miRNA levels are associated with body mass index in endometrial cancer and may have implications for therapy.

Endometrial cancer (EC) is the most prevalent gynecological cancer in high-income countries. Its incidence is skyrocketing due to the increase in risk factors such as obesity, which represents a true pandemic. This study aimed to evaluate microRNA (miRNA) expression in obesity-related EC to identify potential associations between this specific cancer type and obesity. miRNA levels were analyzed in 84 EC patients stratified based on body mass index (BMI; ≥30 or <30) and nine noncancer women with obesity. The data were further tested in The Cancer Genome Atlas (TCGA) cohort, including 384 EC patients, 235 with BMI ≥30 and 149 with BMI <30. Prediction of miRNA targets and analysis of their expression were also performed to identify the potential epigenetic networks involved in obesity modulation. In the EC cohort, BMI ≥30 was significantly associated with 11 deregulated miRNAs. The topmost deregulated miRNAs were first analyzed in 84 EC samples by single miRNA assay and then tested in the TCGA dataset. This independent validation provided further confirmation about the significant difference of three miRNAs (miR-199a-5p, miR-449a, miR-449b-5p) in normal-weight EC patients versus EC patients with obesity, resulting significantly higher expressed in the latter. Moreover, the three miRNAs were significantly correlated with grade, histological type, and overall survival. Analysis of their target genes revealed that these miRNAs may regulate obesity-related pathways. In conclusion, we identified specific miRNAs associated with BMI that are potentially involved in modulating obesity-related pathways and that may provide novel implications for the clinical management of obese EC patients.

KIT/PDGFRA Variant Allele Frequency as Prognostic Factor in Gastrointestinal Stromal Tumors (GISTs): Results From a Multi-Institutional Cohort Study.

BACKGROUND: The patient selection for optimal adjuvant therapy in gastrointestinal stromal tumors (GISTs) is provided by nomogram based on tumor size, mitotic index, tumor location, and tumor rupture. Although mutational status is not currently used to risk assessment, tumor genotype showed a prognostic influence on natural history and tumor relapse. Innovative measures, such as KIT/PDGFRA-mutant-specific variant allele frequency (VAF) levels detection from next-generation sequencing (NGS), may act as a surrogate of tumor burden and correlate with prognosis and overall survival of patients with GIST, helping the choice for adjuvant treatment. PATIENTS AND METHODS: This was a multicenter, hospital-based, retrospective/prospective cohort study to investigate the prognostic role of KIT or PDGFRA-VAF of GIST in patients with radically resected localized disease. In the current manuscript, we present the results from the retrospective phase of the study. RESULTS: Two-hundred (200) patients with GIST between 2015 and 2022 afferent to 6 Italian Oncologic Centers in the EURACAN Network were included in the study. The receiver operating characteristic (ROC) curves analysis was used to classify "low" vs. "high" VAF values, further normalized on neoplastic cellularity (nVAF). When RFS between the low and high nVAF groups were compared, patients with GIST with KIT/PDGFRA nVAF > 50% showed less favorable RFS than patients in the group of nVAF ≤ 50% (2-year RFS, 72.6% vs. 93%, respectively; P = .003). The multivariable Cox regression model confirmed these results. In the homogeneous sub-population of intermediate-risk, patients with KIT-mutated GIST, the presence of nVAF >50% was statistically associated with higher disease recurrence. CONCLUSION: In our study, we demonstrated that higher nVAF levels were independent predictors of GIST prognosis and survival in localized GIST patients with tumors harboring KIT or PDGFRA mutations. In the cohort of intermediate-risk patients, nVAF could be helpful to improve prognostication and the use of adjuvant imatinib.

Beta-Catenin Alterations in Postchemotherapy Yolk Sac Tumor, Postpubertal-Type With Enteroblastic Features.

Postchemotherapy postpubertal-type yolk sac tumors (YST) with glandular and solid phenotypes are aggressive and commonly resistant to systemic chemotherapy. These neoplasms show morphologic features that significantly overlap with those of somatic carcinomas with "enteroblastic" or "fetal" phenotype (the preferred terminology depends on the site of origin). They often present as late or very late recurrences, and their diagnosis is challenging because they frequently affect patients in an age group at risk for carcinomas of somatic origin. Recently, we incidentally identified examples of postchemotherapy glandular and solid YST with "enteroblastic" phenotypes and nuclear expression of beta-catenin, prompting us to further evaluate the prevalence of this phenomenon. We found nuclear expression of beta-catenin in 10 (29%) of 34 such tumors. A subset of cases with nuclear beta-catenin expression was further analyzed with a DNA sequencing panel (n = 6) and fluorescence in situ hybridization for isochromosome 12p [i(12p); n = 5]. Sequencing identified exon 3 CTNNB1 variants in 3 (50%) of 6 analyzed cases, and fluorescence in situ hybridization was positive for i(12p) in 5 of 5 cases. In conclusion, a significant subset of postchemotherapy YST with glandular or solid architecture and "enteroblastic" phenotype demonstrates beta-catenin alterations, suggesting that activation of Wnt signaling may play a role in the progression of these neoplasms. Moreover, nuclear beta-catenin expression in these tumors represents a potential diagnostic pitfall given that carcinomas of true somatic origin with overlapping morphology may also be positive for this marker.

Spatial Cancer-Immune phenotypes predict shorter recurrence free survival in the NSMP molecular subtype of endometrial carcinoma.

Compartmentation of the immune response into three main spatial cancer-immune phenotypes (SCIs) - inflamed, excluded, and desert - has been proposed as the main predictor of response to immune checkpoint inhibitors in solid tumors. The objective of the study is to define and characterize the SCI in a consecutive series of 213 endometrial carcinomas (ECs) by correlating it with molecular subtypes, clinicopathologic features, and prognosis. Immunohistochemistry (IHC) and Next-Generation Sequencing (NGS) were used to assign surrogate molecular EC subtypes: POLE mutant (POLE), mismatch repair deficient (MMRd), TP53 mutant (p53abn), and no specific molecular profile (NSMP). Immune cell markers (CD20, CD3, CD8, CD68, PD-L1) were assessed by IHC on whole sections and quantified by digital image analysis to define the three SCIs. ECs were stratified into four molecular subtypes: 17 (8.0%) POLE, 68 (31.9%) MMRd, 42 (19.7%) p53abn, and 86 (40.4%) NSMP. SCI determination showed 105 (49.3%) inflamed, 62 (29.1%) desert, and 46 (25.6%) excluded tumors. The inflamed phenotype was more prevalent in MMRd (64.7%) and POLE (76.5%) subtypes compared to NSMP (45.3%) and p53abn (21.4%). SCI revealed a strong correlation with DFS in NSMP tumors: inflamed 96.2%, desert 83.2% and excluded 40.5%. The SCI prognostic impact was also maintained in NSMP cases treated with adjuvant therapy resulting in a significant difference in recurrence between the inflamed and excluded phenotypes. To simplify SCI determination, a subset of immune cell markers was selected as appropriate to define the three SCI patterns: high intraepithelial CD8 for the inflamed phenotype; CD68, CD20, and PD-L1 to discriminate between desert and excluded tumors. The integration of SCI into molecular classification could be a promising opportunity to improve the prognostic risk stratification of patients and may guide the therapeutic approach, particularly in the NSMP subtype. Thus, the different patterns of immune response are a new prognostic parameter in the NSMP subtype.

ß-Catenin alterations in testicular Leydig cell tumour: a immunohistochemical and molecular analysis.

BACKGROUND: Testicular Leydig cell tumours (LCTs) are the most common type of sex cord-stromal tumour in men, representing 1%-3% of all testicular neoplasms. Among testicular sex cord-stromal tumours, CTNNB1 mutations and nuclear expression of ß-catenin have been typically associated with Sertoli cell tumour. Recent genomic analyses have shown that CTNNB1 variants are also identified in a subset of LCTs; however, the frequency and clinicopathologic associations of ß-catenin alterations remain incompletely understood in this tumour type. METHODS: In this study, we evaluated 32 LCTs (five malignant/metastasizing, 27 nonmetastasizing) using ß-catenin immunohistochemistry and DNA sequencing. RESULTS: Immunohistochemistry revealed focal or multifocal nuclear ß-catenin expression in 47% of the tumours. Diffuse nuclear ß-catenin expression (in >50% of the tumour cells) was not detected in any of the cases analysed herein. Comparison of ß-catenin-positive and ß-catenin-negative cases did not show significant differences in the frequency of adverse histopathologic findings or malignant clinical behaviour. DNA sequencing performed de novo on a subset of seven cases revealed the presence of exon 3 CTNNB1 variants in four of them (4/7, 57%), with variant allele frequencies (VAF) ranging from 7 to 33%. Two additional ß-catenin-positive cases that had been sequenced as part of a previous study harboured exon 3 CTNNB1 variants at VAF of 28% and 7%, respectively. CONCLUSION: These results demonstrate that ß-catenin alterations are relatively common in LCT, most likely occurring as subclonal events that are not enriched in cases with aggressive features. Further studies are needed to clarify the oncogenic role of ß-catenin in this tumour type.

Accuracy of World Health Organisation-grade parameters (necrosis and mitotic activity) and foci of vascular invasion in predicting prognosis of papillary thyroid carcinoma. A case-control validation study.

AIMS: Tumour necrosis and/or increased mitoses define high-grade papillary thyroid carcinoma (PTC). It is unclear whether angioinvasion is prognostic for PTC. Cut-offs at five or more mitoses/2 mm2 and four or more angioinvasive foci have been empirically defined based upon data from all forms of aggressive non-anaplastic thyroid carcinomas. Performance of tumour necrosis, mitoses and vascular invasion in predicting distant metastases when specifically applied to PTC is undefined. METHODS: We analysed 50 consecutive PTC cases with distant metastases (DM-PTC): 16 synchronous and 34 metachronous. A total of 108 non-metastatic PTC (N-DM-PTC, 15.0-year median follow-up) were used as controls. Invasive encapsulated follicular variant PTC was excluded. Necrosis, mitoses and angioinvasion were quantified. Receiver operating characteristics (ROC) and area under the curve (AUC) analyses determined best sensitivity and specificity cut-offs predictive of distant metastases. RESULTS: Metastases correlated with necrosis (any extent = 43.8% all DM-PTC, 53.1% metachronous DM-PTC versus 5% N-DM-PTC; P < 0.001), mitoses (P < 0.001) and angioinvasion (P < 0.001). Mitoses at five or more per 2 mm2 was the best cut-off correlating with distant metastases: sensitivity/specificity 42.9%/97.2% all DM-PTC (AUC = 0.78), 18.8%/97.2% synchronous DM-PTC (AUC = 0.63), 54.6%/97.2% metachronous DM-PTC (AUC = 0.85). Angioinvasive foci at five or more was the best cut-off correlating with distant metastases: sensitivity/specificity 36.2%/91.7% all DM-PTC (AUC = 0.75), 25%/91.7% synchronous DM-PTC (AUC = 0.79) and 41.9%/91.7% metachronous DM-PTC (AUC = 0.73). Positive/negative predictive values (PPV/NPV) were: necrosis 22.6%/98.2%; five or more mitoses 32.3%/98.2%; five or more angioinvasive foci 11.8%/97.9%. After multivariable analysis, only necrosis and mitotic activity remained associated with DM-PTC. CONCLUSION: Our data strongly support PTC grading, statistically validating World Health Organisation (WHO) criteria to identify poor prognosis PTC. Angioinvasion is not an independent predictor of DM-PTC.

The role of adjuvant chemotherapy in patients with H3K27 altered diffuse midline gliomas: a multicentric retrospective study.

PURPOSE: Adult Diffuse midline glioma (DMG) is a very rare disease. DMGs are currently treated with radiotherapy and chemotherapy even if only a few retrospective studies assessed the impact on overall survival (OS) of these approaches. METHODS: We carried out an Italian multicentric retrospective study of adult patients with H3K27-altered DMG to assess the effective role of systemic therapy in the treatment landscape of this rare tumor type. RESULTS: We evaluated 49 patients from 6 Institutions. The median age was 37.3 years (range 20.1-68.3). Most patients received biopsy as primary approach (n = 30, 61.2%) and radiation therapy after surgery (n = 39, 79.6%). 25 (51.0%) of patients received concurrent chemotherapy and 26 (53.1%) patients received adjuvant temozolomide. In univariate analysis, concurrent chemotherapy did not result in OS improvement while adjuvant temozolomide was associated with longer OS (21.2 vs. 9.0 months, HR 0.14, 0.05-0.41, p < 0.001). Multivariate analysis confirmed the role of adjuvant chemotherapy (HR 0.1, 95%CI: 0.03-0.34, p = 0.003). In patients who progressed after radiation and/or chemotherapy the administration of a second-line systemic treatment had a significantly favorable impact on survival (8.0 vs. 3.2 months, HR 0.2, 95%CI 0.1-0.65, p = 0.004). CONCLUSION: In our series, adjuvant treatment after radiotherapy can be useful in improving OS of patients with H3K27-altered DMG. When feasible another systemic treatment after treatment progression could be proposed.

Morphologic, phenotypic, and genotypic similarities between primary tumors and corresponding 3D cell cultures grown in a repeatable system-preliminary results.

BACKGROUND: Three-dimensional (3D) cell cultures are the new frontier for reproducing the tumor micro-environment in vitro. The aims of the study were (1) to establish primary 3D cell cultures from canine spontaneous neoplasms and (2) to demonstrate the morphological, phenotypic and genotypic similarities between the primary canine neoplasms and the corresponding 3D cultures, through the expression of tumor differentiation markers. RESULTS: Seven primary tumors were collected, including 4 carcinomas and 3 soft tissue sarcomas. 3D cell cultures reproduced the morphological features of the primary tumors and showed an overlapping immunophenotype of the primary epithelial tumors. Immunohistochemistry demonstrated the growth of stromal cells and macrophages admixed with the neoplastic epithelial component, reproducing the tumor microenvironment. Mesenchymal 3D cultures reproduced the immunophenotype of the primary tumor completely in 2 out of 3 examined cases while a discordant expression was documented for a single marker in one case. No single nucleotide variants or small indel were detected in TP53 or MDM2 genes, both in primary tumors and in 3D cell cultures specimens. In one sample, MDM2 amplicons were preferentially increased in number compared to TP53 ones, indicating amplification of MDM2, detectable both in the primary tumor and in the corresponding cell culture specimen. CONCLUSION: Here we demonstrate a good cell morphology, phenotype and genetic profile overlap between primary tumors and the corresponding 3D cultures grown in a repeatable system.

Expanding the Spectrum of BRAF Non-V600E Mutations in Thyroid Nodules: Evidence-Based Data from a Tertiary Referral Centre.

The BRAF p.V600E mutation represents the most specific marker for papillary thyroid carcinoma and is potentially related to aggressive behavior and persistent disease. BRAF alterations other than the p.V600E are less common in thyroid carcinoma and represent an alternative mechanism of BRAF activation with unclear clinical significance. The study aims to describe the frequency and clinicopathologic characteristics of BRAF non-V600E mutations in a large cohort (1654 samples) of thyroid lesions characterized by next-generation sequencing. BRAF mutations have been found in 20.3% (337/1654) of thyroid nodules, including classic (p.V600E) mutation in 19.2% (317/1654) of samples and non-V600E variants in 1.1% of cases (19/1654). BRAF non-V600E alterations include 5 cases harboring p.K601E, 2 harboring p.V600K substitutions, 2 with a p.K601G variant, and 10 cases with other BRAF non-V600E alterations. BRAF non-V600E mutations have been reported in one case of follicular adenoma, three cases of conventional papillary thyroid carcinoma, eight cases of follicular variant of papillary carcinomas, one case of columnar cell variant papillary thyroid carcinoma, one case of oncocytic follicular carcinoma, and two bone metastasis of follicular thyroid carcinoma. We confirm that BRAF non-V600E mutations are uncommon and typically found in indolent follicular-patterned tumors. Indeed, we show that BRAF non-V600E mutations can be found in tumors with metastatic potential. However, in both aggressive cases, the BRAF mutations were concomitant with other molecular alterations, such as TERT promoter mutation.

Multi-Gene Next-Generation Sequencing Panel for Analysis of BRCA1/BRCA2 and Homologous Recombination Repair Genes Alterations Metastatic Castration-Resistant Prostate Cancer.

Despite significant therapeutic advances, metastatic CRPC (mCRPC) remains a lethal disease. Mutations in homologous recombination repair (HRR) genes are frequent in mCRPC, and tumors harboring these mutations are known to be sensitive to PARP inhibitors. The aim of this study was to verify the technical effectiveness of this panel in the analysis of mCRPC, the frequency and type of mutations in the BRCA1/BRCA2 genes, as well as in the homologous recombination repair (HRR) genes. A total of 50 mCRPC cases were analyzed using a multi-gene next-generation sequencing panel evaluating a total of 1360 amplicons in 24 HRR genes. Of the 50 cases, 23 specimens (46.0%) had an mCRPC harboring a pathogenic variant or a variant of uncertain significance (VUS), whereas in 27 mCRPCs (54.0%), no mutations were detected (wild-type tumors). BRCA2 was the most commonly mutated gene (14.0% of samples), followed by ATM (12.0%), and BRCA1 (6.0%). In conclusion, we have set up an NGS multi-gene panel that is capable of analyzing BRCA1/BRCA2 and HRR alterations in mCRPC. Moreover, our clinical algorithm is currently being used in clinical practice for the management of patients with mCRPC.