Biomarkers Found in the Tumor Interstitial Fluid may Help Explain the Differential Behavior Among Keratinocyte Carcinomas.

Basal cell carcinomas (BCCs) and cutaneous squamous cell carcinomas (SCCs) are the most frequent types of cancer, and both originate from the keratinocyte transformation, giving rise to the group of tumors called keratinocyte carcinomas (KCs). The invasive behavior is different in each group of KC and may be influenced by their tumor microenvironment. The principal aim of the study is to characterize the protein profile of the tumor interstitial fluid (TIF) of KC to evaluate changes in the microenvironment that could be associated with their different invasive and metastatic capabilities. We obtained TIF from 27 skin biopsies and conducted a label-free quantitative proteomic analysis comparing seven BCCs, 16 SCCs, and four normal skins. A total of 2945 proteins were identified, 511 of them quantified in more than half of the samples of each tumoral type. The proteomic analysis revealed differentially expressed TIF proteins that could explain the different metastatic behavior in both KCs. In detail, the SCC samples disclosed an enrichment of proteins related to cytoskeleton, such as Stratafin and Ladinin-1. Previous studies found their upregulation positively correlated with tumor progression. Furthermore, the TIF of SCC samples was enriched with the cytokines S100A8/S100A9. These cytokines influence the metastatic output in other tumors through the activation of NF-kB signaling. According to this, we observed a significant increase in nuclear NF-kB subunit p65 in SCCs but not in BCCs. In addition, the TIF of both tumors was enriched with proteins involved in the immune response, highlighting the relevance of this process in the composition of the tumor environment. Thus, the comparison of the TIF composition of both KCs provides the discovery of a new set of differential biomarkers. Among them, secreted cytokines such as S100A9 may help explain the higher aggressiveness of SCCs, while Cornulin is a specific biomarker for BCCs. Finally, the proteomic landscape of TIF provides key information on tumor growth and metastasis, which can contribute to the identification of clinically applicable biomarkers that may be used in the diagnosis of KC, as well as therapeutic targets.

CRABP2 - A novel biomarker for high-risk endometrial cancer.

OBJECTIVE: Investigate the clinical and functional implications of elevated CRABP2 expression in endometrial cancer (EC) patients. METHODS: Patients were stratified into high and low CRABP2 expression groups using a decision tree classifier. Univariate and multivariate statistical analyses determined the prognostic and clinicopathological consequences of increased CRABP2 expression. A CRABP2 gene signature was generated using differential expression analysis, and analyzed using network-based approaches. The findings were validated in The Clinical Proteomic Tumor Analysis Consortium (CPTAC), a newly generated cohort of 120 endometrial tissues, and The Cancer Dependency Map (DepMap). RESULTS: 60 (11%) patients in TCGA had high CRABP2 expression, whilst 468 (89%) had low expression. High expression was associated with serous EC, reduced overall survival, advanced stage and grade. Downstream retinoic acid receptors (RARG and RARA) were correlated with CRABP2 expression and were associated with worse prognosis in serous EC. The CRABP2 gene signature was enriched for Polycomb target gene sets, and was regulated by ELP3 and BMP7. BMP7 expression was increased in the CRABP2-high group, was associated with worse prognosis, and CRISPR-Cas9 screens revealed correlations in its cell-fitness score with CRABP2 following gene knockout. The opposite was true for ELP3, suggesting opposing effects from both master regulators. CONCLUSIONS: CRABP2 expression is associated with poor prognosis and advanced EC. The expression of RARA and RARG correlates with CRABP2 and are associated with worse prognosis in advanced histological subtypes. Polycomb target gene sets and two master regulators, ELP3 and BMP7, were identified as functionally relevant mechanisms driving aberrant CRABP2 expression.

Diagnosis and management of an endometrial cancer patient with Cowden syndrome.

Somatic PTEN alterations are common in endometrial carcinoma (EC), but in rare cases PTEN mutations are associated with inherited syndromes. Here, we present a case of Cowden syndrome-associated EC. We discuss clinical, pathologic and molecular features of her tumor and PTEN-mutated EC, inherited syndromes predisposing to EC and PTEN-targeted therapies.

Differential Immunoexpression of BRAF/V600E, Senescence Markers, PTEN, and T-type Calcium Channels in Acquired Naevi According to their Histopathological and Dermoscopic Classification.

BRAF/V600E mutation and other cell growth/growth-control mechanisms are involved in naevogenesis and melanomagenesis. Immunoexpression of BRAF/V600E and other molecules (p16, phosphatase and tensin homologue (PTEN), Ki67, hTERT and Cav3.1 and 3.2 calcium channels) were investigated in 80 histopatho-logically and dermoscopically classified acquired naevi. Regarding BRAF/V600E, dysplastic naevi showed lower immunostaining than common naevi, which was significant in comparison with intradermal naevi, which showed the highest BRAF/V600E histoscore. Junctional naevi showed the lowest BRAF/V600E levels. Globular/cobblestone and reticular dermoscopic patterns were consistently associated with high and low BRAF/V600E immunoexpression, respectively, but Zalaudek's peripheral globule pattern (CR/PG) showed the highest BRAF/V600E immunoexpression. Among global patterns, the previously not investigated multicomponent pattern showed the lowest BRAF/V600E immunoexpression. Regarding the remaining biomarkers, new immunohistochemical features were found, in particular p16 and PTEN low expression in multicomponent pattern; and Ki67, hTERT and Cav.3.1 high expression in CR/PG. In conclusion, histopathology and dermoscopy provide complementary information regarding the biology of melanocytic naevi.

Detection of somatic mutations in peritoneal lavages and plasma of endometrial cancer patients: A proof-of-concept study.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Genomic profiling of primary and recurrent adult granulosa cell tumors of the ovary.

Adult-type granulosa cell tumor (aGCT) is a rare malignant ovarian sex cord-stromal tumor, harboring recurrent FOXL2 c.C402G/p.C134W hotspot mutations in 97% of cases. These tumors are considered to have a favorable prognosis, however aGCTs have a tendency for local spread and late recurrences, which are associated with poor survival rates. We sought to determine the genetic alterations associated with aGCT disease progression. We subjected primary non-recurrent aGCTs (n = 7), primary aGCTs that subsequently recurred (n = 9) and their matched recurrences (n = 9), and aGCT recurrences without matched primary tumors (n = 10) to targeted massively parallel sequencing of ≥410 cancer-related genes. In addition, three primary non-recurrent aGCTs and nine aGCT recurrences were subjected to FOXL2 and TERT promoter Sanger sequencing analysis. All aGCTs harbored the FOXL2 C134W hotspot mutation. TERT promoter mutations were found to be significantly more frequent in recurrent (18/28, 64%) than primary aGCTs (5/19, 26%, p = 0.017). In addition, mutations affecting TP53, MED12, and TET2 were restricted to aGCT recurrences. Pathway annotation of altered genes demonstrated that aGCT recurrences displayed an enrichment for genetic alterations affecting cell cycle pathway-related genes. Analysis of paired primary and recurrent aGCTs revealed that TERT promoter mutations were either present in both primary tumors and matched recurrences or were restricted to the recurrence and absent in the respective primary aGCT. Clonal composition analysis of these paired samples further revealed that aGCTs display intra-tumor genetic heterogeneity and harbor multiple clones at diagnosis and relapse. We observed that in a subset of cases, recurrences acquired additional genetic alterations not present in primary aGCTs, including TERT, MED12, and TP53 mutations and CDKN2A/B homozygous deletions. Albeit harboring relatively simple genomes, our data provide evidence to suggest that aGCTs are genetically heterogeneous tumors and that TERT promoter mutations and/or genetic alterations affecting other cell cycle-related genes may be associated with disease progression and recurrences.

Intratumour heterogeneity in endometrial serous carcinoma assessed by targeted sequencing and multiplex ligation-dependent probe amplification: a descriptive study.

AIMS: Endometrial serous carcinoma (ESC) represents the most aggressive subtype of endometrial carcinoma (EC). According to The Cancer Genome Atlas (TCGA), ESC exhibits a genomic profile characterised by frequent TP53 mutations and somatic copy-number alterations (SCNA). Several studies have suggested the role of intratumour heterogeneity (ITH) in tumour progression and therapy resistance, highlighting ITH as a challenge for personalised medicine. ITH is described as the co-existence of clonal and subclonal cellular populations within a single tumour. To date, the extent and prevalence of ITH in ESC have not been fully evaluated. The aim of this study was to address ITH analysis in ESC. We performed a descriptive integrated molecular approach using targeted sequencing and multiplex ligation-dependent probe amplification (MLPA) to identify mutations and SCNA patterns, respectively. METHODS AND RESULTS: Eight ESC were examined, selecting three tumour regions per case and their corresponding normal tissue. For targeted sequencing a gene panel of 40 genes based on TCGA and other survey data was performed. For MLPA different probe mixes were used to detect SCNA in 106 genes. Analysis of mutations and SCNA were performed in each sample and comparative analysis of the three tumour regions was also conducted. Targeted sequencing showed that mutations in TP53, PIK3CA and PPP2R1A were ubiquitous in all tumour regions. Moreover, MLPA results demonstrated a high frequency of SCNA, according to the already known presence of genomic instability in ESC. Unlike the homogeneous distribution of somatic mutations, SCNA exhibited ITH affecting targetable genes such as ERBB2. CONCLUSIONS: Our study suggests that somatic gene copy-number alterations are the main source of ITH in ESC.

Cytoplasmic cyclin D1 regulates glioblastoma dissemination.

Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4-retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor suppressive function of E2F-1 on PTEN-induced serrated colorectal carcinogenesis.

Many human cancers present Phosphatase and tensin homolog (PTEN) deficiency and between 20 and 30% of colorectal tumors show PTEN loss. The transcription factor, E2 promoter binding factor 1 (E2F-1), exhibits tumor promoter or suppressive functions depending on cellular type and tissue context, but its role in the progression and development of colorectal carcinogenesis was largely unknown. Here, using a tamoxifen-inducible PTEN knockout mouse model, we have demonstrated that loss of PTEN leads to the development of colorectal tumorigenesis through the serrated pathway. Next, we studied PTEN loss-driven colorectal lesions in the context of E2F-1 deficiency in vivo. Our results revealed that monoallelic and biallelic absence of E2F-1 led to an increased incidence and progression of serrated tumorigenesis induced by PTEN loss. Finally, we investigated the mechanisms by which double PTEN/E2F-1 deficiency leads to enhanced tumorigenesis. We found that colorectal tumors from PTEN/E2F-1 double knockout mice and the human colorectal carcinoma cell line HT29 with shRNA-mediated downregulation of PTEN and E2F-1 exhibit hyperactivation of the RAS-MAPK pathway, accumulation of DNA damage and resistance to apoptosis. To date, this is the first preclinical study evaluating the effect of genetic deletion of E2F-1 in colorectal malignancies driven by PTEN deficiency. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

EV-associated miRNAs from peritoneal lavage as potential diagnostic biomarkers in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide. Current systematic methods for diagnosing have inherent limitations so development of a minimally-invasive diagnosis, based on the identification of sensitive biomarkers in liquid biopsies could therefore facilitate screening among population at risk. METHODS: In this study, we aim to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the peritoneal lavage as a source of potential miRNA diagnostic biomarkers. We isolated EVs by ultracentrifugation from 25 ascitic fluids and 25 peritoneal lavages from non-cancer and CRC patients, respectively. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 371 miRNAs. RESULTS: 210 miRNAs were significantly dysregulated (adjusted p value < 0.05 and abs(logFC) ≥ 1). The top-10 miRNAs, which had the AUC value higher than 0.95, were miRNA-199b-5p, miRNA-150-5p, miRNA-29c-5p, miRNA-218-5p, miRNA-99a-3p, miRNA-383-5p, miRNA-199a-3p, miRNA-193a-5p, miRNA-10b-5p and miRNA-181c-5p. CONCLUSIONS: This finding opens the avenue to the use of EV-associated miRNA of peritoneal lavages as an untapped source of biomarkers for CRC.

Epithelial cell adhesion molecule (EpCAM) from pleural fluid cell lysates is a highly accurate diagnostic biomarker of adenocarcinomatous effusions.

BACKGROUND AND OBJECTIVE: The discovery of highly accurate pleural fluid (PF) biomarkers of malignancy remains elusive. We assessed the operating characteristics of the PF epithelial cell adhesion molecule (EpCAM), claudin 4 (CL4) and human epididymis protein 4 (HE4) as potential markers of epithelial malignancies. METHODS: The three markers were quantified by immunoassays in the supernatants (s) and cell lysates (cl) of 175 PF samples. The cut-off values with 100% specificity were selected for malignant-benign discrimination. An immunocytochemical staining index score for each marker was also evaluated on PF cell blocks. The resulting best biomarker was further validated in two independent populations of 73 and 48 patients with pleural effusions (PE). RESULTS: An EpCAM(cl) >98 pg/g total lysate protein yielded 75% sensitivity, 100% specificity, negative likelihood ratio of 0.25 and area under the curve of 0.94 for labelling adenocarcinomatous effusions. Sensitivity reached 88% if EpCAM(cl) was combined with EpCAM immunostaining. One-third or more of the malignant effusions exhibiting a false-negative cytological fluid examination were correctly classified by EpCAM(cl) concentrations. Immunoassays for CL4 and HE4 were diagnostically useless. CONCLUSION: EpCAM(cl) is a new biomarker of adenocarcinomatous PE with meaningful discriminating properties.

Deletion of Pten in CD45-expressing cells leads to development of T-cell lymphoblastic lymphoma but not myeloid malignancies.

Since its discovery in the late 1990s, Pten has turned out to be one of the most important tumor suppressor genes. Pten loss results in increased activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, which is associated with increased proliferation, survival, and neoplastic growth. Here, we have addressed the effects of conditional deletion of Pten in hematopoietic cells by crossing Pten conditional knockout mice with a knock-in mouse expressing the Cre recombinase in the CD45 locus. CD45 is also known as leukocyte common antigen, and it is expressed in virtually all white cells and in hematopoietic stem cells. Using a reporter mouse, we demonstrate that CD45:Cre mouse displays recombinase activity in both myeloid and lymphoid cells. However, deletion of Pten in CD45-expressing cells induces development of T-cell acute lymphoblastic leukemia and lymphoma, but not other hematologic malignancies.

Tumor Heterogeneity in Endometrial Carcinoma: Practical Consequences.

Endometrial carcinoma (EC) shows intertumor heterogeneity, with several different histologic types differing in their morphologic and molecular features. There is also intratumoral morphologic heterogeneity, with different neoplastic cell components within the same tumor, with different morphologic and molecular features. In this article, we discuss the consequences of tumor heterogeneity in EC at the morphologic and molecular levels, by describing some illustrative examples produced by the research team. They are (1) morphologic heterogeneity in conventional EC and mixed tumors, (2) EC with microsatellite instability, (3) branched evolution as shown by exome sequencing analysis, (4) morphologic, molecular, and metabolomic differences between the tumor surface and myometrial invasion front, (5) tumor heterogeneity at the microenviromental level, (6) the sensitivity of endometrial aspirates to detect tumor heterogeneity in EC, and (7) sampling strategies to detect tumor heterogeneity in hysterectomy specimens. Tumor heterogeneity may have an important clinical impact, since it can be challenging to identify minor tumor cell populations that may have an impact on diagnosis, prognosis, and therapeutic decisions for patients with EC.

Palbociclib has antitumour effects on Pten-deficient endometrial neoplasias.

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1-CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD-332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen-inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten-deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D-CDK4/6 activity in their development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oral intake of genetically engineered high-carotenoid corn ameliorates hepatomegaly and hepatic steatosis in PTEN haploinsufficient mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Here we show that a mouse model of haploinsufficiency in the lipid and protein phosphatase and tensin homolog protein (PTEN(+/-)) exhibits hepatomegaly, increased liver lipogenic gene expression (SREBP-1C and PPARγ) and hepatic lesions analogous to human NAFLD. The livers of PTEN(+/-) mice also contained lower levels of retinoic acid (RA) than normal, similarly to human NAFLD patients. The RA signaling pathway thus offers a novel therapeutic target for the treatment of NAFLD although the impact of nutrition in this context is unclear. We therefore fed PTEN(+/-) mice for 36weeks a diet containing genetically engineered high-carotenoid corn (HCAR) to investigate its potential beneficial effects on the hepatic symptoms of NAFLD. The HCAR diet reduced hepatomegaly and promoted the repartitioning of fatty acids in the liver, away from triacylglycerol storage. At the molecular level, the HCAR diet clearly reduced lipogenic gene expression, boosted catabolism, and increased hepatic RA levels. These results set the stage for human trials to evaluate the use of high-carotenoid foods for the reduction or prevention of steatosis in NAFLD.

Chromatin remodelling and DNA repair genes are frequently mutated in endometrioid endometrial carcinoma.

In developed countries, endometrial carcinoma is the most common cancer that affects the female genital tract. Endometrial carcinoma is divided into two main histological types, type I or endometrioid and type II or non-endometrioid, each of which have characteristic, although not exclusive, molecular alterations and mutational profiles. Nevertheless, information about the implication and relevance of some of these genes in this disease is lacking. We sought here to identify new recurrently mutated genes in endometrioid cancers that play a role in tumourigenesis and that influence the clinical outcome. We focused on low-grade, non-ultramutated tumours as these tumours have a worse prognosis than the ultramutated POLE-positive endometrioid endometrial carcinomas (EECs). We performed exome-sequencing of 11 EECs with matched normal tissue and subsequently validated 15 candidate genes in 76 samples. For the first time, we show that mutations in chromatin remodelling-related genes (KMT2D, KMT2C, SETD1B and BCOR) and in DNA-repair-related genes (BRCA1, BRCA2, RAD50 and CHD4) are frequent in this subtype of endometrial cancer. The alterations to these genes occurred with frequencies ranging from 35.5% for KMT2D to 10.5% for BRCA1 and BCOR, with some showing a tendency toward co-occurrence (RAD50-KMT2D and RAD50-SETD1B). All these genes harboured specific mutational hotspots. In addition, the mutational status of KMT2C, KMT2D and SETD1B helps to predict the degree of myometrial invasion, a critical prognostic feature. These results highlight the possible implication of these genes in this disease, creating opportunities for new therapeutic approaches.

Genetic analysis of uterine aspirates improves the diagnostic value and captures the intra-tumor heterogeneity of endometrial cancers.

Endometrial cancer is the most common cancer of the female genital tract in developed countries. Although the majority of endometrial cancers are diagnosed at early stages and the 5-year overall survival is around 80%, early detection of these tumors is crucial to improve the survival of patients given that the advanced tumors are associated with a poor outcome. Furthermore, correct assessment of the pre-clinical diagnosis is decisive to guide the surgical treatment and management of the patient. In this sense, the potential of targeted genetic sequencing of uterine aspirates has been assessed as a pre-operative tool to obtain reliable information regarding the mutational profile of a given tumor, even in samples that are not histologically classifiable. A total of 83 paired samples were sequenced (uterine aspirates and hysterectomy specimens), including 62 endometrioid and non-endometrioid tumors, 10 cases of atypical hyperplasia and 11 non-cancerous endometrial disorders. Even though diagnosing endometrial cancer based exclusively on genetic alterations is currently unfeasible, mutations were mainly found in uterine aspirates from malignant disorders, suggesting its potential in the near future for supporting the standard histologic diagnosis. Moreover, this approach provides the first evidence of the high intra-tumor genetic heterogeneity associated with endometrial cancer, evident when multiple regions of tumors are analyzed from an individual hysterectomy. Notably, the genetic analysis of uterine aspirates captures this heterogeneity, solving the potential problem of incomplete genetic characterization when a single tumor biopsy is analyzed.

Molecular approaches for classifying endometrial carcinoma.

Endometrial carcinoma is the most common cancer of the female genital tract. This review article discusses the usefulness of molecular techniques to classify endometrial carcinoma. Any proposal for molecular classification of neoplasms should integrate morphological features of the tumors. For that reason, we start with the current histological classification of endometrial carcinoma, by discussing the correlation between genotype and phenotype, and the most significant recent improvements. Then, we comment on some of the possible flaws of this classification, by discussing also the value of molecular pathology in improving them, including interobserver variation in pathologic interpretation of high grade tumors. Third, we discuss the importance of applying TCGA molecular approach to clinical practice. We also comment on the impact of intratumor heterogeneity in classification, and finally, we will discuss briefly, the usefulness of TCGA classification in tailoring immunotherapy in endometrial cancer patients. We suggest combining pathologic classification and the surrogate TCGA molecular classification for high-grade endometrial carcinomas, as an option to improve assessment of prognosis.

Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis.

BACKGROUND & AIMS: The pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD) is still incompletely understood. Several nuclear receptors play a role in liver lipid metabolism and can promote hepatosteatosis, but the possible role of vitamin D receptor (VDR) in NAFLD has not been investigated. METHODS: The expression of liver VDR was investigated in apolipoprotein E knockout (apoE(-/-)) mice on a high fat diet, in wild-type mice on methionine and choline deficient diet and in NAFLD patients with hepatosteatosis and non-alcoholic steatohepatitis. The relevance of VDR was assessed in apoE(-/-) mice by deletion of VDR or paricalcitol treatment and in human HepG2 cells by VDR transfection or silencing. The role of VDR in fibrosis was also determined in VDR knockout mice (VDR(-/-)) treated with thioacetamide. RESULTS: Expression of liver VDR was markedly induced in two mouse models of NAFLD, as well as in patients with hepatosteatosis, but decreased in non-alcoholic steatohepatitis. VDR deletion in high fat diet-fed apoE(-/-) mice protected against fatty liver, dyslipidemia and insulin resistance, and caused a decrease in taurine-conjugated bile acids, but did not influence fibrosis by thioacetamide. apoE(-/-)VDR(-/-) mouse livers showed decreased gene expression of CD36, DGAT2, C/EBPα and FGF21, and increased expression of PNPLA2, LIPIN1 and PGC1α. Treatment of apoE(-/-) mice on high fat diet with paricalcitol had modest opposite effects on steatosis and gene expression. Finally, this set of genes showed concordant responses when VDR was overexpressed or silenced in HepG2 cells. CONCLUSIONS: Induced hepatocyte VDR in NAFLD regulates key hepatic lipid metabolism genes and promotes high fat diet-associated liver steatosis. Therapeutic inhibition of liver VDR may reverse steatosis in early NAFLD. LAY SUMMARY: The amount of vitamin D receptor is induced early in the livers of mice and humans when they develop non-alcoholic fatty liver disease. If the gene for the vitamin D receptor is deleted, hepatic lipid metabolism changes and mice do not accumulate fat in the liver. We conclude that the vitamin D receptor can contribute to the fatty liver disease promoted by a high fat diet.

Practical issues in the diagnosis of serous carcinoma of the endometrium.

Serous carcinoma (SC) represents ~10% of endometrial carcinomas, but is responsible for almost 40% of cancer deaths. This article reviews the main pathological features, differential diagnosis, and the usefulness of molecular pathology and immunohistochemistry in its diagnosis. Most helpful features for the diagnosis include: irregularly shaped and sized papillae, slit-like spaces, cell stratification and budding, highly atypical cells, architectural and cytological discordance in pseudoglandular tumors, as well as lack of endometrioid features. SC shows typically a predominant papillary growth, which is also found in some subtypes of endometrioid carcinoma of the endometrium (EEC). Distinction is easy when attention is paid to the presence of diffuse marked nuclear pleomorphism, but also to the complex papillary architecture. SC may also show a solid or pseudoglandular patterns, and in these cases differential diagnosis may be difficult with EEC grade 3. Moreover, a high proportion of SC may exhibit clear cells, and, thus, may be confused with clear cell carcinoma. Finally, it is sometimes difficult to distinguish mixed SC-EEC, from SC that combines papillary and pseudoglandular growths. Although there is not a single immunohistochemical marker for distinguishing SC from its mimickers, some antibodies are useful (p53, p16, IMP2, and IMP3), particularly when used in combination. Diagnosis of SC may be even more problematic in small biopsies; a diagnosis of high-grade endometrial carcinoma, SC component can not be excluded, is acceptable as a managerial approach, so it could be taken into account at the time of final surgery.