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The identification of mismatch repair deficient (dMMR) and microsatellite unstable (MSI) endometrial cancers (ECs) is important in screening, diagnosis, and therapeutic stratification of patients. We compared the diagnostic performance of 4 MSI molecular tests based on fragment length assay in capillary electrophoresis (OncoMate™ MSI assay, Promega) and in microcapillary electrophoresis (TapeStation 4200, Agilent); with high-resolution melting (HRM) analysis approaches (Idylla™ MSI Test, Biocartis; EasyPGX® ready MSI, Diatech Pharmacogenetics) on a series of 56 ECs, which was well characterized for MMR status with immunohistochemical approach (IHC, nonmolecular reference test). The concordance of fluorescence capillary electrophoresis with IHC (AUC 0.98) was higher respect to the other molecular methodologies. Otherwise, HRM approaches and microcapillary electrophoresis platform failed to detect MSI-ECs showing minimal microsatellite shifts. In conclusion, in colorectal site, several technologies are eligible for MSI test, whereas in ECs, MSI test should be based on fluorescent capillary electrophoresis as it identifies a higher proportion of cases that could be misdiagnosed with other strategies.

Angiogenesis is crucial for cancer progression. While several anti-angiogenic drugs are in use for cancer treatment, their clinical benefits are unsatisfactory. Thus, a deeper understanding of the mechanisms sustaining cancer vessel growth is fundamental to identify novel biomarkers and therapeutic targets. Alternative splicing (AS) is an essential modifier of human proteome diversity. Nevertheless, AS contribution to tumor vasculature development is poorly known. The Neuro-Oncological Ventral Antigen 2 (NOVA2) is a critical AS regulator of angiogenesis and vascular development. NOVA2 is upregulated in tumor endothelial cells (ECs) of different cancers, thus representing a potential driver of tumor blood vessel aberrancies. Here, we identified novel AS transcripts generated upon NOVA2 upregulation in ECs, suggesting a pervasive role of NOVA2 in vascular biology. In addition, we report that NOVA2 is also upregulated in ECs of gastric cancer (GC), and its expression correlates with poor overall survival of GC patients. Finally, we found that the AS of the Rap Guanine Nucleotide Exchange Factor 6 (RapGEF6), a newly identified NOVA2 target, is altered in GC patients and associated with NOVA2 expression, tumor angiogenesis, and poor patient outcome. Our findings provide a better understanding of GC biology and suggest that AS might be exploited to identify novel biomarkers and therapeutics for anti-angiogenic GC treatments.

Supplementary Data from Subcapsular Sinus Macrophages Promote Melanoma Metastasis to the Sentinel Lymph Nodes via an IL1α–STAT3 Axis

During melanoma metastasis, tumor cells originating in the skin migrate via lymphatic vessels to the sentinel lymph node (sLN). This process facilitates tumor cell spread across the body. Here, we characterized the innate inflammatory response to melanoma in the metastatic microenvironment of the sLN. We found that macrophages located in the subcapsular sinus (SS) produced protumoral IL1α after recognition of tumoral antigens. Moreover, we confirmed that the elimination of LN macrophages or the administration of an IL1α-specific blocking antibody reduced metastatic spread. To understand the mechanism of action of IL1α in the context of the sLN microenvironment, we applied single-cell RNA sequencing to microdissected metastases obtained from animals treated with the IL1α-specific blocking antibody. Among the different pathways affected, we identified STAT3 as one of the main targets of IL1α signaling in metastatic tumor cells. Moreover, we found that the antitumoral effect of the anti-IL1α was not mediated by lymphocytes because Il1r1 knockout mice did not show significant differences in metastasis growth. Finally, we found a synergistic antimetastatic effect of the combination of IL1α blockade and STAT3 inhibition with stattic, highlighting a new immunotherapy approach to preventing melanoma metastasis.

Supplementary Figure from Subcapsular Sinus Macrophages Promote Melanoma Metastasis to the Sentinel Lymph Nodes via an IL1α–STAT3 Axis

Although cancer stem-like cells (CSC) are thought to be the most tumorigenic, metastatic, and therapy-resistant cell subpopulation within human tumors, current therapies target bulk tumor cells while tending to spare CSC. In seeking to understand mechanisms needed to acquire and maintain a CSC phenotype in prostate cancer, we investigated connections between the ETS transcription factor ESE3/EHF, the Lin28/let-7 microRNA axis, and the CSC subpopulation in this malignancy. In normal cells, we found that ESE3/EHF bound and repressed promoters for the Lin28A and Lin28B genes while activating transcription and maturation of the let-7 microRNAs. In cancer cells, reduced expression of ESE3/EHF upregulated Lin28A and Lin28B and downregulated the let-7 microRNAs. Notably, we found that deregulation of the Lin28/let-7 axis with reduced production of let-7 microRNAs was critical for cell transformation and expansion of prostate CSC. Moreover, targeting Lin28A/Lin28B in cell lines and tumor xenografts mimicked the effects of ESE3/EHF and restrained tumor-initiating and self-renewal properties of prostate CSC both in vitro and in vivo. These results establish that tight control by ESE3/EHF over the Lin28/let-7 axis is a critical barrier to malignant transformation, and they also suggest new strategies to antagonize CSC in human prostate cancer for therapeutic purposes. Cancer Res; 76(12); 3629–43. ©2016 AACR.

PDF file - 107K, Primer sets for RT-PCR, qRT-PCR and CHIP

Legend for Supplementary Figures S1-S8.

XLS file - 142K, Genes up- and down- regulated in ESE3low tumors compared to all tumors (Q

PDF file - 7K, Identification of ETS binding site on the promoter of putative ESE3 target genes

Knockdown of ESE3/EHF and let-7 miRNAs in immortalized human prostate epithelial cells (S1); Lin28 protein expression in xenografts of human prostate cancer cell lines (S2); Ablation and forced expression of Lin28A and Lin28B affect the phenotype of normal and prostate cancer cells (S3); Consequences of miR-let7b expression in prostate cancer cells (S4); Predicted ETS binding sites (EBS) in the promoters of five let-7 family members (S5); ESE3/EHF rescues the effects of forced expression of Lin28A on the CSC compartment in normal prostate epithelial cells (S6); Lin28A and Lin28B downregulation affect the phenotype of human prostate cancer cells (S7); Efficiency of Lin28B downregulation by the Lin28B-2 siRNA in DU145 cells (S8).

PDF file - 432K, Supplementary Figure 1 Histopathological and clinical parameters of primary prostate cancer patients in the two patient cohorts evaluated by TMAs. Supplementary Figure 2 ESE3/EHF knockdown induces epithelial to mesenchymal transition Supplementary Figure 3 Transient downregulation of ESE3/EHF induces a mesenchymal phenotype Supplementary Figure 4 In vivo formation of lung metastasis Supplementary Figure 5 Prostato-sphere formation by prostate epithelial cells Supplementary Figure 6 Transient downregulation of ESE3/EHF in PrECs and RWPE-1 cells induces stem-like properties Supplementary Figure 7 Prostato-sphere formation and stem-like properties in DU145 cells Supplementary Figure 8 Overall survival and PSA biochemical relapse in prostate cancer patients

Primer sets for qRT-PCR and ChIP experiments.

PDF file - 782K, ESE1/ELF3 overexpression in primary prostate tumors (S1); Generation of stable ESE-1/ELF3 over-expressing prostate cancer cell lines (S2); Level of ESE1/ELF3 in prostate tumor xenografts (S3); Network interactions between ESE1/ELF3-induced genes and ETS transcription factors (S4); Position of ETS binding sites identified by computational analysis using Motiviz (S5); Convergence between the transcriptional program induced in IL-1B transgenic mice and ESE1/ELF3 overexpressing 22RVI cells (S6); Reciprocal regulatory loops between ESE1/ELF3 and NFKB (S7); Immunofluorescence microscopy for detection of ESE1/ELF3, p50, p65, and nuclei (S8); Kaplan-Meyer analysis of overall survival of prostate cancer patients from the TMA cohort subdivided according to ESE1/ELF3 and p50/p65 nuclear expression (S9).

XLS file - 156K, Genes up- and down- regulated in ESE3low tumors compared to all tumors (Q

XLS file - 168K, Genes up- and down- regulated in ESE3low tumors compared to all tumors (Q

PDF file - 65K, Primer sets for RT-PCR, qRT-PCR and ChIP.

XLSX file - 2556K, Genes induced in 22RV1ESE1 vs 22RV1control.

PDF file - 105K

PDF file - 94K, Supplementary materials contain detailed description of the bioinformatic approaches such as Functional Annotation, Transcription factor interactome analysis, Gene Set Enrichment analysis and Survival analysis.

PDF file - 1MB, Genes differentially expressed in ESE3KD-PrECs compared to control PrECs

Objectives Poorly cohesive carcinomas (PCCs) are neoplasms defined by a predominantly dyshesive growth pattern with single cell or cord-like stromal infiltration. The ­distinctive clinicopathologic and prognostic features of small bowel PCCs (SB-PCCs) in comparison with conventional-type small intestinal adenocarcinomas have only recently been characterized. However, as SB-PCCs’ genetic profile is still unknown, we aimed to analyze the molecular landscape of SB-PCCs. Methods A next-generation sequencing analysis through Trusight Oncology 500 on a series of 15 nonampullary SB-PCCs was performed. Results The most frequently found gene alterations were TP53 (53%) and RHOA (13%) mutations and KRAS amplification (13%), whereas KRAS, BRAF, and PIK3CA mutations were not identified. Most SB-PCCs (80%) were associated with Crohn disease, including both RHOA-mutated SB-PCCs, which featured a non-SRC-type histology, and showed a peculiar appendiceal-type, low-grade goblet cell adenocarcinoma (GCA)–like component. Rarely, SB-PCCs showed high microsatellite instability, mutations in IDH1 and ERBB2 genes, or FGFR2 amplification (one case each), which are established or promising therapeutic targets in such aggressive cancers. Conclusions SB-PCCs may harbor RHOA mutations, which are reminiscent of the diffuse subtype of gastric cancers or appendiceal GCAs, while KRAS and PIK3CA mutations, commonly involved in colorectal and small bowel adenocarcinomas, are not typical of such cancers.

Introduction: Poorly differentiated neuroendocrine carcinomas (NECs) are characterized by aggressive clinical course and poor prognosis. No reliable prognostic markers have been validated to date; thus, the definition of a specific NEC prognostic algorithm represents a clinical need. This study aimed to analyze a large NEC case series to validate the specific prognostic factors identified in previous studies on gastro-entero-pancreatic and lung NECs and to assess if further prognostic parameters can be isolated. Methods: A pooled analysis of four NEC retrospective studies was performed to evaluate the prognostic role of Ki-67 cut-off, the overall survival (OS) according to primary cancer site, and further prognostic parameters using multivariable Cox proportional hazards model and machine learning random survival forest (RSF). Results: 422 NECs were analyzed. The most represented tumor site was the colorectum (n = 156, 37%), followed by the lungs (n = 111, 26%), gastroesophageal site (n = 83, 20%; 66 gastric, 79%) and pancreas (n = 42, 10%). The Ki-67 index was the most relevant predictor, followed by morphology (pure or mixed/combined NECs), stage, and site. The predicted RSF response for survival at 1, 2, or 3 years showed decreasing survival with increasing Ki-67, pure NEC morphology, stage III–IV, and colorectal NEC disease. Patients with Ki-67 Conclusion: The most effective parameters to predict OS for NEC patients could be Ki-67, pure or mixed/combined morphology, stage, and site.

Poorly cohesive carcinomas (PCCs) are neoplasms characterized by a dyshesive cell invasion pattern featuring single-cell or cord-like stromal infiltration. Although they have been extensively studied in the stomach and other digestive system organs, limited data regarding nonampullary small bowel poorly cohesive carcinomas (SB-PCCs) are hitherto available. The aims of our study were to analyze the clinicopathologic and immunophenotypical features of SB-PCCs (PCC pattern accounting for >50% of the neoplasm) and to compare them with small bowel adenocarcinomas (SBAs), not otherwise specified (SBAs-NOS) and with cancers with a histologically distinct PCC component accounting for 10% to 50% of the neoplasm (mixed-poorly-cohesive-glandular-SBAs). Fifteen SB-PCCs were identified and compared with 95 SBAs-NOS and 27 mixed-poorly-cohesive-glandular-SBAs. Most SB-PCCs (67%) were composed of

Background Endometrial carcinoma represents a sentinel cancer for Lynch syndrome (LS) identification. It is crucial to highlight how other types of tumors can arise in the gynecological tract acting as sentinel tumors in LS patients.Up to now, no established LS patient management strategy has incorporated the presence of these additional candidate sentinel tumors to improve the prevention and management of LS tumors. Methods In order to investigate the involvement of the most frequent gynecological cancers in gynecological cancers, we studied different subsets of gynecological cancers using both somatic approaches, including mismatch repair (MMR) gene immunohistochemical expression, microsatellite instability, and germline analyses ofMSH2, MSH6, MLH1, PMS2 and EPCAM genes.A total of 261 patients referring to the Cancer Genetic Counselling Service of our institution were included in the study. In detail, our series was composed of 131 patients affected by uterus cancers including endometrial, isthmus and non-HPV endocervical carcinomas, 113 patients affected by ovarian cancers and 17 patients affected by synchronous endometrial/ovarian carcinomas (SEOC).In addition, we studied 115 cases of endometrial cancers identified by 2 years of universal testing (endometrial cancers/UTs) using IHC analysis of four MMR proteins. Results and conclusions The incidence of MMR defective gynecological cancers ranged from 7.1 to 47.1% depending on cancer site and selection. LS patients carriers of pathogenetic MMR variants were identified in 19.8% of uterus cancers, 35.3% of SEOC, 4.4% of ovarian cancers. In addition, pathogenetic MMR variants were identified in 4.3% of endometrial cancers/universal testing investigated with universal screening.In conclusion, gynecological cancers are heavily involved in LS and our study shows that MMR screening using immunohistochemical pattern and MSI analysis of endometrial and ovarian cancers as well as of rare entities such as non-HPV related endocervical cancers and synchronous endometrial and ovarian cancers are sentinels for LS.Tumor testing approach improves early identification of MMR defective gynecological cancers and this is an effective strategy to detect high-risk patients and to offer them and their relatives personalized cancer prevention.

Fibromuscular dysplasia is a non-atherosclerotic, non-inflammatory arteriopathy, considered a rare cause of coronary artery disease. Although familial cases have been described, no specific gene association has been detected so far. When the coronary vessels are involved, the main clinical scenarios are stable angina, acute coronary syndromes, left ventricular dysfunction, and sudden death. Specific clinical and angiographic findings may suggest this as the underlying disease, but certain diagnosis histological. The involvement of the lower and upper limbs is unusual; however, it may have decisive clinical implications for the most appropriate revascularization method and the selection of the arterial graft to be used.

The main hypothesis of this study is that gene expression profiles (GEPs) integrating both tumor antigenicity and a pre-existing adaptive immune response can be used to generate distinct immune-related signatures of BRAF mutant colorectal cancers (BRAF-CRCs) to identify actionable biomarkers predicting response to immunotherapy. GEPs of 89 immunotherapy-naïve BRAF-CRCs were generated using the Pan-Cancer IO 360 gene expression panel and the NanoString nCounter platform and were correlated with microsatellite instability (MSI) status and with CD8+ tumor-infiltrating lymphocyte (TIL) content. Hot/inflamed profiles were found in 52% of all cases, and high scores of Tumor Inflammation Signature were observed in 42% of the metastatic BRAF-CRCs. A subset of MSI tumors showed a cold profile. Antigen Processing Machinery (APM) signature was not differentially expressed in MSI tumors compared with MSS cases. By contrast, the APM signature was significantly upregulated in CD8+ BRAF-CRCs versus CD8− tumors. Our study demonstrates that a significant fraction of BRAF-CRCs may be a candidate for immunotherapy and that the simultaneous analysis of MSI status and CD8+ TIL content increases accuracy in identifying patients who can potentially benefit from immune checkpoint inhibitors. GEPs may be very useful in expanding the spectrum of patients with BRAF-CRCs who can benefit from immune checkpoint blockade.

Objectives Sinonasal mucosal melanoma (SNMM) is an extremely rare and challenging sinonasal malignancy with a poor prognosis. Standard treatment involves complete surgical resection, but the role of adjuvant therapy remains unclear. Crucially, our understanding of its clinical presentation, course, and optimal treatment remains limited, and few advancements in improving its management have been made in the recent past. Methods We conducted an international multicenter retrospective analysis of 505 SNMM cases from 11 institutions across the United States, United Kingdom, Ireland, and continental Europe. Data on clinical presentation, diagnosis, treatment, and clinical outcomes were assessed. Results One-, three-, and five-year recurrence-free and overall survival were 61.4, 30.6, and 22.0%, and 77.6, 49.2, and 38.3%, respectively. Compared with disease confined to the nasal cavity, sinus involvement confers significantly worse survival; based on this, further stratifying the T3 stage was highly prognostic (p Conclusions We present findings from the largest cohort of SNMM reported to date. We demonstrate the potential utility of further stratifying the T3 stage by sinus involvement and present promising data on the benefit of immune checkpoint inhibitors for recurrent, persistent, or metastatic disease with implications for future clinical trials in this field.

Introduction. Natural Killer (NK) cells are innate lymphoid cells involved in tumour recognition/elimination. NK cells are altered in their phenotype and functions in diverse tumors, including prostate cancer (PCa). We demonstrated that PCa circulating NK cells (TANKs) acquire the pro-angiogenic/decidual-like CD56brightCD9+CD49a+ phenotype, release IL-8 and MMP-9, functionally support endothelial cell activation and secrete monocyte-recruiting/M2-like macrophage-polarizing factors. Materials and methods. Here, we characterized the phenotype of tumour infiltrating NKs (TINKs) and tumour-associated (TANKs) in PCa patients and evaluated the contribution of STAT3, as possible driver of NK cell polarization. PCa TINKs and TANKs were characterized by multicolour flow cytometry (FC) for decidual-like surface markers (CD9, CD49a) and degranulation capabilities (CD107a). STAT3 activation, was investigated in circulating PCa NK cells, by FC. Using a drug-repurposing approach with the antipsychotic agent Pimozide (a chemical inhibitor of STAT3), we modulated STAT3 activation, ex vivo, in PCa TANKs and monitored their secretome changes, by commercially available protein membrane arrays together with their capability to degranulate and produce perforin/GranzymeB. Results and discussion. We observed that PCa TINKs acquire the same CD9+CD49a+ decidual-like NK cell phenotype, as found in PCa TANKs. We detected the presence of CD56brightCD9+CD49a+ decidual-like NK cell also in peripheral blood of subjects with benign prostatic hyperplasia (BPH), but in a lower frequency, compared to those from PCa TANKs. Sera from PCa patients were enriched in IL-4, IL-6, IL-8 and IL-10, all cytokines able to activate STAT3 signalling. We detected increased phosphorylation of STAT3 in PCa TANKs, compared to NK cells from healthy controls, that was reduced following 24 hours of stimulation by Pimozide. This treatment resulted in decreased capabilities of PCa TANKs to secrete pro-angiogenic factors (IL-8, IL-6), molecules involved in monocytes recruitment/M2-like macrophage polarization (CCL-2, CCL5, GM-CSF, IL-10), together with increased degranulation, augmented secretion of (IFN-γ and TNF-α) and increased production of Perforin and Granzyme. Conclusions. Our results suggest that STAT3 inhibition can be envisaged as a potential strategy to limit the generation of pro-angiogenic/decidual-like NKs, while contributing to NK cell re-education in PCa. Citation Format: Matteo Gallazzi, Maria Teresa Palano, Martina Cucchiara, Federico Dehò, Paolo Capogrosso, Francesca Franzi, Fausto Sessa, Angelo Naselli, Lorenzo Mortara, Antonino Bruno. STAT-3 chemical inhibition modulates decidual-like polarization in NK cells from PCa patients and restore their anti-tumor activities. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4595.

Glioblastoma multiforme (GBM) remains one of the tumors with the worst prognosis. In recent years, a better overall survival (OS) has been described in cases subjected to Gross Total Resection (GTR) that were presenting hypermethylation of Methylguanine-DNA methyltransferase (MGMT) promoter. Recently, also the expression of specific miRNAs involved in MGMT silencing has been related to survival. In this study, we evaluate MGMT expression by immunohistochemistry (IHC), MGMT promoter methylation and miRNA expression in 112 GBMs and correlate the data to patients’ clinical outcomes. Statistical analyses demonstrate a significant association between positive MGMT IHC and the expression of miR-181c, miR-195, miR-648 and miR-767.3p between unmethylated cases and the low expression of miR-181d and miR-648 and between methylated cases and the low expression of miR-196b. Addressing the concerns of clinical associations, a better OS has been described in presence of negative MGMT IHC, in methylated patients and in the cases with miR-21, miR-196b overexpression or miR-767.3 downregulation. In addition, a better progression-free survival (PFS) is associated with MGMT methylation and GTR but not with MGMT IHC and miRNA expression. In conclusion, our data reinforce the clinical relevance of miRNA expression as an additional marker to predict efficacy of chemoradiation in GBM.

Fibromatosis-like metaplastic breast carcinoma is an uncommon variety of breast cancer whose diagnosis could be challenging. The reported case underlines the diagnostic difficulties if abundant inflammatory cells and no in situ carcinoma or epithelioid features are present in the sample. The broad spectrum of differential diagnosis, including benign and malignant lesions, requires immunohistochemical characterization. Cytokeratin expression is mandatory. The case clearly represents the indolent behavior of this entity whose treatment algorithm differs from that of all other breast cancers. As a curative treatment, our patient was offered a multi-modal approach, which cannot be considered a standard option for other triple-negative breast cancers. The overall benefit in disease-free survival was surprisingly strong, despite some prognostic negative markers, such as Ki67 index and p53 nuclear accumulation. The presence of tumor infiltrating lymphocytes opens a width of novel therapeutic options that are yet to be assessed in this subgroup of breast cancers.