First Transcriptome Analysis of Hepatoblastoma in Brazil: Unraveling the Pivotal Role of Noncoding RNAs and Metabolic Pathways.

Hepatoblastoma stands as the most prevalent liver cancer in the pediatric population. Characterized by a low mutational burden, chromosomal and epigenetic alterations are key drivers of its tumorigenesis. Transcriptome analysis is a powerful tool for unraveling the molecular intricacies of hepatoblastoma, shedding light on the effects of genetic and epigenetic changes on gene expression. In this study conducted in Brazilian patients, an in-depth whole transcriptome analysis was performed on 14 primary hepatoblastomas, compared to control liver tissues. The analysis unveiled 1,492 differentially expressed genes (1,031 upregulated and 461 downregulated), including 920 protein-coding genes (62%). Upregulated biological processes were linked to cell differentiation, signaling, morphogenesis, and development, involving known hepatoblastoma-associated genes (DLK1, MEG3, HDAC2, TET1, HMGA2, DKK1, DKK4), alongside with novel findings (GYNG4, CDH3, and TNFRSF19). Downregulated processes predominantly centered around oxidation and metabolism, affecting amines, nicotinamides, and lipids, featuring novel discoveries like the repression of SYT7, TTC36, THRSP, CCND1, GCK and CAMK2B. Two genes, which displayed a concordant pattern of DNA methylation alteration in their promoter regions and dysregulation in the transcriptome, were further validated by RT-qPCR: the upregulated TNFRSF19, a key gene in the embryonic development, and the repressed THRSP, connected to lipid metabolism. Furthermore, based on protein-protein interaction analysis, we identified genes holding central positions in the network, such as HDAC2, CCND1, GCK, and CAMK2B, among others, that emerged as prime candidates warranting functional validation in future studies. Notably, a significant dysregulation of non-coding RNAs (ncRNAs), predominantly upregulated transcripts, was observed, with 42% of the top 50 highly expressed genes being ncRNAs. An integrative miRNA-mRNA analysis revealed crucial biological processes associated with metabolism, oxidation reactions of lipids and carbohydrates, and methylation-dependent chromatin silencing. In particular, four upregulated miRNAs (miR-186, miR-214, miR-377, and miR-494) played a pivotal role in the network, potentially targeting multiple protein-coding transcripts, including CCND1 and CAMK2B. In summary, our transcriptome analysis highlighted disrupted embryonic development as well as metabolic pathways, particularly those involving lipids, emphasizing the emerging role of ncRNAs as epigenetic regulators in hepatoblastomas. These findings provide insights into the complexity of the hepatoblastoma transcriptome and identify potential targets for future therapeutic interventions.

Evidence of the Role of Inflammation and the Hormonal Environment in the Pathogenesis of Adrenal Myelolipomas in Congenital Adrenal Hyperplasia.

Adrenal myelolipomas (AML) are composed of mature adipose and hematopoietic components. They represent approximately 3 percent of adrenal tumors and are commonly found in patients with congenital adrenal hyperplasia (CAH). CAH provides a unique environment to explore AML pathogenesis. We aimed to evaluate the role of the immune system and hormones that accumulate in poorly controlled CAH in the development of AML. When compared to normal adrenal tissue, CAH-affected adrenal tissue and myelolipomas showed an increased expression of inflammatory cells (CD68, IL2Rbeta), stem cells (CD117) B cells (IRF4), and adipogenic markers (aP2/FABP4, AdipoQ, PPARγ, Leptin, CideA), and immunostaining showed nodular lymphocytic accumulation. Immunohistochemistry staining revealed a higher density of inflammatory cells (CD20, CD3, CD68) in CAH compared to non-CAH myelolipomas. In vitro RNA-sequencing studies using NCI-H295R adrenocortical cells with exogenous exposure to ACTH, testosterone, and 17-hydroxyprogesterone hormones, showed the differential expression of genes involved in cell cycle progression, phosphorylation, and tumorigenesis. Migration of B-lymphocytes was initiated after the hormonal treatment of adrenocortical cells using the Boyden chamber chemotaxis assay, indicating a possible hormonal influence on triggering inflammation and the development of myelolipomas. These findings demonstrate the important role of inflammation and the hormonal milieu in the development of AML in CAH.

Expanding the clinicopathological spectrum of succinate dehydrogenase-deficient renal cell carcinoma with a focus on variant morphologies: a study of 62 new tumors in 59 patients.

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.

Neuromuscular Choristoma: Report of Five Cases With CTNNB1 Sequencing.

Neuromuscular choristoma (NMC) are lesions of the peripheral nervous system characterized by an admixture of skeletal muscle fibers and nerves fascicles that are frequently associated with desmoid fibromatosis (DF). Mutations in CTNNB1, the gene for ß-catenin protein, are common in DF and related to its pathogenesis. They are restricted to exon 3, with 3 point mutations: T41A, S45F, and S45P. To understand the pathogenesis of NMC, we tested CTNNB1 status in 5 cases of NMC whether or not they were associated with DF. The screening of mutations in CTNNB1 gene was based on amplicon deep sequencing using the ION Proton platform. Three patients had the S45F mutation; in 2 the mutation was common to both lesions and in one the DF was wild type while the NMC had the S45F mutation. One patient had a T41A mutation in the NMC and no associated DF. In the last patient, the DF lesion had a T41A mutation; there was no lesion with the S45P mutation. The presence of similar CTNNB1 mutations in NMC/DF-associated lesions and sporadic DF reinforces the relationship between both lesions and points to a common pathogenic mechanism.

Morphologic and Molecular Characterization of Adrenals and Adrenal Rest Affected by Congenital Adrenal Hyperplasia.

Introduction: Adrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues. Patients/Methods: CAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses. Results: Based on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue. Conclusions: CAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

Prognostic Impact of Loss of SETD2 in Clear Cell Renal Cell Carcinoma.

PURPOSE: To evaluate the prognostic impact of immunohistochemical expression of SETD2 in patients with clear cell renal cell carcinoma (ccRCC). PATIENTS AND METHODS: A total of 662 patients with primary or metastatic ccRCC were evaluated. Two genitourinary pathologist reviewed all of the cases for uniform reclassification and determined the selection of the most representative tumor areas for construction of the tissue microarray (TMA). RESULTS: SETD2 nuclear staining showed that 101 areas (15.3%) had negative expression, and 561 areas (84,7%) had positive expression of SETD2. The protein expression of SETD2 was associated with clinical stage (P < .001), pathological stage (P < .001), tumor size (P < .001), perinephric fat invasion (P < .001), Eastern Cooperative Oncology Group status (P = .004), surgery type (P < .001), International Society of Urologic Pathologists grade (P < .001), and tumor necrosis (P < .001). SETD2 influenced disease-specific survival (DSS) and overall survival (OS). DSS rates in patients with positive and negative expression of SETD2 were 90.2% and 58.4%, respectively (P < .001). OS rates in patients with positive and negative expression of SETD2 were 87% and 55.4%, respectively (P < .001). In a multivariate Cox analysis, low SETD2 expression was an independent predictor of DSS (hazard ratio [HR], 1.690; 95% confidence interval [CI], 1.0582.700; P = .031) and OS (HR, 1.641; 95% CI, 1.039-2.593; P = .037). CONCLUSION: Our study showed that the negative expression of SETD2 was associated with a worse prognosis, and it was an independent predictor of survival in patients with ccRCC. We believe that the protein expression of SETD2 is an important biomarker in the management of patients with ccRCC.

New developments in existing WHO entities and evolving molecular concepts: The Genitourinary Pathology Society (GUPS) update on renal neoplasia.

The Genitourinary Pathology Society (GUPS) reviewed recent advances in renal neoplasia, particularly post-2016 World Health Organization (WHO) classification, to provide an update on existing entities, including diagnostic criteria, molecular correlates, and updated nomenclature. Key prognostic features for clear cell renal cell carcinoma (RCC) remain WHO/ISUP grade, AJCC/pTNM stage, coagulative necrosis, and rhabdoid and sarcomatoid differentiation. Accrual of subclonal genetic alterations in clear cell RCC including SETD2, PBRM1, BAP1, loss of chromosome 14q and 9p are associated with variable prognosis, patterns of metastasis, and vulnerability to therapies. Recent National Comprehensive Cancer Network (NCCN) guidelines increasingly adopt immunotherapeutic agents in advanced RCC, including RCC with rhabdoid and sarcomatoid changes. Papillary RCC subtyping is no longer recommended, as WHO/ISUP grade and tumor architecture better predict outcome. New papillary RCC variants/patterns include biphasic, solid, Warthin-like, and papillary renal neoplasm with reverse polarity. For tumors with 'borderline' features between oncocytoma and chromophobe RCC, a term "oncocytic renal neoplasm of low malignant potential, not further classified" is proposed. Clear cell papillary RCC may warrant reclassification as a tumor of low malignant potential. Tubulocystic RCC should only be diagnosed when morphologically pure. MiTF family translocation RCCs exhibit varied morphologic patterns and fusion partners. TFEB-amplified RCC occurs in older patients and is associated with more aggressive behavior. Acquired cystic disease (ACD) RCC-like cysts are likely precursors of ACD-RCC. The diagnosis of renal medullary carcinoma requires a negative SMARCB1 (INI-1) expression and sickle cell trait/disease. Mucinous tubular and spindle cell carcinoma (MTSCC) can be distinguished from papillary RCC with overlapping morphology by losses of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22. MTSCC with adverse histologic features shows frequent CDKN2A/2B (9p) deletions. BRAF mutations unify the metanephric family of tumors. The term "fumarate hydratase deficient RCC" ("FH-deficient RCC") is preferred over "hereditary leiomyomatosis and RCC syndrome-associated RCC". A low threshold for FH, 2SC, and SDHB immunohistochemistry is recommended in difficult to classify RCCs, particularly those with eosinophilic morphology, occurring in younger patients. Current evidence does not support existence of a unique tumor subtype occurring after chemotherapy/radiation in early childhood.

Novel, emerging and provisional renal entities: The Genitourinary Pathology Society (GUPS) update on renal neoplasia.

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in renal neoplasia, particularly focusing on the newly accumulated evidence post-2016 World Health Organization (WHO) classification. In the era of evolving histo-molecular classification of renal neoplasia, morphology is still key. However, entities (or groups of entities) are increasingly characterized by specific molecular features, often associated either with recognizable, specific morphologies or constellations of morphologies and corresponding immunohistochemical profiles. The correct diagnosis has clinical implications leading to better prognosis, potential clinical management with targeted therapies, may identify hereditary or syndromic associations, which may necessitate appropriate genetic testing. We hope that this undertaking will further facilitate the identification of these entities in practice. We also hope that this update will bring more clarity regarding the evolving classification of renal neoplasia and will further reduce the category of "unclassifiable renal carcinomas/tumors". We propose three categories of novel entities: (1) "Novel entity", validated by multiple independent studies; (2) "Emerging entity", good compelling data available from at least two or more independent studies, but additional validation is needed; and (3) "Provisional entity", limited data available from one or two studies, with more work required to validate them. For some entities initially described using different names, we propose new terminologies, to facilitate their recognition and to avoid further diagnostic dilemmas. Following these criteria, we propose as novel entities: eosinophilic solid and cystic renal cell carcinoma (ESC RCC), renal cell carcinoma with fibromyomatous stroma (RCC FMS) (formerly RCC with leiomyomatous or smooth muscle stroma), and anaplastic lymphoma kinase rearrangement-associated renal cell carcinoma (ALK-RCC). Emerging entities include: eosinophilic vacuolated tumor (EVT) and thyroid-like follicular renal cell carcinoma (TLFRCC). Finally, as provisional entities, we propose low-grade oncocytic tumor (LOT), atrophic kidney-like lesion (AKLL), and biphasic hyalinizing psammomatous renal cell carcinoma (BHP RCC).

Practice patterns related to prostate cancer grading: results of a 2019 Genitourinary Pathology Society clinician survey.

PURPOSE: To survey urologic clinicians regarding interpretation of and practice patterns in relation to emerging aspects of prostate cancer grading, including quantification of high-grade disease, cribriform/intraductal carcinoma, and impact of magnetic resonance imaging-targeted needle biopsy. MATERIALS AND METHODS: The Genitourinary Pathology Society distributed a survey to urology and urologic oncology-focused societies and hospital departments. Eight hundred and thirty four responses were collected and analyzed using descriptive statistics. RESULTS: Eighty percent of survey participants use quantity of Gleason pattern 4 on needle biopsy for clinical decisions, less frequently with higher Grade Groups. Fifty percent interpret "tertiary" grade as a minor/<5% component. Seventy percent of respondents would prefer per core grading as well as a global/overall score per set of biopsies, but 70% would consider highest Gleason score in any single core as the grade for management. Seventy five percent utilize Grade Group terminology in patient discussions. For 45%, cribriform pattern would affect management, while for 70% the presence of intraductal carcinoma would preclude active surveillance. CONCLUSION: This survey of practice patterns in relationship to prostate cancer grading highlights similarities and differences between contemporary pathology reporting and its clinical application. As utilization of Gleason pattern 4 quantification, minor tertiary pattern, cribriform/intraductal carcinoma, and the incorporation of magnetic resonance imaging-based strategies evolve, these findings may serve as a basis for more nuanced communication and guide research efforts involving pathologists and clinicians.

The 2019 Genitourinary Pathology Society (GUPS) White Paper on Contemporary Grading of Prostate Cancer.

CONTEXT.­: Controversies and uncertainty persist in prostate cancer grading. OBJECTIVE.­: To update grading recommendations. DATA SOURCES.­: Critical review of the literature along with pathology and clinician surveys. CONCLUSIONS.­: Percent Gleason pattern 4 (%GP4) is as follows: (1) report %GP4 in needle biopsy with Grade Groups (GrGp) 2 and 3, and in needle biopsy on other parts (jars) of lower grade in cases with at least 1 part showing Gleason score (GS) 4 + 4 = 8; and (2) report %GP4: less than 5% or less than 10% and 10% increments thereafter. Tertiary grade patterns are as follows: (1) replace "tertiary grade pattern" in radical prostatectomy (RP) with "minor tertiary pattern 5 (TP5)," and only use in RP with GrGp 2 or 3 with less than 5% Gleason pattern 5; and (2) minor TP5 is noted along with the GS, with the GrGp based on the GS. Global score and magnetic resonance imaging (MRI)-targeted biopsies are as follows: (1) when multiple undesignated cores are taken from a single MRI-targeted lesion, an overall grade for that lesion is given as if all the involved cores were one long core; and (2) if providing a global score, when different scores are found in the standard and the MRI-targeted biopsy, give a single global score (factoring both the systematic standard and the MRI-targeted positive cores). Grade Groups are as follows: (1) Grade Groups (GrGp) is the terminology adopted by major world organizations; and (2) retain GS 3 + 5 = 8 in GrGp 4. Cribriform carcinoma is as follows: (1) report the presence or absence of cribriform glands in biopsy and RP with Gleason pattern 4 carcinoma. Intraductal carcinoma (IDC-P) is as follows: (1) report IDC-P in biopsy and RP; (2) use criteria based on dense cribriform glands (>50% of the gland is composed of epithelium relative to luminal spaces) and/or solid nests and/or marked pleomorphism/necrosis; (3) it is not necessary to perform basal cell immunostains on biopsy and RP to identify IDC-P if the results would not change the overall (highest) GS/GrGp part per case; (4) do not include IDC-P in determining the final GS/GrGp on biopsy and/or RP; and (5) "atypical intraductal proliferation (AIP)" is preferred for an intraductal proliferation of prostatic secretory cells which shows a greater degree of architectural complexity and/or cytological atypia than typical high-grade prostatic intraepithelial neoplasia, yet falling short of the strict diagnostic threshold for IDC-P. Molecular testing is as follows: (1) Ki67 is not ready for routine clinical use; (2) additional studies of active surveillance cohorts are needed to establish the utility of PTEN in this setting; and (3) dedicated studies of RNA-based assays in active surveillance populations are needed to substantiate the utility of these expensive tests in this setting. Artificial intelligence and novel grading schema are as follows: (1) incorporating reactive stromal grade, percent GP4, minor tertiary GP5, and cribriform/intraductal carcinoma are not ready for adoption in current practice.

Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

Mutational Portrait of Lung Adenocarcinoma in Brazilian Patients: Past, Present, and Future of Molecular Profiling in the Clinic.

Objectives: Approximately 60% of lung adenocarcinomas (LAs) carry mutations that can guide treatment with tyrosine-kinase inhibitors (TKI) and other targeted therapies. Data on activating mutations in EGFR and other tyrosine-kinase receptor (TKR) genes in highly admixed populations, such as that of Brazil, are scarce. In this study, we comprehensively analyzed the actionable alteration profile of LA in Brazilian patients. Materials and Methods: EGFR driver mutation data were collected from a large Brazilian LA cohort covering an 8-year period of molecular testing in a single institution. Tests were performed using three distinct methods, and demographic and histopathological data were analyzed. For a subset of patients, driver mutations in KRAS, NRAS, and BRAF and gene fusions involving TKR genes (before TKI treatment) and EGFR T790M (after TKI treatment) were assessed. Results: EGFR mutations were detected in 25% of 1,316 LAs evaluated, with exon 19 deletions and exon 21 L858R TKI sensitizing mutations representing 72.5% of all mutations. Mutation rates were higher in women and non-smokers (p < 0.001). Next-generation sequencing was very sensitive, with a lower rate of inconclusive results compared with Sanger sequencing and pyrosequencing. EGFR/RAS/BRAF hotspot gene panels were applied in 495 LA cases and detected oncogenic mutations in 51.3% of samples, most frequently in EGFR (22.4%) and KRAS (26.9%). In subgroups of 36 and 35 patients, gene fusions were detected in 11.1% of tumors and EGFR T790M resistance mutations were detected in 59% of plasma samples from patients previously treated with TKI, respectively. Conclusion: This report provides the first comprehensive actionable alteration portrait of LA in Brazil. The high rate of actionable alterations in EGFR and other driver genes in LA reinforces the need to incorporate TKI guided by molecular diagnostics into clinical routines for patients in both public and private healthcare systems.

Loss of 5'-Methylthioadenosine Phosphorylase (MTAP) is Frequent in High-Grade Gliomas; Nevertheless, it is Not Associated with Higher Tumor Aggressiveness.

The 5'-methylthioadenosine phosphorylase (MTAP) gene is located in the chromosomal region 9p21. MTAP deletion is a frequent event in a wide variety of human cancers; however, its biological role in tumorigenesis remains unclear. The purpose of this study was to characterize the MTAP expression profile in a series of gliomas and to associate it with patients' clinicopathological features. Moreover, we sought to evaluate, through glioma gene-edited cell lines, the biological impact of MTAP in gliomas. MTAP expression was evaluated in 507 glioma patients by immunohistochemistry (IHC), and the expression levels were associated with patients' clinicopathological features. Furthermore, an in silico study was undertaken using genomic databases totalizing 350 samples. In glioma cell lines, MTAP was edited, and following MTAP overexpression and knockout (KO), a transcriptome analysis was performed by NanoString Pan-Cancer Pathways panel. Moreover, MTAP's role in glioma cell proliferation, migration, and invasion was evaluated. Homozygous deletion of 9p21 locus was associated with a reduction of MTAP mRNA expression in the TCGA (The Cancer Genome Atlas) - glioblastoma dataset (p < 0.01). In addition, the loss of MTAP expression was markedly high in high-grade gliomas (46.6% of cases) determined by IHC and Western blotting (40% of evaluated cell lines). Reduced MTAP expression was associated with a better prognostic in the adult glioblastoma dataset (p < 0.001). Nine genes associated with five pathways were differentially expressed in MTAP-knockout (KO) cells, with six upregulated and three downregulated in MTAP. Analysis of cell proliferation, migration, and invasion did not show any significant differences between MTAP gene-edited and control cells. Our results integrating data from patients as well as in silico and in vitro models provide evidence towards the lack of strong biological importance of MTAP in gliomas. Despite the frequent loss of MTAP, it seems not to have a clinical impact in survival and does not act as a canonic tumor suppressor gene in gliomas.

Immunohistochemical negative expression of ezrin predicts poor prognosis in clear cell renal cell carcinoma.

PURPOSE: To analyze the immunohistochemical expression of ezrin and moesin in clear cell renal cell carcinoma (ccRCC). These proteins, as part of the ezrin-radixin-moesin complex link the cell membrane to the actin cytoskeleton, affecting such processes as cell adhesion, cell survival, cell motility, and signal transduction. Our aim was to examine the impact of their expression on clinical outcomes and survival rates. PATIENTS AND METHODS: Five hundred seventy-five consecutive patients who had been treated surgically for ccRCC in a single center between 1985 and 2016 were selected. A single pathologist reviewed all cases to perform a uniform reclassification and determined the most representative tumor areas for construction of a tissue microarray. RESULTS: Of all ccRCC specimens, 106 (18.3%) were negative for ezrin, and 469 (81.7%) had positive ezrin expression; 16 (2.8%) were negative and 559 (97.2%) were positive for moesin, respectively. Ezrin expression was associated with pT stage (P < 0.001), clinical stage (P = 0.012), synchronic metastasis (P < 0.001), incidental tumors (P = 0.007), and International Society of Urological Pathology histological grade (P = 0.025). There was a correlation between moesin expression and clinical stage (P = 0.027), pT stage (P = 0.025), and pN stage (P = 0.007). Ezrin expression significantly influenced tumor-related deaths. By multivariate analysis, negative ezrin expression was an independent risk factor for disease-specific survival (HR 1.89; 95% CI 1.11-3.20). CONCLUSIONS: Negativity for ezrin in ccRCC patients significantly impacts survival rates. We encourage further prospective studies to analyze ezrin analysis to evaluate its significance in the prognosis of ccRCC.

Immunohistochemical expression of renin is a prognostic factor for recurrence in nonmetastatic renal cell carcinoma.

PURPOSE: To analyze the intratumoral immunohistochemical expression of renin and its value as a prognostic factor for recurrence in nonmetastatic clear cell renal cell carcinoma (ccRCC). METHODS: A total of 498 patients with nonmetastatic ccRCC from the Latin American Renal Cancer Group database who underwent partial or radical nephrectomy between 1990 and 2016 were selected. All cases were revised, and 2 distinct samples were obtained for tissue microarray construction. Ten years of follow-up was assessed, and disease-free survival rates (DFS) were analyzed. Renin expression was classified qualitatively as negative or positive. For the quantitative analysis, a cutoff was estimated using the maximum of the standardized log-rank statistic. RESULTS: Nuclear renin was qualitatively positive in 360 cases (72%) and negative in 138 (28%), whereas quantitatively, an equal number of cases had ≤35% or >35% renin-positive nuclei. The absence of renin expression was associated with high-grade tumors (by ISUP and Fuhrman classification, both P < 0.001), greater microscopic venous invasion (P = 0.046), and renal vein invasion (P = 0.026). In the multivariate analyses, qualitatively negative renin expression was an unfavorable prognostic factor for DFS (RR = 2.923, P < 0.001). With regard to quantitative renin expression, a cutoff of ≤35 was associated with worse DFS (RR = 4.085, P < 0.001). CONCLUSIONS: The intratumoral immunohistochemical expression of renin in patients with ccRCC provides valuable prognostic data regarding the likelihood of recurrence.

TET Upregulation Leads to 5-Hydroxymethylation Enrichment in Hepatoblastoma.

Hepatoblastoma is an embryonal liver tumor carrying few genetic alterations. We previously disclosed in hepatoblastomas a genome-wide methylation dysfunction, characterized by hypermethylation at specific CpG islands, in addition to a low-level hypomethylation pattern in non-repetitive intergenic sequences, in comparison to non-tumoral liver tissues, shedding light into a crucial role for epigenetic dysregulation in this type of cancer. To explore the underlying mechanisms possibly related to aberrant epigenetic modifications, we evaluated the expression profile of a set of genes engaged in the epigenetic machinery related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L, UHRF1, TET1, TET2, and TET3), as well as the 5-hydroxymethylcytosine (5hmC) global level. We observed in hepatoblastomas a general disrupted expression of these genes from the epigenetic machinery, mainly UHRF1, TET1, and TET2 upregulation, in association with an enrichment of 5hmC content. Our findings support a model of active demethylation by TETs in hepatoblastoma, probably during early stages of liver development, which in combination with UHRF1 overexpression would lead to DNA hypomethylation and an increase in overall 5hmC content. Furthermore, our data suggest that decreased 5hmC content might be associated with poor survival rate, highlighting a pivotal role of epigenetics in hepatoblastoma development and progression.

External validation of nomogram to predict inguinal lymph node metastasis in patients with penile cancer and clinically negative lymph nodes.

INTRODUCTION: Penile cancer (PC) occurs less frequently in Europe and in the United States than in South America and parts of Africa. Lymph node (LN) involvement is the most important prognostic factor, and inguinal LN (ILN) dissection can be curative; however, ILN dissection has high morbidity. A nomogram was previously developed based on clinicopathological features of PC to predict ILN metastases. Our objective was to conduct an external validation of the previously developed nomogram based on our population. MATERIALS AND METHODS: We included men with cN0 ILNs who underwent ILN dissection for penile carcinoma between 2000 and 2014. We performed external validation of the nomogram considering three different external validation methods: k-fold, leave-oneout, and bootstrap. We also analyzed prognostic variables. Performance was quantified in terms of calibration and discrimination (receiver operator characteristic curve). A logistic regression model for positive ILNs was developed based on clinicopathological features of PC. RESULTS: We analyzed 65 men who underwent ILN dissection (cN0). The mean age was 56.8 years. Of 65 men, 24 (36.9%) presented with positive LNs. A median 21 ILNs were removed. Considering the three different methods used, we concluded that the previously developed nomogram was not suitable for our sample. CONCLUSIONS: In our study, the previously developed nomogram that was applied to our population had low accuracy and low precision for correctly identifying patients with PC who have positive ILNs.

Loss of BAP1 expression in metastatic tumor tissue is an event of poor prognosis in patients with metastatic clear cell renal cell carcinoma.

PURPOSE: To evaluate the prognostic impact of the protein expression of both PBRM1 and BAP1 in metastatic tissue of patients with metastatic clear cell renal cell carcinoma (ccRCC). PATIENTS AND METHODS: In all 124 consecutive cases of metastatic ccRCC, who underwent metastasectomy or biopsy of metastatic tumor tissue between 2007 and 2016 were selected from the medical records of our institution. Additionally, 38 paired cases with tissue from the primary tumor involving radical or partial nephrectomy for ccRCC were also selected. All cases were reviewed for uniform reclassification and the most representative tumor areas were selected for the construction of a tissue microarray. RESULTS: PBRM1 nuclear staining of the 124-immunostained metastases of ccRCC specimens showed that 98 (79.0%) had negative expression and 26 (21.0%) positive expression of PBRM1. Regarding BAP1 expression, we observed that 77 (62.1%) specimens were negative and 47 (37.9%) showed positive nuclear staining. When we compared the expression of both markers on primary tumor and tumor metastasis, we found disagreement in half of the cases. Five-year overall survival rates in patients with positive expression and negative expression of BAP1 were 53.2% and 35.1%, respectively (P = 0.004). Five-year progression-free survival rates in patients with positive expression and negative expression of BAP1 were 14.9% and 3.9%, respectively (P = 0.003). Conversely, PBRM1 expression did not significantly influence either overall survival or progression-free survival rates. In multivariate analysis, negative expression of BAP1 tumors also presented higher risks of death (hazard ratio (HR) = 1.913, P = 0.041) and disease progression (HR = 1.656, P = 0.021). CONCLUSION: The use of prognostic biomarkers identified in the primary tumor tissue might be not reliable in the metastatic disease scenario. Patients with metastatic ccRCC that present loss of BAP1 expression in metastatic tissue demonstrated poor survival rates and represent a relevant risk group for tumor recurrence and death.

The Role of Immunohistochemical Analysis as a Tool for the Diagnosis, Prognostic Evaluation and Treatment of Prostate Cancer: A Systematic Review of the Literature.

Background: Prostate cancer (PCa) is a heterogeneous disease that lends itself toward numerous therapeutic options depending on its risk stratification. One of the greatest challenges in PCa urologic practice is to select patients who should be referred for biopsy and, for those patients who are diagnosed with cancer, to differentiate between patients with indolent disease from those with an unfavorable prognosis and, to determine ideal patient management and avoid unnecessary interventions. Accordingly, there is a growing body of literature reporting immunohistochemical studies with the objective of determining a prostate cancer prognosis. Among the most frequent biomarkers studied are Ki-67, p53, PTEN, MYC, and ERG. Based on these findings, we systematically reviewed articles that assessed the role of these main prognostic markers in prostate cancer. Methods: Consistent with PRISMA guidelines, we performed a systematic literature search throughout the Web of Science and PubMed Medline databases. We considered all types of studies evaluating the role of Ki-67, p53, PTEN, MYC, and ERG immunohistochemical analysis in prostate cancer until July 2017. Results: We identified 361 articles, 44 of which were summarized in this review. Diagnostically, no single immunohistochemical marker was able to define a tumor as benign or malignant. Prognostically, Ki-67, p53, and MYC were related to the tumor grade given by Gleason score and to the tumor stage (higher levels related to higher tumor grade). Furthermore, Ki-67 was also related to higher PSA levels, shorter disease-free intervals and shorter tumor-specific survival; the latter was also related to p53. The loss of PTEN protein expression showed a higher association with biochemical recurrence and with a worse prognosis, beyond that predicted by the Gleason score and tumor stage. ERG staining also showed a strong association with biochemical recurrence. Conclusion: There are several studies relating immunohistochemical markers with clinical-laboratorial outcomes in prostate cancer, the most frequent being Ki-67, p53, ERG, PTEN, and MYC. However, none of these markers have been validated by literary consensus to be routinely applied in medical practice.