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none 33 no This report contains clinically oriented guidelines for the diagnostic work-up and follow-up of cystic pancreatic neoplasms in patients fit for treatment. The statements were elaborated by working groups of experts by searching and analysing the literature, and then underwent a consensus process using a modified Delphi procedure. The statements report recommendations regarding the most appropriate use and timing of various imaging techniques and of endoscopic ultrasound, the role of circulating and intracystic markers and the pathologic evaluation for the diagnosis and follow-up of cystic pancreatic neoplasms open Italian Association of Hospital Gastroenterologists and Endoscopists; Italian Association for the Study of the Pancreas; Buscarini E; Pezzilli R; Cannizzaro R; De Angelis C; Gion M; Morana G; Zamboni G; Arcidiacono P; Balzano G; Barresi L; Basso D; Bocus P; Calculli L; Capurso G; Canzonieri V; Casadei R; Crippa S; D'Onofrio M; Frulloni L; Fusaroli P; Manfredi G; Pacchioni D; Pasquali C; Rocca R; Ventrucci M; Venturini S; Villanacci V; Zerbi A; Falconi M; Cystic Pancreatic Neoplasm Study Group Italian Association of Hospital Gastroenterologists and Endoscopists; Italian Association for the Study of the Pancreas; Buscarini E; Pezzilli R; Cannizzaro R; De Angelis C; Gion M; Morana G; Zamboni G; Arcidiacono P; Balzano G; Barresi L; Basso D; Bocus P; Calculli L; Capurso G; Canzonieri V; Casadei R; Crippa S; D'Onofrio M; Frulloni L; Fusaroli P; Manfredi G; Pacchioni D; Pasquali C; Rocca R; Ventrucci M; Venturini S; Villanacci V; Zerbi A; Falconi M; Cystic Pancreatic Neoplasm Study Group

ObjectiveTo screen and characterize germline variants for E-cadherin (CDH1) in non-hereditary gastric cancer (GC) patients and in subjects at risk of GC.Methods59 GCs, 59 first degree relatives (FDRs) of GC, 20 autoimmune metaplastic atrophic gastritis (AMAGs) and 52 blood donors (BDs) were analyzed for CDH1 by direct sequencing, structural modelling and bioinformatics. Functional impact on splicing was assessed for intronic mutations. E-cadherin/β-catenin immunohistochemical staining and E-cadherin mRNA quantification using RT-PCR were performed.ResultsIn GCs, 4 missense variants (p.G274S; p.A298T; p.T470I; p.A592T), 1 mutation in the 5'UTR (-71C>G) and 1 mutation in the intronic IVS12 (c.1937-13T>C) region were found. First pathogenic effect of p.A298T mutation was predicted by protein 3D modelling. The novel p.G274S mutation showed a no clear functional significance. Moreover, first, intronic IVS12 (c.1937-13T>C) mutation was demonstrated to lead to an aberrant CDH1 transcript with exon 11 deletion. This mutation was found in 2 GCs and in 1 BD. In FDRs, we identified 4 variants: the polymorphic (p.A592T) and 3 mutations in untranslated regions with unidentified functional role except for the 5'UTR (-54G>C) that had been found to decrease CDH1 transcription. In AMAGs, we detected 2 alterations: 1 missense (p.A592T) and 1 novel variant (IVS1 (c.48+7C>T)) without effect on CDH1 splicing. Several silent and polymorphic substitutions were found in all the groups studied.ConclusionsOverall our study improves upon the current characterization of CDH1 mutations and their functional role in GC and in individuals at risk of GC. Mutations found in untranslated regions and data on splicing effects deserve a particular attention like associated with a reduced E-cadherin amount. The utility of CDH1 screening, in addition to the identification of other risk factors, could be useful for the early detection of GC in subjects at risk (i.e. FDRs and AMAGs), and warrants further study.

Background. In women with cancer-related hysterectomy, the vaginal vault cytology has a low efficacy - when performed by conventional methods - for the early detection of vaginal recurrence. The amount of exfoliated cells collected is generally low because of atrophy, and the vaginal vault corners can be so narrow that the commonly used Ayres spatula cannot often penetrate deeply into them. This prospective study aimed at identifying the advantages obtained in specimens collection using the cytobrush, as compared to the Ayres's spatula. Patients and methods. 141 gynaecologic cancer patients were studied to compare samplings collected with Ayre's spatula or with cytobrush. In a pilot setting of 15 patients, vaginal cytology samples obtained by both Ayre's spatula and cytobrush were placed at the opposite sites of a single slide for quali-quantitative evaluation. Thereafter, the remaining 126 consecutive women were assigned to either group A (spatula) or B (cytobrush) according to the order of entry. The same gynaecologist performed all the procedures. Results. In all 15 pilot cases, the cytobrush seemed to collect a higher quantity of material. The comparative analysis of the two complete groups indicated that the cytobrush technique was more effective than the spatula one. The odds ratio (OR) for an optimal cytology using the cytobrush was 2.8 (95% confidence interval -C. I. 1.3-6.2; chi-square test, p=0.008). Conclusions. Vaginal vault cytology with cytobrush turned out to better perform than the traditional Ayre's spatula to obtain an adequate sampling in gynecological cancer patients.

Pathological complete response after neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients is related to a favorable prognosis. The identification of early biomarkers predictive of pathological complete response would help optimize the multimodality management of the patients. A panel of 11 tumor-related proteins was investigated by immunohistochemistry in the pretreatment biopsy of a group of locally advanced rectal cancer patients to identify early biomarkers of pathological complete response to neoadjuvant chemoradiotherapy. A mono-institutional retrospective cohort of 95 stage II/III locally advanced rectal cancer patients treated with neoadjuvant chemoradiotherapy and surgery was selected based on clinicalpathological characteristics and the availability of a pretreatment tumor biopsy. Eleven selected protein marker expression (MLH1, GLUT1, Ki67, CA-IX, CXCR4, COX2, CXCL12, HIF1, VEGF, CD44, and RAD51) was investigated. The optimal cutoff values were calculated by receiver operating characteristic curve analysis. Classification and regression tree analysis was performed to investigate the biomarker interaction. Patients presenting either Ki-67 or HIF1 or RAD51 below the cutoff value, or CXCR4 or COX2 above the cutoff value, were more likely to get a pathological complete response. Classification and regression tree analysis identified three groups of patients resulting from the combination of Ki-67 and CXCR4 expression. Patients with high expression of Ki-67 had the lowest chance to get a pathological complete response (18%), as compared to patients with low expression of both Ki-67 and CXCR4 (29%), and patients with low Ki-67 and high CXCR4 expression (70%). Pretreatment Ki-67, CXCR4, COX2, HIF1, and RAD51 in tumor biopsies are associated with pathological complete response after neoadjuvant chemoradiotherapy in locally advanced rectal cancer. A combined evaluation of Ki-67 and CXCR4 would increase their predictive potential. If validated, their optimal cutoff could be used to select patients for a tailored multimodality treatment.

Background Small bowel adenocarcinoma is a relatively rare cancer, often diagnosed in an advanced stage. In localized and resectable disease, surgery alone or in combination with adjuvant chemotherapy is the mainstay of treatment. In the recently published National Comprehensive Cancer Network Clinical Practice guidelines, criteria for selecting patients with stage II small bowel adenocarcinoma to receive adjuvant chemotherapy are provided, and they are mainly extrapolated from studies on colorectal cancer. Patients and Methods In the present study, we aimed to verify whether mismatch repair deficiency phenotype, high-risk pathologic features (including T4, positive resection margins and a low number of lymph nodes harvested), as well as tumor histologic subtype, were associated with cancer-specific survival in 66 stage II non-ampullary small bowel adenocarcinoma patients, collected through the Small Bowel Cancer Italian Consortium. A central histopathology review was performed. Mismatch repair deficiency was tested by immunohistochemistry for MLH1, MSH2, MSH6 and PMS2, and confirmed by polymerase chain reaction for microsatellite instability. Results We identified mismatch repair deficiency, glandular/medullary histologic subtype, and celiac disease as significant predictors of favorable cancer-specific survival using univariable analysis with retained significance in bivariable models adjusted for pT stage. Among the high-risk features, only T4 showed a significant association with an increased risk of death; however, its prognostic value was not independent of mismatch repair status. Conclusions Mismatch repair protein expression, histologic subtype, association with celiac disease, and, in the mismatch repair proficient subset only, T stage, may help identify patients who may benefit from adjuvant chemotherapy. Graphic Abstract

Objective High grade serous ovarian carcinoma (HGSOC) is the most common type of malignant ovarian neoplasm and the main cause of ovarian cancer related deaths worldwide. Although novel biomarkers such as homologous recombination deficiency testing have been implemented into the clinical decision-making algorithm since diagnosis, morphological classification and immunohistochemistry analysis are essential for diagnostic purpose. This study aims at identifying histologic and clinical features that can be predictive of patients' prognosis. Methods Morphological and architectural characterization including SET (Solid-Endometroid-Transitional)/Classic features was carried out in a cohort of 234 patients analyzing 695 slides. From each slide tumor infiltrating lymphocyte (TILs), the presence of necrosis, the number of mitoses, the presence of psammoma bodies, giant cells and atypical mitoses were recorded. Morphological heterogeneity was quantified by the Shannon's diversity index (SDI) considering the percentage of each architectural pattern per patient's slide. Results The frequency of architectural patterns and morphological variables varied with respect of the surgical strategy (primary debulking surgery vs interval surgery after neoadjuvant chemotherapy). HGSOCs exhibiting SET features had a longer overall as well as progression free survival. Among SET features, pseudo-endometrioid and transitional like patterns had the best outcome, while it was heterogenous for solid pattern, that had better outcome for BRCA 1 negative and less heterogeneous tumors. In patients submitted to neoadjuvant chemotherapy a higher intratumor heterogeneity as defined by SDI was a negative independent prognostic factor. Conclusions A comprehensive histological examination considering architectural patterns and their heterogeneity can help in prognostication of HGSOCs.

High-grade serous ovarian cancer (HGSOC) is one of the major life-threatening cancers in women, with a survival rate of less than 50%. So far, chemotherapy is the main therapeutic tool to cure this lethal disease; however, in many cases, it fails to cure HGSOC even with severe side effects. Self-therapeutic nanomaterials could be an effective alternative to chemotherapy, facilitated by their diverse physicochemical properties and the ability to generate reactive species for killing cancer cells. Herein, inorganic cobalt hydroxide nanosheets (Co(OH)2 NS) were synthesized by a simple solution process at room temperature, and morphological, spectroscopic, and crystallographic analyses revealed the formation of Co(OH)2 NS with good crystallinity and purity. The as-prepared Co(OH)2 NS showed excellent potency, comparable to the FDA-approved cisplatin drug to kill ovarian cancer cells. Flow cytometric analysis (nnexin V) revealed increased cellular apoptosis for Co(OH)2 NS than cobalt acetate (the precursor). Tracking experiments demonstrated that Co(OH)2 NS are internalized through the lysosome pathway, although relocalization in the cytoplasm has been observed. Hence, Co(OH)2 NS could be an effective self-therapeutic drug and open up an area for the optimization of self-therapeutic properties of cobalt nanomaterials for cancer treatment.

Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Small bowel adenocarcinomas (SBAs) are often associated with poor prognosis and have limited therapeutic options. Programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway blockade is an effective treatment in many microsatellite instability-high (MSI-H) solid tumors. We aimed at investigating PD-L1 and PD-1 expression in non-hereditary, non-ampullary SBAs, associated with celiac disease (CeD), Crohn’s disease (CrD), or sporadic, recruited through the Small Bowel Cancer Italian Consortium. We assessed PD-L1 and PD-1 by immunohistochemistry in a series of 121 surgically resected SBAs, including 34 CeD-SBAs, 49 CrD-SBAs, and 38 sporadic SBAs. PD-L1 and PD-1 expression was correlated with several clinico-pathological features, such as the etiology, microsatellite instability status, and tumor-infiltrating lymphocyte (TIL) density. The prevalence of PD-L1 positivity according to combined positive score (CPS) was 26% in the whole cohort of SBAs, with significantly (p = 0.001) higher percentage (35%) in both CeD-SBAs and CrD-SBAs in comparison with sporadic SBAs (5%). CPS ≥ 1 SBAs were significantly (p = 0.013) more frequent in MSI-H cases (41%) than in non-MSI-H ones (18%); however, 15 CPS ≥ 1 microsatellite stable SBAs were also identified. CPS ≥ 1 SBAs showed higher TIL and PD-1+ immune cell density, more frequently medullary histotype, as well as a better outcome in comparison with CPS < 1 cases. This study demonstrates an increased proportion of PD-L1+ cases in both CeD-SBAs and CrD-SBAs in comparison with sporadic SBAs. In addition, the identification of a subset of PD-L1+ microsatellite stable SBAs supports the need to ascertain additional biomarkers of response to immune checkpoint inhibitors along with MSI-H.

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK–a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epithelial ovarian cancer (EOC) is an infrequent but highly lethal disease, almost invariably treated with platinum‐based therapies. Improving the response to platinum represents a great challenge, since it could significantly impact on patient survival. Here, we report that silencing or pharmacological inhibition of CDK6 increases EOC cell sensitivity to platinum. We observed that, upon platinum treatment, CDK6 phosphorylated and stabilized the transcription factor FOXO3, eventually inducing ATR transcription. Blockage of this pathway resulted in EOC cell death, due to altered DNA damage response accompanied by increased apoptosis. These observations were recapitulated in EOC cell lines in vitro , in xenografts in vivo , and in primary tumor cells derived from platinum‐treated patients. Consistently, high CDK6 and FOXO3 expression levels in primary EOC predict poor patient survival. Our data suggest that CDK6 represents an actionable target that can be exploited to improve platinum efficacy in EOC patients. As CDK4/6 inhibitors are successfully used in cancer patients, our findings can be immediately transferred to the clinic to improve the outcome of EOC patients.

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C > T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C > T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs., Graphical Abstract Image 1, Highlights • A modest mutator phenotype in MUTYH-defective colorectal cancer • Novel mutational signature of G>T mutations from persisting 8-oxoguanine:A mispair • This mutational signature is present in oncogenes/tumor suppressor associated with colorectal cancer • The mutational signature allows identifying a role for 8-oxoguanine-related mutations in other types of human cancer. Viel et al. identifies by exome-sequencing a mutational signature in colorectal cancer defective in the MUTYH DNA glycosylase, a DNA repair gene preventing 8-oxoguanine-related mutagenesis. A strong sequence dependence of G > T transversions characterizes this mutational fingerprint and unveils the contribution of persistent 8-oxoG:A mismatches in human cancer.

Background and aims An increased risk of small bowel carcinoma [SBC] has been reported in coeliac disease [CD] and Crohn's disease [CrD]. We explored clinico-pathological, molecular, and prognostic features of CD-associated SBC [CD-SBC] and CrD-associated SBC [CrD-SBC] in comparison with sporadic SBC [spo-SBC]. Methods A total of 76 patients undergoing surgical resection for non-familial SBC [26 CD-SBC, 25 CrD-SBC, 25 spo-SBC] were retrospectively enrolled to investigate patients' survival and histological and molecular features including microsatellite instability [MSI] and KRAS/NRAS, BRAF, PIK3CA, TP53, HER2 gene alterations. Results CD-SBC showed a significantly better sex-, age-, and stage-adjusted overall and cancer-specific survival than CrD-SBC, whereas no significant difference was found between spo-SBC and either CD-SBC or CrD-SBC. CD-SBC exhibited a significantly higher rate of MSI and median tumour-infiltrating lymphocytes [TIL] than CrD-SBC and spo-SBC. Among the whole SBC series, both MSI─which was the result of MLH1 promoter methylation in all but one cases─and high TIL density were associated with improved survival at univariable and stage-inclusive multivariable analysis. However, only TILs retained prognostic power when clinical subgroups were added to the multivariable model. KRAS mutation and HER2 amplification were detected in 30% and 7% of cases, respectively, without prognostic implications. Conclusions In comparison with CrD-SBC, CD-SBC patients harbour MSI and high TILs more frequently and show better outcome. This seems mainly due to their higher TIL density, which at multivariable analysis showed an independent prognostic value. MSI/TIL status, KRAS mutations and HER2 amplification might help in stratifying patients for targeted anti-cancer therapy.

Preoperative neoadjuvant chemoradiotherapy (nCRT) is the gold standard in locally advanced rectal cancer, only 10-30% of patients achieving benefits. Currently, there is a need of a reliable selection of markers for the identification of poor or non-responders prior to therapy. In this work, we compared protein profiles before therapy of patients differing in their responses to nCRT to find novel predictive markers of response to therapy. Patients were grouped into 3 groups according to their tumor regression grading (TRG) after surgery: 'TRG 1-2', good responders, 'TRG 3' and 'TRG 4', poor responders. Paired surgical specimens of rectal cancer and healthy (histologically confirmed) rectal tissues from 15 patients were analysed before nCRT by two dimensional difference in gel electrophoresis followed by mass spectrometry. Thirty spots were found as differentially expressed (p < 0.05). Among them, 3 spots (spots 471, 683 and 684) showed an increased amount of protein in poor responders compared with good responders, and they were more tumor associated compared with healthy tissues. Proteins of these spots were identified as fibrinogen beta chain fragment D, actin isoforms, B9 and B5 serpins, cathepsin D isoforms and peroxiredoxin-4. In an independent validation set of 20 rectal carcinomas we validated the increased fibrinogen beta chain abundance before nCRT in poor responders by immunoblotting. In conclusion, we propose a risk-stratification tool in predicting the response to nCRT treatment in rectal cancer based on the quantity of these proteins.

Colorectal cancer (CRC) represents one of the most commonly diagnosed cancers worldwide. It is the second leading cause of cancer death in Western Countries. In the last decade, the survival of patients with metastatic CRC has improved dramatically. Due to the advent of new drugs (irinotecan and oxaliplatin) and target therapies (i.e. bevacizumab, cetuximab, panitumab, aflibercept and regorafenib), the median overall survival has risen from about 12 mo in the mid nineties to 30 mo recently. Molecular studies have recently widened the opportunity for testing new possible markers, but actually, only few markers can be recommended for practical use in clinic. In the next future, the hope is to have a complete panel of clinical biomarkers to use in every setting of CRC disease, and at the same time: 1) to receive information about prognostic significance by their expression and 2) to be oriented in the choice of the adequate treatment. Moreover, molecular analyses have shown that the natural history of all CRCs is not the same. Individual patients with same stage tumors may have different long-term prognosis and response to therapy. In addition, some prognostic variables are likely to be more important than others. Here we review the role of serum and tissue markers according to the recently published English literature. This paper is an extension of the article "Biological and clinical markers in colorectal cancer: state of art" by Cappellani A published in Jan 2010.

Background Inherited epimutations of Mismatch Repair (MMR) genes are responsible for Lynch Syndrome (LS) in a small, but well defined, subset of patients. Methylation of the MSH2 promoter consequent to the deletion of the upstream EPCAM gene is found in about 1%–3% of the LS patients and represents a classical secondary, constitutional and tissue‐specific epimutation. Several different EPCAM deletions have been reported worldwide, for the most part representing private variants caused by an Alu‐mediated recombination. Methods 712 patients with suspected LS were tested for MMR mutation in our Institute. EPCAM deletions were detected by multiplex ligation‐dependent probe amplification (MLPA) and then defined by Long‐Range polymerase chain reaction (PCR)/Sanger sequencing. A comprehensive molecular characterization of colorectal cancer (CRC) tissues was carried out by immunohistochemistry of MMR proteins, Microsatellite Instability (MSI) assay, methylation specific MLPA and transcript analyses. In addition, somatic deletions and/or variants were investigated by MLPA and next generation sequencing (NGS). Results An EPCAM deletion was found in five unrelated probands in Italy: variants c.556‐490_*8438del and c.858+1193_*5826del are novel; c.859‐1430_*2033del and c.859‐670_*530del were previously reported. All probands were affected by CRC at young age; tumors showed MSI and abnormal MSH2/MSH6 proteins expression. MSH2 promoter methylation, as well as aberrant in‐frame or out‐of‐frame EPCAM/MSH2 fusion transcripts, were detected in CRCs and normal mucosae. Conclusion An EPCAM deletion was the causative variant in about 2% of our institutional series of 224 LS patients, consistent with previously estimated frequencies. Early age and multiple CRCs was the main clinical feature of this subset of patients.

PIN1 is a member of a family of peptidylprolyl isomerases that bind phosphoproteins and catalyze the rapid cis-trans isomerization of proline peptidyl bonds, resulting in an alteration of protein structure, function, and stability. PIN1 is overexpressed in human cancers, suggesting it promotes tumorigenesis, but depending on the cellular context, it also acts as a tumor suppressor. Here, we review the role of PIN1 in cancer and the regulation of PIN1 expression, and catalog the single nucleotide polymorphisms, and mutations in PIN1 gene associated with cancer. In addition, we provide a 3D model of the protein to localize the mutated residues.

STARD3 is a cellular protein that represents an attractive target for cancer therapy, being overexpressed in breast cancer and implied in the development of colorectal, gastric, and prostate cancers. Unfortunately, no STARD3 inhibitor has been identified yet. In this work, an in silico strategy was applied to predict a reliable binding mode of cholesterol into STARD3 and to develop a pharmacophore-based virtual screening protocol that allowed the identification of the first STARD3 inhibitor ever reported. The identified compound VS1 binds STARD3 with micromolar affinity (IC 50 = 35 μM) and shows antiproliferative activity in breast (MCF7 and MDA- MB-231) and colon (HCT-116) cancer cell lines in the same concentration range (IC 50 = 49.7-105.5 μM). Although VS1 has a moderate potency, we demonstrated that it specifically targets STARD3 in the cells and induces its degradation. Overall, the results confirm the reliability of the computational strategies herein applied and the identification of the first hit compound for the development of novel potent STARD3 inhibitors. © 2019 American Chemical Society.

Probe based confocal laser endomicroscopy (pCLE) is an advanced technique which provides imaging of gastrointestinal mucosa at subcellular resolution and, importantly, a valid tool for the evaluation of microvasculature during endoscopic examination. In order to assess intratumoral vascularization and the efficiency of blood flow in locally advanced gastric cancer, we examined 57 patients through pCLE imaging. The vascular alterations in gastric cancer were mainly characterized by leakage and by the presence of tortuous and large size vessels. Defects in blood flow were detected very rarely. No association between the angiogenic score and the gastric tumor site or histological type was observed. Interestingly, no correlation was also found with the tumor grading indicating that the vascular angiogenic anomalies in gastric cancer represent an early pathological event to be observed and detected. The majority of patients displayed unchanged vascular alterations following neoadjuvant chemotherapy and this positively correlated with stable or progressive disease, suggesting that an unaltered angiogenic score could per se be indicative of poor therapeutic efficacy. Different vascular parameters were evaluated by immunofluorescence using bioptic samples and the vessel density did not correlate with clinical staging, site or histologic type. Interestingly, only CD105, Multimerin-2 and GLUT1 were able to discriminate normal from tumoral gastric mucosa. Taken together, these findings indicate that functional and structural angiogenic parameters characteristic of tumor blood network were fully detectable by pCLE. Moreover, the evaluation of tumor vasculature by real-time assessment may provide useful information to achieve tailored therapeutic interventions for gastric cancer patients.

The potential role of the probe-based confocal laser endomicroscopy (pCLE) has been analyzed in different pathologic conditions of the gastrointestinal tract. Here, we analyzed a case of extrapulmonary high grade neuroendocrine rectal carcinoma (HGNEC) using, for the first time, the pCLE system. A 72-year old man was diagnosed with an 8 cm diameter rectal HGNEC by standard colonoscopy integrated with the pCLE system. The diagnosis of neuroendocrine carcinoma was confirmed by immunohistochemical analyses. By using the pCLE system, we well defined and resolved vascular structures and mucosal architecture. An altered mucosal pattern and vascular defects, peculiar for HGNEC, were observed at high magnification, allowing the identification of a pattern which was quite different from that observed in poorly differentiated adenocarcinomas (PDA) where tissues appear darker, very irregular, even if glandular structures can still be recognized. This underlines the usefulness of pCLE in discriminating HGNECs from PDAs. In conclusion, pCLE could represent a valid and helpful method for in vivo HGNEC diagnosis, allowing prompt and careful management of the patient.

Aim: To demonstrate that exosomes (exo) could increase the therapeutic index of doxorubicin (DOX). Materials & methods: Exosomes were characterized by nanoparticle tracking analysis and western blot. Tissue toxicity was evaluated by histopathological analysis and drug efficacy by measuring tumor volume. DOX biodistribution was analyzed by MS. Results: Exosomal doxorubicin (exoDOX) avoids heart toxicity by partially limiting the crossing of DOX through the myocardial endothelial cells. For this reason, mice can be treated with higher concentration of exoDOX thus increasing the efficacy of DOX as demonstrated in breast and ovarian mouse tumors. Conclusion: ExoDOX is safer and more effective than free DOX. Importantly, the first spontaneous transformed syngeneic model of high-grade serous ovarian cancer was utilized for providing a new therapeutic opportunity.

Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1−/− mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel ‘ECM’ pharmacological approach to assessing new lymphoedema treatments.

Gastric cancer (GC) is a common malignant neoplasm worldwide and one of the main cause of cancer-related deaths. Despite some advances in therapies, long-term survival of patients with advanced disease remains poor. Different types of classification have been used to stratify patients with GC for shaping prognosis and treatment planning. Based on new knowledge of molecular pathways associated with different aspect of GC, new pathogenetic classifications for GC have been and continue to be proposed. These novel classifications create a new paradigm in the definition of cancer biology and allow the identification of relevant GC genomic subsets by using different techniques such as genomic screenings, functional studies and molecular or epigenetic characterization. An improved prognostic classification for GC is essential for the development of a proper therapy for a proper patient population. The aim of this review is to discuss the state-of-the-art on combining histological and molecular classifications of GC to give an overview of the emerging therapeutic possibilities connected to the latest discoveries regarding GC.

Elevated plasma fibrinogen levels and tumor progression in patients with gastric cancer (GC) have been largely reported. However, distinct fibrinogen chains and domains have different effects on coagulation, inflammation, and angiogenesis. The aim of this study was to characterize fibrinogen beta chain (FGB) in GC tissues. Retrospectively we analyzed the data of matched pairs of normal (N) and malignant tissues (T) of 28 consecutive patients with GC at diagnosis by combining one- and two-dimensional electrophoresis (1DE and 2DE) with immunoblotting and mass spectrometry together with two-dimensional difference in gel electrophoresis (2D-DIGE). 1DE showed bands of the intact FGB at 50 kDa and the cleaved forms containing the fragment D at similar to 37-40 kDa, which corresponded to 19 spots in 2DE. In particular, spot 402 at similar to 50 kDa and spots 526 and 548 at similar to 37 kDa were of interest by showing an increased expression in tumor tissues. A higher content of spot 402 was associated with stomach antrum, while spots 526 and 548 amounts correlated with corpus and high platelet count (>208 x 10(9)/L). The quantification of FGB and cleaved products may help to further characterize the interconnections between GC and platelet/coagulation pathways.

Background Extra-mammary Paget’s disease of the vulva (EMPDV) is an infrequent chronic disease that often recurs. The aim of the study was to assess the presence of neoangiogenesis and the expression of epithelial-mesenchymal transition (EMT) markers in EMPDV, and their potential correlation with stromal invasion. Methods All the women consecutively treated for EMPDV at our Institute, between January 2011 and December 2014, were studied for neoangiogenesis, analysed by microvessel density (MVD) using antibodies against CD31 and CD34. Immunohistochemical expression of E- / N-cadherin, β-catenin and SLUG was also evaluated. In each slide, three fields with the highest number of capillaries and small venules were identified at low power. In these three fields, the highest vessel density (HVD) and the average vessel density (AVD) at 200× and 400× magnification were counted. Immunohistochemical reactions for non-vascular markers were semiquantitatively scored by two pathologists, using a three-tier scale. Results Seventeen cases of EMPDV (including 10 cases of invasive disease) were included. The AVD at 200× and 400× and the HVD at 400× magnification were significantly associated with invasive EMPDV (p = 0.02, 0.03, 0.03 respectively). No significant correlation between MVD, EMT-markers expression and risk of recurrence was detected. Conclusion These results indicate that MVD, as a measure of neoangiogenesis, may be associated with histological progression of EMPDV. EMT could also be linked to an invasive potential of EMPDV but larger series are required to confirm this hypothesis.

The effectiveness of new personalized treatment procedures in oncology is based on the fact that certain tumors exhibit specific molecular features. More in detail, neoplastic tissues of patients should display a specific biomarker, most often a specific genetic alteration and/or under/overexpression of a definite protein, that could be the target of its respective drug. Immunohistochemical and molecular analyses, which usually include examination of nucleic acids from either tissues or fluids, are common tests to define the status of a tumor. This review focuses on the pathologist’s role in carefully controlling pre- analytic procedures and standard operating procedures that are a crucial prerequisite to reach reliable and reproducible results. Six paradigmatic applications of targeted therapy, for which pathological diagnosis plays a fundamental role, are summarized. Traditional and next-generation sequencing are also addressed from the pathologist's perspective as well as the importance pathologists have in this shift to more accurate definition of disease risk and prognostication of therapy response in the personalized medicine era.

Lynch syndrome (LS) and familial adenomatous polyposis (FAP) are autosomal dominant hereditary diseases caused by germline mutations leading to the development of colorectal cancer. Moreover, these mutations result in the development of a spectrum of different tumors, including gastric cancers (GCs). Since the clinical characteristics of GCs associated with LS and FAP are not well known, we investigated clinical and molecular features of GCs occurring in patients with LS and FAP attending our Institution. The Hereditary Tumor Registry was established in 1994 at the Department of Oncologic Gastroenterology, CRO Aviano National Cancer Institute, Italy. It includes 139 patients with LS and 86 patients with FAP. Patients were recruited locally for prospective surveillance. Out of 139 LS patients, 4 developed GC&mdash, 3 in the presence of helicobacter pylori infection and 1 on the background of autoimmune diseases. All GCs displayed a high microsatellite instability (MSI-H) and loss of related mismatch repair (MMR) protein. One of the FAP patients developed a flat adenoma, displaying low-grade dysplasia at the gastric body, and another poorly differentiated adenocarcinoma with signet ring cells like Krukenberg without HP infection. LS carriers displayed a risk of GC. The recognition of HP infection and autoimmune diseases would indicate those at higher risk for an endoscopic surveillance. Regarding FAP, the data suggested the need of suitable endoscopic surveillance in long survivals with diffuse fundic gland polyps.

AimThe aim of this study was to evaluate if the lymph node count from inguinofemoral lymphadenectomy impacted the risk of isolated groin recurrence in patients with node-negative squamous cell vulvar cancer.Materials and MethodsThis is a retrospective cohort study of women with squamous cell vulvar cancer (stage IB–II according to the 2009 Revised International Federation of Gynecology and Obstetrics staging system) who underwent primary radical vulvar surgery and groin lymphadenectomy between January 2005 and December 2014. Patients' sociodemographic characteristics, the disease characteristics, the number of nodes removed from each groin, and the oncologic outcome were evaluated. A cutoff value of at least 6 nodes removed from each groin was used to define the adequacy of inguinofemoral dissection.ResultsSeventy-six patients, fulfilling the study inclusion criteria, were considered. The mean number of nodes removed (bilaterally) was 14.5 (±5.3, SD), with a range of 2 to 29 nodes. Thirty-three women (43.4%) had less than 6 nodes removed from each groin. In the whole study cohort, 4 cases of isolated groin recurrence (5.3%) were detected, and all these recurrences developed in patients with less than 6 nodes removed. Considering the demographic, clinical, and histopathological characteristics potentially related to the risk of groin recurrence, only the number of nodes removed showed a significant correlation.ConclusionsWomen treated for vulvar cancer in which less than 6 nodes are removed from each groin are at higher risk of groin recurrence.

Somatic mutations in TP53 are a hallmark of high-grade serous ovarian cancer (HGSOC), although their prognostic and predictive value as markers is not well defined. Next-generation sequencing (NGS) can identify novel mutations with high sensitivity, that may be repurposed as potential druggable anti-cancer targets and aid in therapeutic decisions. Here, a commercial NGS cancer panel comprising 26 genes, including TP53, was used to identify new genetic markers of platinum resistance and patient prognosis in a retrospective set of patients diagnosed with epithelial ovarian cancer. Six novel TP53 somatic mutations in untreated tumors from six distinct patients diagnosed with HGSOC were identified: TP53 c.728_739delTGGGCGGCATGA (p.Met243_Met247del, in-frame insertion or deletion (INDEL); TP53 c.795_809delGGGACGGAACAGCTT (p.Gly266_Phe270del, in-frame INDEL); TP53 c.826_827GC>AT (p.Ala276Ile, missense); TP53 c.1022insT (p.Arg342Profs*5, frameshift INDEL); TP53 c.1180delT (p.Ter394Aspfs*28, frameshift INDEL); and TP53 c.573insT (p.Gln192Serfs*17, frameshift INDEL). Novel TP53 variants were validated by classical sequencing methods and their impact on protein expression in tumors explored by immunohistochemistry. Further insights into the potential functional effect of the mutations were obtained by different in silico approaches, bioinformatics tools, and structural modeling. This discovery of previously unreported TP53 somatic mutations provides an opportunity to translate NGS technology into personalized medicine and identify new potential targets for therapeutic applications.

Over the last decade, our understanding of the mechanisms underlying immune modulation has greatly improved, allowing for the development of multiple therapeutic approaches that are revolutionizing the treatment of cancer. Immunotherapy for gastric cancer (GC) is still in the early phases but is rapidly evolving. Recently, multi-platform molecular analyses of GC have proposed a new classification of this heterogeneous group of tumors, highlighting subset-specific features that may more reliably inform therapeutic choices, including the use of new immunotherapeutic drugs. The clinical benefit and improved survival observed in GC patients treated with immunotherapeutic strategies and their combination with conventional therapies highlighted the importance of the immune environment surrounding the tumor. A thorough investigation of the tumor microenvironment and the complex and dynamic interaction between immune cells and tumor cells is a fundamental requirement for the rational design of novel and more effective immunotherapeutic approaches. This review summarizes the pre-clinical and clinical results obtained so far with immunomodulatory and immunotherapeutic treatments for GC and discusses the novel combination strategies that are being investigated to improve the personalization and efficacy of GC immunotherapy.

Gastrointestinal (GI) tumors are among the leading cause of death in cancer patients worldwide. Particularly, gastric cancer (GC) is the third cause of cancer deaths, whereas esophageal neoplasm is the eighth leading most common cancer worldwide and its incidence, especially adenocarcinoma type, is continuously increasing. Also, Hepatocellular carcinoma, Cholangiocarcinoma and pancreatic cancer represent a very interesting model to multidisciplinary approach and recently new drugs are used in their treatment. Currently, new clinical trials are designed including classic chemotherapy in association with either small molecule inhibitors (i.e. Tyrosine Kinase inhibitors) and/or monoclonal antibody (i.e. anti-EGFR antibody). Moreover, a comprehensive list of new molecules for target therapy is included in this issue. The development of new treatment modalities (multidisciplinary approach) and targeted therapy approaches have contributed to improving the outcome in these cancer diseases. During the past few years, remarkable progress in molecular biology of malignancy, the discovery of specific targets, and the resulting development of systemic drugs that block critical kinases and several molecular pathways have all contributed to progress in cancer treatment, also in GI non-colorectal cancer treatment.

Germline mutations in CDH1, the gene coding for the E-cadherin adhesion protein, are known to cause hereditary diffuse gastric cancer. We identified a new truncating germline mutation (p.Asp538Thrfs*19) in exon 11 of the CDH1 gene in a 41-year-old male with a diffuse gastric cancer. Although he had no parental history of gastric cancer, the co-segregation study in the family detected the same mutation in his healthy 31-year-old brother. The mutation affects one of the extracellular repeat (CAD repeats) domains which is essential for the homophilic binding specificity that directs “E-cadherin” to bind with itself each others. In this case, immunohistochemical analysis showed no expression of E-cadherin in the tumor sample and was a useful prescreening tool to genetic testing. This finding was associated with a poor response to trastuzumab-based treatment.

Colorectal polyps are small clumps of cells and they are classified on the basis of their histological characteristics. Until recently, they have been divided into two major groups: neoplastic and non-neoplastic mucosal polyps. Adenomatous polyps contain epithelial neoplasia and their size and histology correlate with the risk of progression to carcinoma. Non-neoplastic polyps, instead, can be divided into several distinct and unrelated categories including hyperplastic, mucosal, juvenile, Peutz-Jeghers, and inflammatory. Adenomas are recognized as the precursor lesions for colorectal carcinoma and recently also some hyperplastic lesions, with serrated morphology, have been reported to display a significant risk of neoplastic progression through the so-called serrated pathway. The sequence adenoma-carcinoma is caused by different molecular pathways. The most frequent are those of chromosomal instability pathway; the microsatellite instability pathway; the serrated pathway. Molecular complexity can explain the morphological heterogeneity and the timing of neoplastic progression.

Pin1, a prolyl isomerase that sustains tumor progression, is overexpressed in different types of malignancies. Functional inactivation of Pin1 restrains tumor growth and leaves normal cells unaffected making it an ideal pharmaceutical target. Although many studies on Pin1 have focused on malignancies that are influenced by sex hormones, studies in ovarian cancer have lagged behind. Here, we show that Pin1 is an important therapeutic target in high-grade serous epithelial ovarian cancer. Knock down of Pin1 in ovarian cancer cell lines induces apoptosis and restrains tumor growth in a syngeneic mouse model. Since specific and non-covalent Pin1 inhibitors are still limited, the first liposomal formulation of a Pin1 inhibitor was designed. The drug was efficiently encapsulated in modified cyclodextrins and remotely loaded into pegylated liposomes. This liposomal formulation accumulates preferentially in the tumor and has a desirable pharmacokinetic profile. The liposomal inhibitor was able to alter Pin1 cancer driving-pathways trough the induction of proteasome-dependent degradation of Pin1 and was found to be effective in curbing ovarian tumor growth in vivo.

Background Endometrial stromal sarcomas arising in extrauterine and extraovarian sites, in the absence of a primary uterine lesion are quite rare, especially in the absence of endometriosis. They usually present as an abdominal or pelvic mass lesion. Case presentation In 2007, a 45-year-old woman underwent total hysterectomy for in situ squamous cell carcinoma of the cervix. In 2014, an upper left pulmonary lobectomy was performed for a mass, which was provisionally diagnosed as primary carcinosarcoma of the lung. A second histological revision of the lung surgical specimen was performed in the Pathology Unit of our Institute. After extensive immunohistochemical analyses, the preferred diagnosis was spindle-cell sarcoma, consistent with high-grade extragenital endometrial stromal sarcoma (EESS). A review of all slides of the hysterectomy specimen confirms the original diagnosis: no evidence of stromal tumor was found. Afterwards, the patient developed multiple and metachronous pulmonary lesions and a scapular soft tissue mass, which showed the same morphophenotypic features of the first lung mass. The patient was treated with antiblastic therapy, surgical resection and radioablation, when appropriate. To date, the patient has no signs or symptoms. Conclusions The authors present the first case of primary EESS arising in the lung with no association with endometriosis published to date. Detailed clinical history and follow-up are also described. Moreover, extensive literature review is reported, along with differential diagnoses, immunohistochemical and molecular findings, pathogenetic hypotheses and treatment options. The knowledge of EESS potential extrauterine location and of its peculiar morphophenotypic aspects are required for a correct diagnosis, and for choosing the most suitable treatment.

Background Helicobacter pylori (H. pylori) represents a key factor in the etiology of autoimmune atrophic gastritis (AAG), duodenal ulcer (DU) and gastric cancer (GC). The aim of this study was to characterize the differential protein expression of H. pylori isolated from gastric biopsies of patients affected by either AAG, DU or GC. Methods The H. pylori strains were isolated from endoscopic biopsies from the stomach of patients with gastric disease. Protein profiles of H. pylori were compared by two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) for the identification of significantly different spots (Student t-test, p 1.5). These spots corresponded to 35 unique proteins. The identity of 7 protein spots was validated after one-dimensional electrophoresis and MS/MS analyses of excised gel portions. In H. pylori isolated from DU-patients a significant increase in proteins with antioxidant activity emerged (AroQ, AspA, FldA, Icd, OorA and ScoB), together with a higher content of proteins counteracting the high acid environment (KatA and NapA). In H. pylori isolated from AAG-patients proteins neutralizing hydrogen concentrations through organic substance metabolic processes decreased (GroL, TrxB and Tuf). In addition, a reduction of bacterial motility (FlhA) was found to be associated with AAG-H. pylori isolates. In GC-H. pylori strains it was found an increase in nucleic acid-binding proteins (e.g. DnaG, Tuf, RpoA, RplU) which may be involved in a higher demand of DNA- and protein-related processes. Conclusion Our data suggest the presence of specific protein signatures discriminating among H. pylori isolated from either AAG, DU or GC. Changes in protein expression profiles evaluated by DIGE succeeded in deciphering part of the molecular scenarios associated with the different H. pylori-related gastric diseases.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Probe-based Confocal Laser Endomicroscopy (pCLE) is a powerful imaging technique that allows to perform gastrointestinal endomicroscopy at subcellular resolution. The aim of this study was to assess the use of pCLE to evaluate tumor angiogenesis in rectal and gastric cancers. A total of 35 consecutive patients with gastric and 91 with rectal carcinomas underwent endoscopy and pCLE during the same examination. Vascular assessment was based on vessel shape and size, vessel permeability and blood flow, and allowed the creation of an angiogenic score ranging from 0, for normal vasculature, to 4, for aberrant vasculature. A significant difference for the presence of vessels with large diameter and defective blood flow was found between rectal and gastric cancers. Overall, rectal cancers displayed a higher angiogenic score compared to gastric cancers. Conventional therapy induced a striking reduction in the angiogenic score only in rectal cancer patients. Taken together, our findings suggest that the pCLE technology is suitable for the evaluation of the tumor microvasculature abnormalities. Therefore, the real-time assessment of the vasculature status may represent a promising approach to predict the efficacy of the treatments and improve the clinical management of patients with gastric or rectal carcinomas.

Background: Germline mutations of the oncosuppressor gene breast cancer 1-associated protein 1 (BAP1) were recently related to an autosomal-dominant tumor predisposition syndrome (BAP1-TPDS), characterized by uveal melanoma, malignant mesothelioma (MM), cutaneous melanoma, and other malignancies. The demonstration that BAP1 mutations are strongly associated with MM has provided a real breakthrough in the study of genetic predisposition in MM, that may explain why only a fraction of asbestos-exposed individuals go on to develop MM. Materials and Methods: To evaluate the possible role of BAP1 mutations in the epidemiology of sporadic MM, and their relationship with asbestos exposure, we determined the prevalence of germline BAP1 mutations by the Sanger method in a group of 29 asbestos-exposed patients, 21 of which were diagnosed with MM. They were residents of Trieste, a ship-building town in Northeast Italy with a very high incidence of mesothelioma. Results: We identified non-obviously pathogenetic germline sequence variants of BAP1 in 3/29 patients and in 2/21 MM cases (10%). Conclusion: Non obviously pathogenic germline sequence variants of BAP1 were found. Nevertheless, limitations of predictive web tools allowed us to comment on some interesting peculiarities of our findings.

Angiogenesis is a crucial process occurring under physiological and pathological conditions, including cancer. The development of blood vessels is tightly regulated by a plethora of cytokines, endothelial cell (EC) receptors and extracellular matrix (ECM) components. In this context, we have shown that Multimerin 2 (MMRN2), an ECM molecule specifically secreted by ECs, exerts angiostatic functions by binding VEGFA and other pro-angiogenic cytokines. Here, we demonstrate that during angiogenic stimuli MMRN2 mRNA levels significantly decrease. Furthermore, we provide evidence that MMRN2 is processed by matrix metalloproteinases (MMPs) including MMP-9 and, to a lesser degree, by MMP-2. This proteolytic cleavage correlates with an increased migration of ECs. Accordingly, MMRN2 down-regulation is associated with an increased number of EC pseudopodia at the migrating front and this effect is attenuated using specific MMP-9 inhibitors. The down-modulation of MMRN2 occurs also in the context of tumor-associated angiogenesis. Immunofluorescence performed on tumor sections indicate a broad co-localization of MMP-9 and MMRN2, suggesting that the molecule may be extensively remodeled during tumor angiogenesis. Given the altered expression in tumors and the key role of MMRN2 in blood vessel function, we postulate that analyses of its expression may serve as a marker to predict the efficacy of the treatments. In conclusion, these data further support the role of MMRN2 as a key molecule regulating EC function and sprouting angiogenesis. (C) 2017 Published by Elsevier B.V.

The CDH1 gene, coding for the E-cadherin protein, is linked to gastric cancer (GC) susceptibility and tumor invasion. The human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in a portion of GC. HER2 is an established therapeutic target in metastatic GC (mGC). Trastuzumab, in combination with various chemotherapeutic agents, is a standard treatment for these tumors leading to outcome improvement. Unfortunately, the survival benefit is limited to a fraction of patients. The aim of this study was to improve knowledge of the HER2 and the E-cadherin alterations in the context of GC to characterize subtypes of patients that could better benefit from targeted therapy. An association between the P7-CDH1 haplotype, including two polymorphisms (rs16260A-rs1801552T) and a subset of HER2-positive mGC with better prognosis was observed. Results indicated the potential evaluation of CDH1 haplotypes in mGC to stratify patients that will benefit from trastuzumab-based treatments. Moreover, data may have implications to understanding the HER2 and the E-cadherin interactions in vivo and in response to treatments.

Nanomedicine requires intelligent and non-toxic nanomaterials for real clinical applications. Carbon materials possess interesting properties but with some limitations due to toxic effects. Interest in carbon nanoparticles (CNPs) is increasing because they are considered green materials with tunable optical properties, overcoming the problem of toxicity associated with quantum dots or nanocrystals, and can be utilized as smart drug delivery systems. Using black tea as a raw material, we synthesized CNPs with a narrow size distribution, tunable optical properties covering visible to deep red absorption, non-toxicity and easy synthesis for large-scale production. We utilized these CNPs to label subcellular structures such as exosomes. More importantly, these new CNPs can escape lysosomal sequestration and rapidly distribute themselves in the cytoplasm to release doxorubicin (doxo) with better efficacy than the free drug. The release of doxo from CNPs was optimal at low pH, similar to the tumour microenvironment. These CNPs were non-toxic in mice and reduced the tumour burden when loaded with doxo due to an improved pharmacokinetics profile. In summary, we created a new delivery system that is potentially useful for improving cancer treatments and opening a new window for tagging microvesicles utilized in liquid biopsies.

// Massimiliano Berretta 1 , Carla Cavaliere 2 , Lara Alessandrini 3,* , Brigida Stanzione 1,* , Gaetano Facchini 4 , Luca Balestreri 5 , Tiziana Perin 3 and Vincenzo Canzonieri 3 1 Department of Medical Oncology, National Cancer Institute, Aviano (PN), Italy 2 Department of Onco-Ematology Medical Oncology, S.G. Moscati Hospital of Taranto Taranto, Italy 3 Division of Pathology, National Cancer Institute, Aviano (PN), Italy 4 Department of Medical Oncology, National Cancer Institute, “G. Pascale” Foundation, Naples, Italy 5 Department of Radiology, National Cancer Institute, Aviano (PN), Italy * These authors have contributed equally to this work Correspondence to: Massimiliano Berretta, email: // Keywords : hepatocellular carcinoma, cholangiocarcinoma, serum markers, tissue markers, prognosis, metastasis Received : September 16, 2016 Accepted : October 28, 2016 Published : December 14, 2016 Abstract HCC represents the sixth most common cancer worldwide and the second leading cause of cancer-related death. Despite the high incidence, treatment options for advanced HCC remain limited and unsuccessful, resulting in a poor prognosis. Despite the major advances achieved in the diagnostic management of HCC, only one third of the newly diagnosed patients are presently eligible for curative treatments. Advances in technology and an increased understanding of HCC biology have led to the discovery of novel biomarkers. Improving our knowledge about serum and tissutal markers could ultimately lead to an early diagnosis and better and early treatment strategies for this deadly disease. Serum biomarkers are striking potential tools for surveillance and early diagnosis of HCC thanks to the non-invasive, objective, and reproducible assessments they potentially enable. To date, many biomarkers have been proposed in the diagnosis of HCC. Cholangiocarcinoma (CCA) is an aggressive malignancy, characterized by early lymph node involvement and distant metastasis, with 5-year survival rates of 5%-10%. The identification of new biomarkers with diagnostic, prognostic or predictive value is especially important as resection (by surgery or combined with a liver transplant) has shown promising results and novel therapies are emerging. However, the relatively low incidence of CCA, high frequency of co-existing cholestasis or cholangitis (primary sclerosing cholangitis –PSC- above all), and difficulties with obtaining adequate samples, despite advances in sampling techniques and in endoscopic visualization of the bile ducts, have complicated the search for accurate biomarkers. In this review, we attempt to analyze the existing literature on this argument.

// Erika Maria Parasido 1,2 , Alessandra Silvestri 1 , Vincenzo Canzonieri 3 , Claudio Belluco 4 , Maria Grazia Diodoro 5 , Massimo Milione 6 , Flavia Melotti 6 , Ruggero De Maria 7 , Lance Liotta 1 , Emanuel F. Petricoin 1,* and Mariaelena Pierobon 1,* 1 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA 2 Department of Experimental Oncology, CRO-National Cancer Institute, Aviano, Italy 3 Department of Pathology, CRO-National Cancer Institute, Aviano, Italy 4 Department of Surgical Oncology, CRO-National Cancer Institute, Aviano, Italy 5 Department of Pathology, Istituto Nazionale Tumori Regina Elena, Roma, Italy 6 Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy 7 Department of Pathology, Sacred Heart Catholic University of Rome, Roma, Italy * These authors have contributed equally to this work Correspondence to: Mariaelena Pierobon, email: // Keywords : intra-tumor heterogeneity, personalized therapy, kinase signaling, laser capture microdissection, reverse phase protein microarray Received : August 16, 2016 Accepted : December 09, 2016 Published : December 15, 2016 Abstract Introduction: Recent studies indicated tumors may be comprised of heterogeneous molecular subtypes and incongruent molecular portraits may emerge if different areas of the tumor are sampled. This study explored the impact of intra-tumoral heterogeneity in terms of activation/phosphorylation of FDA approved drug targets and downstream kinase substrates. Material and methods: Two independent sets of liver metastases from colorectal cancer were used to evaluate protein kinase-driven signaling networks within different areas using laser capture microdissection and reverse phase protein array. Results: Unsupervised hierarchical clustering analysis indicated that the signaling architecture and activation of the MAPK and AKT-mTOR pathways were consistently maintained within different regions of the same biopsy. Intra-patient variability of the MAPK and AKT-mTOR pathway were

Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III&ndash, IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD &lt, 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor.

Helicobacter pylori (H. pylori) represents an independent risk factor for Gastric Cancer (GC). First Degree Relatives (FDR) of GC subjects and Autoimmune Gastritis (AG) patients are both at increased risk for GC. H. pylori genetic heterogeneity within the gastric niche of FDR and AG individuals has been little explored. To understand whether they exploit an increased H. pylori stability and virulence, 14 AG, 25 FDR, 39 GC and 13 dyspeptic patients (D) were investigated by a cultural PCR-based approach characterizing single colonies-forming-units. We chose three loci within the Cytotoxin-associated gene-A Pathogenicity Island (CagPAI) (cagA,cagE,virB11), vacA, homA and homB as markers of virulence with reported association to GC. Inflammatory/precancerous lesions were staged according to Sydney System. When compared to D, FDR, similarly to GC patients, were associated to higher atrophy (OR = 6.29, 95% CI):1.23&ndash, 31.96 in FDR, OR = 7.50, 95% CI:1.67&ndash, 33.72 in GC) and a lower frequency of mixed infections (OR = 0.16, 95% CI:0.03&ndash, 0.81 in FDR, OR = 0.10, 95% CI:0.02&ndash, 0.48 in GC). FDR presented also an increased neutrophil infiltration (OR = 7.19, 95% CI:1.16&ndash, 44.65). Both FDR and GC carried a higher proportion of CagPAI+vacAs1i1mx+homB+ profiles (OR = 2.71, 95% CI: 1.66&ndash, 4.41 and OR = 3.43, 95% CI: 2.16&ndash, 5.44, respectively). Conversely, AG patients presented a lower frequency of subtypes carrying a stable CagPAI and vacAs1i1mx. These results underline different H. pylori plasticity in FDR and AG individuals, and thus, a different host-bacterium interaction capacity that should be considered in the context of eradication therapies.

Background Risk of relapse or progression remains high in the treatment of most patients with epithelial ovarian cancer, and development of a molecular predictor could be a valuable tool for stratification of patients by risk. We aimed to develop a microRNA (miRNA)-based molecular classifier that can predict risk of progression or relapse in patients with epithelial ovarian cancer.Methods We analysed miRNA expression profiles in three cohorts of samples collected at diagnosis. We used 179 samples from a Multicenter Italian Trial in Ovarian cancer trial (cohort OC179) to develop the model and 263 samples from two cancer centres (cohort OC263) and 452 samples from The Cancer Genome Atlas epithelial ovarian cancer series (cohort OC452) to validate the model. The primary clinical endpoint was progression-free survival, and we adapted a semi-supervised prediction method to the miRNA expression profile of OC179 to identify miRNAs that predict risk of progression. We assessed the independent prognostic role of the model using multivariable analysis with a Cox regression model.Findings We identified 35 miRNAs that predicted risk of progression or relapse and used them to create a prognostic model, the 35-miRNA-based predictor of Risk of Ovarian Cancer Relapse or progression (MiROvaR). MiROvaR was able to classify patients in OC179 into a high-risk group (89 patients; median progression-free survival 18 months [95% CI 15-22]) and a low-risk group (90 patients; median progression-free survival 38 months [24-not estimable]; hazard ratio [HR] 1.85 [1.29-2.64], p=0.00082). MiROvaR was a significant predictor of progression in the two validation sets (OC263 HR 3.16, 95% CI 2.33-4.29, p

// Eva Dreussi 1 , Salvatore Pucciarelli 2 , Antonino De Paoli 3 , Jerry Polesel 4 , Vincenzo Canzonieri 5 , Marco Agostini 2,6,7 , Maria Luisa Friso 8 , Claudio Belluco 9 , Angela Buonadonna 10 , Sara Lonardi 11 , Chiara Zanusso 1 , Elena De Mattia 1 , Giuseppe Toffoli 1 and Erika Cecchin 1 1 Experimental and Clinical Pharmacology, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy 2 Department of Surgical, Oncological and Gastroenterological Sciences, Section of Surgery, University of Padova, Padua, Italy 3 Radiation Oncology, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy 4 Epidemiology and Biostatistics, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy 5 Pathology, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy 6 Nano Inspired Biomedicine Laboratory, Istituto di Ricerca Pediatrica, Citta della Speranza, Padua, Italy 7 Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, Texas, USA 8 Radiation Oncology, Istituto Oncologico Veneto, IRCCS, Padova, Italy 9 Surgical Oncology, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy 10 Medical Oncology B, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy 11 Medical Oncology 1, Istituto Oncologico Veneto, IRCCS, Padova, Italy Correspondence to: Giuseppe Toffoli, email: // Antonino De Paoli, email: // Keywords : microRNA, polymorphisms, rectal cancer, neoadjuvant therapy Received : November 13, 2015 Accepted : February 16, 2016 Published : February 26, 2016 Abstract In rectal cancer, a pathologic complete response (pCR) to pre-operative treatment is a favourable prognostic marker, but is reported in a minority of the patients. We aimed at identifying microRNA-related host genetic polymorphisms predictive of pCR. A panel of 114 microRNA-related tagging polymorphisms was selected and analyzed on 265 locally advanced rectal cancer patients treated with neoadjuvant chemo-radiotherapy. Patients were stratified in two subgroups according to the radiotherapy dose (50.4Gy for 202 patients, 55.0Gy for 78 patients). Interactions among genetic and clinical-pathological variants were investigated by recursive partitioning analysis. Only polymorphisms with a consistent significant effect in the two subgroups of patients were selected as predictive markers of pCR. The results were validated by bootstrap analysis. SMAD3 -rs744910, SMAD3 -rs745103, and TRBP -rs6088619 were associated to an increased chance of pCR (p=0.0153, p=0.0471, p=0.0125). DROSHA -rs10719 and SMAD3 -rs17228212 had an opposite detrimental effect on pathological tumour response (p=0.0274, p=0.0049). Recursive partitioning analysis highlighted that a longer interval time between the end of radiotherapy and surgery increases the chance of pCR in patients with a specific combination of SMAD3 -rs744910 and TRBP -rs6088619 genotypes. This study demonstrated that microRNA-related host genetic polymorphisms can predict pCR to neo-adjuvant chemo-radiotherapy, and could be used to personalize the interval time between the end of radiotherapy and surgery.

Objective The aim of this study was to analyze women treated with excisional procedures for vaginal high-grade squamous intraepithelial lesions (HSILs). The histopathological upgrading of the lesions previously detected on vaginal biopsy and the presence of occult invasive vaginal cancer in the specimens excised were investigated, to identify a higher risk subset of women.Materials and Methods A retrospective analysis of the medical records of 86 women with a biopsy histopathologic diagnosis of vaginal HSIL (vaginal intraepithelial neoplasias [VaINs]: VaIN2 and VaIN3) and subsequent excisional therapy, consecutively referred to the Aviano National Cancer Institute (Aviano, Italy) from January 1991 to April 2014, was performed.Results Of the 86 patients, 4 cases (4.6%) of occult vaginal cancer were detected, all of them in women previously diagnosed with VaIN3 on biopsy (4/39 cases, 10.3%). Women with diagnosis of VaIN2 on biopsy showed an upgrading of lesions, with diagnosis of VaIN3 on the final specimen in 5 (10.6%) of 47 cases, with no cases of VAIN2 upgraded to invasive cancer. In 33.3% of the women initially diagnosed with VaIN2 and with previous hysterectomy for human papillomavirus-related disease, a final histopathological upgrading of lesions emerged. Furthermore, tobacco use was significantly related to the histopathological upgrading of lesions previously detected on vaginal biopsy.Conclusions Women diagnosed with VaIN3 should be treated with excisional procedures as first-line surgical approach, given the risk of occult invasive disease in 10% of the cases. Women diagnosed with VaIN2 and with previous hysterectomy for human papillomavirus-related cervical diseases should always be carefully evaluated and possibly excised, given the higher risk of histopathological upgrading of lesions and thus the potential risk of occult vaginal cancer. Tobacco users should be considered as high-risk group.

// Francesco Perrone 1,* , Gustavo Baldassarre 2,* , Stefano Indraccolo 3,* , Simona Signoriello 4 , Gennaro Chiappetta 1 , Franca Esposito 5 , Gabriella Ferrandina 6 , Renato Franco 1,15 , Delia Mezzanzanica 7 , Maura Sonego 2 , Elisabetta Zulato 3 , Gian F. Zannoni 6 , Vincenzo Canzonieri 2 , Giovanni Scambia 6 , Roberto Sorio 2 , Antonella Savarese 8 , Enrico Breda 9 , Paolo Scollo 10 , Antonella Ferro 11 , Stefano Tamberi 12 , Antonio Febbraro 13 , Donato Natale 14 , Massimo Di Maio 1,16 , Daniela Califano 1 , Giosue Scognamiglio 1 , Domenica Lorusso 7 , Silvana Canevari 7 , Simona Losito 1 , Ciro Gallo 4,** and Sandro Pignata 1,** 1 Istituto Nazionale per lo Studio e la Cura dei Tumori - Fondazione G.Pascale, IRCCS, Napoli, Italy 2 Centro di Riferimento Oncologico, IRCCS, Aviano (PN), Italy 3 Istituto Oncologico Veneto, IRCCS, Padova, Italy 4 Dipartimento di Salute Mentale, Fisica e Medicina Preventiva, Statistica Medica, Seconda Universita, Napoli, Italy 5 Universita di Napoli Federico II, Napoli, Italy 6 Catholic University, Roma, Italy 7 Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 8 Istituto Nazionale Tumori Regina Elena, IRCCS, Roma, Italy 9 Ospedale S. Giovanni Calibita Fatebenefratelli, Roma, Italy 10 Ospedale Cannizzaro, Catania, Italy 11 Ospedale S. Chiara, Trento, Italy 12 Ospedale Civile, Faenza, Italy 13 Ospedale Fatebenefratelli, Benevento, Italy 14 Ospedale S. Massimo, Penne (PE), Italy 15 Dipartimento di Salute mentale, Fisica e Medicina Preventiva, Anatomia Patologica, Seconda Universita, Napoli Italy 16 Universita degli Studi, Torino, Italy * co-first author ** co-last author Correspondence to: Sandro Pignata, email: // Keywords : ovarian cancer, phase 3 clinical trial, predictive factors, pACC, DNA-PK Received : June 11, 2016 Accepted : August 03, 2016 Published : September 15, 2016 Abstract Background: No biomarker is available to predict prognosis of patients with advanced ovarian cancer (AOC) and guide the choice of chemotherapy. We performed a prospective-retrospective biomarker study within the MITO2 trial on the treatment of AOC. Patients and methods: MITO2 is a randomised multicentre phase 3 trial conducted with 820 AOC patients assigned carboplatin/paclitaxel (carboplatin: AUC5, paclitaxel: 175 mg/m², every 3 weeks for 6 cycles) or carboplatin/PLD-pegylated liposomal doxorubicin (carboplatin: AUC5, PLD: 30 mg/m², every 3 weeks for 6 cycles) as first line treatment. Sixteen biomarkers (pathways of adhesion/invasion, apoptosis, transcription regulation, metabolism, and DNA repair) were studied in 229 patients, in a tissue microarray. Progression-free and overall survival were analysed with multivariable Cox model. Results: After 72 months median follow-up, 594 progressions and 426 deaths were reported; there was no significant difference between the two arms in the whole trial. No biomarker had significant prognostic value. Statistically significant interactions with treatment were found for DNA-dependent protein kinase (DNA-PK) and phosphorylated acetyl-coenzymeA carboxylase (pACC), both predicting worse outcome for patients receiving carboplatin/paclitaxel. Conclusion: These data show that in presence of DNA-PK or pACC overexpression, carboplatin/paclitaxel might be less effective than carboplatin/PLD as first line treatment of ovarian cancer patients. Further validation of these findings is warranted.