐term follow‐up (n = 177). Two different patterns of angiogenesis have been highlighted with CD34 at the front of tumor invasion, termed continuous and discontinuous, respectively. The continuous pattern of angiogenesis showed a complete microvascular network surrounding totally tumor nests. Conversely, the discontinuous pattern displayed an incomplete network around tumor nests. The continuous pattern was associated to shorter 5‐year (p = 0.00064, hazard ratio = 2.8) and 15‐year (p = 0.014, hazard ratio = 1.7) survivals. Cox regression multivariate analysis also showed that the continuous pattern (p = 0.016373) remains a significant variable when considered together with grade (p = 0.001755) and stage (p = 0.000952). These findings support the notion that a continuous CD34+ pattern of intratumor angiogenesis may be useful for pathologists in predicting tumor behavior in clear cell renal cell carcinomas. Sin financiación 2.026 JCR (2017) Q2, 38/79 Pathology; Q3, 83/125 Microbiology; Q4, 124/155 Inmunology UEM

Clear cell renal cell carcinoma is a complex disease with only partial response to therapy and scarce reliable clinical parameters indicative of progression and survival. Fibroblast activation protein expression has been correlated with prognosis in several malignancies but never in renal cancer. We aim to analyze the immunohistochemical expression of fibroblast activation protein in 208 clear cell renal cell carcinomas and to evaluate its impact on the prognosis and survival. A positive cytoplasmic immunostaining of this protein in the stromal fibroblasts associated to cancer cells is associated with large tumor diameter (≥ 4 cm), high-grade (G3/4) tumors, and high-stage (≥ pT3) tumors. Fibroblast activation protein–positive cases had significantly shorter survivals after 5 (P = .00015), 10 (P = .0000042), and 15 (P = .000043) years of follow-up, with a hazard ratio of 0.31. Multivariate analysis showed that fibroblast activation protein (P = .00117) was stronger than grade and stage in predicting clinical aggressiveness in clear cell renal cell carcinoma. This study confirms the usefulness of fibroblast activation protein detection in the stromal fibroblast associated to cancer in clear cell renal cell carcinoma and adds a new immunohistochemical marker to predict clinical behavior in these patients. Sin financiación 3.014 JCR (2016) Q1, 18/79 Pathology UEM

Clear cell renal cell carcinoma (CCRCC) is a heterogeneous and complex disease that frequently develops distant metastases. Fibroblast activation protein (FAP) is a serine peptidase the expression of which in cancer-associated fibroblasts has been associated with higher risk of metastases and poor survival. The objective of this study was to evaluate the role of FAP in metastatic CCRCC (mCCRCC). A series of 59 mCCRCC retrospectively collected was included in the study. Metastases developed either synchronous (n = 14) or metachronous to renal disease (n = 45). Tumor specimens were obtained from both primary lesion (n = 59) and metastases (n = 54) and FAP expression was immunohistochemically analyzed. FAP expression in fibroblasts from primary tumors correlated with FAP expression in the corresponding metastatic lesions. Also, primary and metastatic FAP expression was correlated with large tumor diameter (>7cm), high grade (G3/4), high stage (pT3/4), tumor necrosis and sarcomatoid transformation. The expression of FAP in primary tumors and in their metastases was associated both with synchronous metastases and also with metastases to the lymph nodes. FAP expression in the primary tumor was correlated with worse 10-year overall survival. Immunohistochemical detection of FAP in the stromal tumor fibroblasts could be a biomarker of early lymph node metastatic status and therefore could account for the poor prognosis of FAP positive CCRCC. This work was partially funded by Grant SAF2013-48812-R from Ministerio de Economia y Competitividad (Spain), IT 8-11/13 from de Basque Government and EHUA14/25 from de University of the Basque Country (UPV/EHU). The current work has been developed as PhD project of PE and MB, who are recipients of a Predoctoral Fellowship from the Basque Government (Exp no PRE_2013_1_762 and PRE_2015_2_0148). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Clear cell renal cell carcinoma (CCRCC) frequently develops distant metastases. However, high-grade primary CCRCC rarely leads to low-grade metastases. Cellular changes occurring during neoplastic progression known as epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions explain this apparent contradiction. Four high-grade CCRCCs, which lead to low-grade metastases, are analyzed in this study, with the focus on epithelial-to-mesenchymal and mesenchymal-to-epithelial processes. Clinicopathologic data have been collected retrospectively and immunohistochemistry has been performed with E-cadherin, N-cadherin, vimentin, and WT-1. Three cases had organ-confined disease (2 pT2 and 1 pT1b). Three cases were G3 and 1 case was G4. Lung (3 cases), bone (2 cases), and pancreas (1 case) were the metastatic organs (2 patients developed multiple metastases). Metastases were G1 in all the cases. Average elapsed time between the primary tumor and the metastasis was 35.5 months. Three patients died of disease after 36, 120, and 180 months of follow-up, respectively. One patient is alive without disease after 75 months of follow-up. E-cadherin and N-cadherin showed concordant immunostaining patterns between primaries and metastases but inverse when correlated with Fuhrman grade. Hence, E-cadherin was positive in G3 cases and negative in G4, whereas N-cadherin was negative in G3 and positive in G4. Vimentin was positive in primaries and metastases only in 2 cases. WT-1 was consistently negative in all cases. In conclusion, pathologists must remember that high-grade CCRCC may develop low-grade metastases. Cadherin switching seems to be related to Fuhrman grade in this group of cases. This preliminary observation must be confirmed in longer studies.

Cervical cancer is the third most common cancer in women worldwide. The hypermethylation of P16, TSLC-1 and TSP-1 genes was analyzed in squamous cell carcinomas (SCC), cervical intraepithelial lesions (CIN) and adenocarcinomas (ADC) of the uterine cervix (total 181 lesions). Additionally human papillomavirus (HPV) type, EPB41L3, RASSF1 and RASSF2 hypermethylation were tested in ADC and the results were compared with those obtained previously by our group in SCC. P16, TSLC-1 and TSP-1 hypermethylation was more frequent in SCCs than in CINs. These percentages and the corresponding ones for EPB41L3, RASSF1 and RASSF2 genes were also higher in SCCs than in ADCs, except for P16. The presence of HPV in ADCs was lower than reported previously in SCC and CIN. Patients with RASSF1A hypermethylation showed significantly longer disease-free survival (P = 0.015) and overall survival periods (P = 0.009) in ADC patients. To our knowledge, this is the first description of the EPB41L3 and RASSF2 hypermethylation in ADCs. These results suggest that the involvement of DNA hypermethylation in cervical cancer varies depending on the histological type, which might contribute to explaining the different prognosis of patients with these types of tumors.

Contribution over time of human papillomavirus (HPV) types in human cancers has been poorly documented. Such data is fundamental to measure current HPV vaccines impact in the years to come. We estimated the HPV type-specific distribution in a large international series of invasive cervical cancer (ICC) over 70 years prior to vaccination. Paraffin embedded ICC cases diagnosed between 1940 and 2007 were retrieved from eleven countries in Central-South America, Asia and Europe. Included countries reported to have low-medium cervical cancer screening uptake. Information on age at and year of diagnosis was collected from medical records. After histological confirmation, HPV DNA detection was performed by SPF-10/DEIA/LiPA25 (version1). Logistic regression models were used for estimating the adjusted relative contributions (RC) of HPV16 and of HPV18 over time. Among 4,771 HPV DNA positive ICC cases, HPV16 and HPV18 were the two most common HPVs in all the decades with no statistically significant variations of their adjusted-RC from 1940–59 to 2000–07 (HPV16—from 61.5 to 62.1%, and HPV18—from 6.9 to 7.2%). As well, the RC of other HPV types did not varied over time. In the stratified analysis by histology, HPV16 adjusted-RC significantly increased across decades in adenocarcinomas. Regarding age, cases associated to either HPV16, 18 or 45 were younger than those with other HPV types in all the evaluated decades. The observed stability on the HPV type distribution predicts a high and stable impact of HPV vaccination in reducing the cervical cancer burden in future vaccinated generations.

A new case is reported of müllerian adenosarcoma, presenting as a 'benign cervical polyp' protruding through the vulva of a 44 year-old woman admitted with abnormal vaginal bleeding. This report emphasizes the importance of a careful examination of the stroma and special features of the entrapped glands in order to contribute to an earlier and proper diagnosis. The literature is reviewed and the probable histogenesis of this tumor and differential diagnosis with embryonal rhabdomysarcoma (sarcoma botryoides), adenofibroma, malignant mesodermal tumor and carcinosarcoma is discussed.

Aims Lichen sclerosus (LS) is a chronic inflammatory disease of the genital skin of unknown aetiology. The role of LS in penile squamous cell carcinogenesis is not well characterized. HPV has been implicated in both, as have epigenetic changes. The presence of HPV and hypermethylation of the MGMT, p16, RASSF1, RASSF2, TSLC1 and TSP1 genes were studied in penile LS; MGMT, RASSF2 and TSLC1 hypermethylation in penile cancer and TSLC1 hypermethylation in vulvar LS and cancer extends previous results reported by our group. Methods and results Thirty-seven HPV genotypes and hypermethylation were evaluated by PCR/reverse-line-blot and methylation-specific PCR respectively, in 27 preputial LS, 24 penile SCC, 30 vulvar SCC, 21 vulvar LS and 22 normal skin cases. HPV66 was present in 3.7% of penile LS cases, and p16 and RASSF2 hypermethylation were more frequent in penile cancer than in penile LS. p16, RASSF1, RASSF2 and TSP1 hypermethylation were similar in penile and vulvar LS. Conclusions Gene hypermethylation is a common event in penile LS, and occurs approximately as frequently as in vulvar LS. Certain genes can be hypermethylated as an early or late event in LS or cancer, respectively. This suggests a possible sequential role for these alterations in the transition from benign to malignant lesions.

The aim of this study was to define the histological spectrum, frequency and significance of nonconventional tumour cells in clear cell renal cell carcinomas (CCRCC). Fifty-one totally sampled CCRCC were studied histologically to evaluate the spectrum of cell morphology variability, its frequency and significance, and their correlation with tumour grade and stage, and other histological parameters of aggressive behaviour like necrosis. Aside from conventional clear/eosinophilic granular cells, three additional cellular types were identified and considered in this study: small clear cells, syncytial cells and rhabdoid cells. Small clear cells were detected in 11 cases (21.5%), syncytial cells in 8 (15.6%) and rhabdoid cells in 5 (9.8%). The presence of syncytial and rhabdoid cells statistically correlated with grade (p = 0.003 and p = 0.006) and stage (p = 0.049 and p = 0.05) in CCRCC. Necrosis correlated with stage (p = 0.018) and grade (p = 0.004), but not with syncytial, rhabdoid or small clear cells. The presence of syncytial and rhabdoid cells in CCRCC is a relatively frequent event that significantly correlates with high-grade tumours and high stage status.

Ras association (RalGDS/AF-6) domain family member 2 (RASSF2) is a gene involved in the progression of several human cancers, including breast, colorectal and lung cancer. The aims of this study were to determine the hypermethylation of the gene in squamous cervical cancer and precursor lesions, along with that of RASSF1 and the recently described EPB41L3, and to analyze the potential prognostic role of these genes. Methylation-specific PCR and bisulfite sequencing were used to analyze the methylation status of RASSF2 and EPB41L3 gene in 60 squamous cervical cancer, 76 cervical intraepithelial neoplasias grade III, 16 grade II, 14 grade I and 13 cases of normal tissue adjacent to cervical intraepithelial neoplasia. RASSF2 expression was evaluated by immunohistochemistry and the re-expression of RASSF2 and EPB41L3 was analyzed by quantitative reverse-transcription PCR in HeLa, SiHa, C33A and A431 cell lines treated with 5-aza-2'-deoxycytidine and/or trichostatin. RASSF1 hypermethylation and human papillomavirus type were also analyzed in all the cases by methylation-specific PCR and reverse line blot, respectively. RASSF2 hypermethylation was predominant in squamous cervical cancer (60.9%) compared with cervical intraepithelial neoplasias (4.2%) and was associated with a lower level of RASSF2 expression and vascular invasion in squamous cervical cancer. EPB41L3 and RASSF1 hypermethylations were also more frequent in cancer than in precursor lesions. Patients with RASSF2 hypermethylation had shorter survival time, independent of tumor stage (hazard ratio: 6.0; 95% confidence interval: 1.5-24.5). Finally, the expressions of RASSF2 and EPB41L3 were restored in several cell lines treated with 5-aza-2'-deoxycytidine. Taken together, our results suggest that RASSF2 potentially functions as a new tumor-suppressor gene that is inactivated through hypermethylation in cervical cancer and is related to the bad prognosis of these patients.

Squamous cell carcinoma (SCC) of the vulva is a heterogeneous disease, associated or not with vulvar lichen sclerosus (LS). The precursor role of LS in vulvar cancer is unclear. We studied the epigenetic alterations of RASSF1A, RASSF2A, p16, TSP-1 and MGMT genes in vulvar SCCs, LS associated with SCC, isolated LS and normal vulvar skin. Gene hypermethylation and human papillomavirus presence were evaluated by methylation-specific PCR and PCR/reverse line blot, respectively. High-risk human papillomavirus types were present in 16.7% of the patients with vulvar SCC. There were increasing percentages of hypermethylation of genes from isolated LS to LS associated with vulvar SCC and vulvar SCC. The genes were hypermethylated more frequently in vulvar SCC associated with LS than in those not associated with LS, MGMT and RASSF2A being unmethylated in LS not associated with vulvar SCC. TSP-1 hypermethylation was related to recurrence in patients with vulvar cancer. Conclusions are as follows: (i) the epigenetic inactivation of genes is a common event in vulvar SCC and is also present in adjacent lesions, implying a possible precursor role for these alterations; (ii) MGMT and RASSF2A hypermethylation are present exclusively in vulvar SCC and LS associated with SCC, and absent from isolated LS; and (iii) TSP-1 hypermethylation is a bad prognosis factor in vulvar SCC.

Purpose: To analyze immunohistochemically morules in endometrioid lesions to show that CD10 is a sensitive marker for morular metaplasia. Experimental Design: Immunohistochemical analysis of 53 instances of morular metaplasia comprising 1 cyclic endometrium and 52 endometrioid lesions associated with focal glandular complexity corresponding to 9 polyps, 4 atypical polypoid adenomyomas, 24 complex endometrial hyperplasias (18 with and 6 without atypia), 12 grade 1 endometrioid adenocarcinomas in early clinical stages of both uterus and ovary, and three ovarian adenofibromas. Immunohistochemistry in paraffin sections was done for CD10, β-catenin, estrogen and progesterone receptors, and cytokeratins 5-6, 7, 8, 13, 18, 19, 20, and 34β-E12. Results: Morules were negative for estrogen and progesterone receptors and had β-catenin–positive nuclei. Cytokeratins 8, 18, 19 were positive; cytokeratins 7 and 20 were negative; and cytokeratins 5-6, 13, and 34β-E12 were weakly positive. All cases revealed strongly positive membranous CD10 staining in morules, which was absent in glands. CD10 positivity allowed easy identification of morules at low power in various types of surgical specimens and in curettings. CD10 also highlighted early morular metaplasia in glandular epithelium. In cases associated with squamous, keratinizing metaplasia, CD10 discriminated between both types of metaplasia. Conclusions: CD10 staining represents a useful marker of morules in endometrioid neoplasms of the female genital tract, permitting identification of lesions usually associated with an attenuated malignancy. Considering the immunohistochemical and genetic similarities of morules in tumors of different organs, it is likely that this marker may be also useful to diagnose morular metaplasia in similar neoplasms of extragenital locations.

A 42 year-old female with a preoperative clinical diagnosis of ovarian cancer underwent laparotomy which revealed leiomyomatosis peritonealis disseminata (LPD) in the peritoneum and omentum and a left ovarian endometriotic cyst associated with a clear cell carcinoma. A grade 1, superfically invasive villoglandular endometrial endometrioid adenocarcinoma was also found. Microscopically, the endometriotic cyst wall contained an extensive peripheral band-like condensation of stromal cells. These cells were strongly positive for alpha inhibin and may have been the hormonal source responsible for the induction of the simultaneous LPD and endometrial adenocarcinoma. It is proposed that endometriosis is not only a precursor of clear cell carcinoma but, through secondary hormonal induction of the surrounding ovarian stroma, may also provide a hormonal stimulus for diverse proliferative processes.

Background: Intratumor heterogeneity may be responsible of the unpredictable aggressive clinical behavior that some clear cell renal cell carcinomas display. This clinical uncertainty may be caused by insufficient sampling, leaving out of histological analysis foci of high grade tumor areas. Although molecular approaches are providing important information on renal intratumor heterogeneity, a focus on this topic from the practicing pathologist' perspective is still pending. Methods: Four distant tumor areas of 40 organ-confined clear cell renal cell carcinomas were selected for histopathological and immunohistochemical evaluation. Tumor size, cell type (clear/granular), Fuhrman's grade, Staging, as well as immunostaining with Snail, ZEB1, Twist, Vimentin, E-cadherin, beta-catenin, PTEN, p-Akt, p110 alpha, and SETD2, were analyzed for intratumor heterogeneity using a classification and regression tree algorithm. Results: Cell type and Fuhrman's grade were heterogeneous in 12.5 and 60 % of the tumors, respectively. If cell type was homogeneous (clear cell) then the tumors were low-grade in 88.57 % of cases. Immunostaining heterogeneity was significant in the series and oscillated between 15 % for p110a and 80 % for Snail. When Snail immunostaining was homogeneous the tumor was histologically homogeneous in 100 % of cases. If Snail was heterogeneous, the tumor was heterogeneous in 75 % of the cases. Average tumor diameter was 4.3 cm. Tumors larger than 3.7 cm were heterogeneous for Vimentin immunostaining in 72.5 % of cases. Tumors displaying negative immunostaining for both ZEB1 and Twist were low grade in 100 % of the cases. Conclusions: Intratumor heterogeneity is a common event in clear cell renal cell carcinoma, which can be monitored by immunohistochemistry in routine practice. Snail seems to be particularly useful in the identification of intratumor heterogeneity. The suitability of current sampling protocols in renal cancer is discussed. The authors thank the excellent immunohistochemical work of Mar Gonzalez, Alicia Esteve, Aida Larranaga and Maria Cruz Andreu, Lab Technicians at the Department of Pathology, Cruces University Hospital, Barakaldo, Bizkaia, Spain. This work was partially funded by grant SAF2013-48812-R from Ministerio de Economia y Competitividad (Spain).