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In preclinical experiments, cyclic fasting-mimicking diets (FMDs) showed broad anticancer effects in combination with chemotherapy. Among different tumor types, triple-negative breast cancer (TNBC) is exquisitely sensitive to FMD. However, the antitumor activity and efficacy of cyclic FMD in TNBC patients remain unclear. Here, we show that a severely calorie-restricted, triweekly, 5-day FMD regimen results in excellent pathologic complete response (pCR) rates (primary endpoint) and long-term clinical outcomes (secondary endpoints) when combined with preoperative chemotherapy in 30 patients with early-stage TNBC enrolled in the phase 2 trial BREAKFAST. Bulk and single-cell RNA sequencing analysis revealed that highly glycolytic cancer cells, myeloid cells, and pericytes from tumors achieving pCR undergo a significant, early downmodulation of pathways related to glycolysis and pyruvate metabolism. Our findings pave the wave for conducting larger clinical trials to investigate the efficacy of cyclic FMD in early-stage TNBC patients and to validate early changes of intratumor glycolysis as a predictor of clinical benefit from nutrient restriction. This study was registered at Clinicaltrials.gov (NCT04248998)., Competing Interests: Declaration of interests The authors declare the following competing interests: G.V.B., speaker/advisory board: Novartis, Eli Lilly, Daichi/Astrazeneca, Roche, MSD, and Seagen; G.P., consulting fees: Roche, Bayer, and Astra Zeneca; C.V., consulting fees/advisory board: Novartis, Pfizer, Eli Lilly, Daiichi Sankyo, and Menarini Stemline; C.V., honoraria as speaker: Novartis, Eli Lilly, Istituto Gentili, Accademia Nazionale di Medicina, MSD, Research grant: Roche; F.d.B., consulting fees/advisory board: Roche, EMD Serono, NMS Nerviano Medical Science, Sanofi, MSD, Novartis, Incyte, BMS, Menarini, Astra Zeneca, Pierre Fabre, Mattioli 1885, MCCann Health, Taiho; F.d.B., IQVIA Speaker Bureau: BMS, Healthcare Research & Pharmacoepidemiology, Merck Group, MSD, Pfizer, Servier, Sanofi, Roche, AMGEN, Incyte, Dephaforum, Seagen, Nadirex, BMS, Ambrosetti, and Itanet; and F.d.B., research grant/funding: Novartis, F.Hoffmann-LaRoche Ltd, BMS, Ignyta Inc., Merck Sharp & Dohme Spa, Kymab, Pfizer, Tesaro, MSD, MedImmune LCC, Exelixis Inc., LOXO Oncology Inc., DAICHI SANKIO Dev. Ltd, Basilea Pharmaceutica International AG, Janssen-Cilag International NV, and Merck KGAA. C.V. and F.d.B. are co-inventors of the FMD regimen used in this study. The Italian patent (no. 102019000009954) has been released and was first deposited on June 24(th), 2019, with an extension granted under PCT WO 2020/261131 on June 24(th), 2020. The European patent (no. 207403399), Canadian patent (no. 2144217), and USA patent are pending., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: The cutoff of <1% positive cells to define estrogen receptor (ER) negativity by immunohistochemistry (IHC) in breast cancer (BC) is debated. We explored the tumor immune microenvironment and gene-expression profile of patients with early-stage HER2-negative ER-low (ER 1%-9%) BC, comparing them to ER-negative (ER <1%) and ER-intermediate (ER 10%-50%) tumors., Methods: Among 921 patients with early-stage I-III, ER ≤50%, HER2-negative BCs, tumors were classified as ER-negative (n = 712), ER-low (n = 128), or ER-intermediate (n = 81). Tumor-infiltrating lymphocytes (TILs) were evaluated. CD8+, FOXP3+ cells, and PD-L1 status were assessed by IHC and quantified by digital pathology. We analyzed 776 BC-related genes in 116 samples. All tests were 2-sided at a <.05 significance level., Results: ER-low and ER-negative tumors exhibited similar median TILs, statistically significantly higher than ER-intermediate tumors. CD8/FOXP3 ratio and PD-L1 positivity rates were comparable between ER-low and ER-negative groups. These groups showed similar enrichment in basal-like intrinsic subtypes and comparable expression of immune-related genes. ER-low and ER-intermediate tumors showed significant transcriptomic differences. High TILs (≥30%) were associated with improved relapse-free survival (RFS) in ER-low (5-year RFS 78.6% vs 66.2%, log-rank P = .033, hazard ratio [HR] 0.37 [95% CI = 0.15 to 0.96]) and ER-negative patients (5-year RFS 85.2% vs 69.8%, log-rank P < .001, HR 0.41 [95% CI = 0.27 to 0.60])., Conclusions: ER-low and ER-negative tumors are similar biological and molecular entities, supporting their comparable clinical outcomes and treatment responses, including to immunotherapy. Our findings contribute to the growing evidence calling for a reevaluation of ER-positive BC classification and management, aligning ER-low and ER-negative tumors more closely., (© The Author(s) 2024. Published by Oxford University Press.)

Background: In resectable gastric/gastroesophageal junction adenocarcinoma, microsatellite instability-high (MSI-H) confers improved survival, but limited benefit from chemotherapy. Immunotherapy may eliminate the need for chemotherapy or surgery., Patients and Methods: INFINITY is a multicenter, multicohort phase II trial (NCT04817826) investigating in cohort 1 the activity and safety of tremelimumab + durvalumab (T300/D) as neoadjuvant treatment of mismatch repair deficient/MSI-H, resectable gastric/gastroesophageal junction adenocarcinoma. Primary endpoint was pathologic complete response (pCR) rate; Secondary endpoints: progression-free survival (PFS), overall survival (OS), quality of life, and translational analyses. In cohort 2, the activity and safety of T300/D was explored as definitive treatment in patients achieving clinical complete response (cCR). Primary endpoint was 2-year cCR rate, and secondary endpoints were PFS, OS, quality of life, gastrectomy-free survival and translational analyses., Results: In cohort 1, 18 patients were recruited and 15 evaluable. pCR and major pathologic response-pCR were 60% and 80%, respectively. Since pCR rate in T4 tumors was 17%, this subgroup of patients was excluded from enrollment in cohort 2. At 28.1 months median follow-up, 24-month gastric cancer-specific PFS and OS rates were 85% and 92%, respectively. In cohort 2, 18 patients were enrolled and 17 assessable, and 13 had cCR and started non-operative management. At 11.5 months median follow-up, one patient had local regrowth and underwent salvage surgery; 12-month gastrectomy-free survival was 64.2%., Conclusions: The INFINITY study provided promising activity results of a chemo-free T300/D combination regimen as preoperative treatment in mismatch repair deficient/MSI gastric/gastroesophageal junction adenocarcinoma and the first available feasibility results of a non-operative management strategy in this disease setting, worthy of further validation in larger cohorts., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: Epithelioid hemangioendothelioma (EHE), an ultra-rare sarcoma, poses therapeutic challenges because of limited efficacy of conventional chemotherapy in advanced cases, necessitating exploration of new treatment avenues and identification of novel aggressive biomarkers. This study aimed at (i) utilizing a patient-derived xenograft model of EHE and its associated cell line to assess the efficacy of sirolimus and (ii) analyzing two distinct patient cohorts to pinpoint circulating biomarkers of EHE aggressiveness., Experimental Design: A patient-derived xenograft model and corresponding cell line were established from a patient with advanced EHE, demonstrating consistency with the original tumor in terms of histomorphology, WWTR1::CAMTA1 fusion presence, and genomic and transcriptomic profiles. Two independent patient series were employed to investigate the association between growth/differentiation factor 15 (GDF-15) serum levels and EHE aggressiveness., Results: ELISA analyses on EHE cell culture medium and blood from EHE-carrying mice revealed the release of GDF-15 by EHE cells. Sirolimus exhibited markedly higher antitumor activity compared with doxorubicin, concurrently reducing GDF-15 expression/release both in vivo and in vitro. This reduction was attributed to the drug-induced inhibition of phosphorylation/activation of 4E-BP1 and subsequent downregulation of the GDF-15 transcription factors ATF4 and ATF5. Blood sample analyses from two independent patient series showed a significant correlation between GDF-15 and EHE aggressiveness., Conclusions: This study identifies GDF-15 as a novel biomarker of EHE aggressiveness and underscores the superior efficacy of sirolimus compared with doxorubicin in our experimental models. The observed inhibition of GDF-15 release by sirolimus suggests its potential as a biomarker for monitoring the drug's activity in patients., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Anthracycline-based neoadjuvant chemotherapy (NAC) may modify tumour immune infiltrate. This study characterized immune infiltrate spatial distribution after NAC in primary high-risk soft tissue sarcomas (STS) and investigate association with prognosis., Methods: The ISG-STS 1001 trial randomized STS patients to anthracycline plus ifosfamide (AI) or a histology-tailored (HT) NAC. Four areas of tumour specimens were sampled: the area showing the highest lymphocyte infiltrate (HI) at H&E; the area with lack of post-treatment changes (highest grade, HG); the area with post-treatment changes (lowest grade, LG); and the tumour edge (TE). CD3, CD8, PD-1, CD20, FOXP3, and CD163 were analyzed at immunohistochemistry and digital pathology. A machine learning method was used to generate sarcoma immune index scores (SIS) that predict patient disease-free and overall survival (DFS and OS)., Findings: Tumour infiltrating lymphocytes and PD-1+ cells together with CD163+ cells were more represented in STS histologies with complex compared to simple karyotype, while CD20+ B-cells were detected in both these histology groups. PD-1+ cells exerted a negative prognostic value irrespectively of their spatial distribution. Enrichment in CD20+ B-cells at HI and TE areas was associated with better patient outcomes. We generated a prognostic SIS for each tumour area, having the HI-SIS the best performance. Such prognostic value was driven by treatment with AI., Interpretation: The different spatial distribution of immune populations and their different association with prognosis support NAC as a modifier of tumour immune infiltrate in STS., Funding: Pharmamar; Italian Ministry of Health [RF-2019-12370923; GR-2016-02362609]; 5 × 1000 Funds-2016, Italian Ministry of Health; AIRC Grant [ID#28546]., Competing Interests: Declaration of interests Sandro Pasquali reports institutional research funds from Pharmamar, Ikena Oncology, and ASTX Pharmaceutical. Cleofe Romagosa reports institutional research funds from Pharmamar. Angelo Paolo Dei Tos reports institutional research funds from Pharmamar. Javier Martin-Broto reports institutional research funds from Pharmamar, Adaptimmune, Amgen, AROG, Bayer, Blueprint, BMS, Celgene, Daiichi Sankyo, Deciphera, Eisai, Forma, GSK, IMMIX Biopharma, Karyopharm, Lilly, LIXTE, Nektar, Novartis, Pfizer, Roche, and PharmaMar and compensations for advisory board or consulting relationship from Tecnofarma. Jean-Yves Blay reports compensations for advisory board or consulting relationship with Merck Sharp & Dohme, Merck Serono, Daiichi-Sankyo, Astellas, Alexo Oncology, Samyang Biopharm, Hanmi, Daewoong and Amgen and has received institutional research funds from Genentech/Roche, Merck Sharp & Dohme, Merck Serono, Daiichi-Sankyo, Astellas and Amgen. Giovanni Grignani reports institutional research funds from Eli Lilly and Company, GlaxoSmithKline, Merk, Novartis, and Pharmamar. Emanuela Palmerini reports compensations for advisory boards from Daiichi Sankyo Company, Daiichi Sankyo Europe GmbH, Deciphera Pharmaceuticals Inc., EUSA Pharma (US) LLC, and SynOx Therapeutics. Paolo Casali received honoraria for speaker, consultancy, or advisory roles from: Bayer, Deciphera, Eisai, Eli Lilly, and Pfizer; his unit received funds from: Advenchen Laboratories, Amgen Dompé, AROG Pharmaceuticals, Bayer, Blueprint Medicines, Daiichi Sankyo, Deciphera, Eisai, Eli Lilly, Epizyme Inc, Glaxo, Karyopharm Pharmaceuticals, Novartis, Pfizer, and PharmaMar. Silvia Stacchiotti reports institutional research funds from: Advenchen Laboratories, Amgen Dompé, AROG Pharmaceuticals, Bayer, Blueprint Medicines, Daiichi Sankyo, Deciphera, Eisai, Eli Lilly, Epizyme Inc, Glaxo Smith Kline, Karyopharm Pharmaceuticals, Novartis, Pfizer, PharmaMar, and SpringWorks; honoraria, consultancy, or advisory role from: Bayer, Bavarian Nordic, Boehringer, Deciphera, Daiichi Sankyo Pharma, Gentili, Glaxo Smith Kline, Inhibrix, Maxivax, PharmaMar, and Servier; travel, accommodations, expenses from: PharmaMar. Alessandro Gronchi reports compensations for advisory boards from Novartis, Pfizer, Bayer, Lilly, PharmaMar, SpringWorks, and Deciphera and institutional research grants from PharmaMar and Nanobiotix. The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Abstract: CD19-directed chimeric antigen receptor (CAR) T cells can induce durable remissions in relapsed/refractory large B-cell lymphomas (R/R LBCLs), but 60% of patients do not respond or relapse. Biological mechanisms explaining lack of response are emerging, but they are largely unsuccessful in predicting disease response at the patient level. Additionally, to maximize the cost-effectiveness of CAR T-cell therapy, biomarkers able to predict response and survival before CAR T-cell manufacturing would be desirable. We performed transcriptomic and functional evaluations of leukapheresis products in 95 patients with R/R LBCL enrolled in a prospective observational study, to identify correlates of response and survival to tisagenlecleucel and axicabtagene ciloleucel. A signature composed of 4 myeloid genes expressed by T cells isolated from leukapheresis products is able to identify patients with a very short progression-free survival (PFS), highlighting the impact of monocytes in CAR T-cell therapy response. Accordingly, response and PFS were also negatively influenced by high circulating absolute monocyte counts at the time of leukapheresis. The combined evaluation of peripheral blood monocytes at the time of leukapheresis and the 4-gene signature represents a novel tool to identify patients with R/R LBCL at very high risk of progression after CAR T-cell therapy and could be used to plan trials evaluating CAR T cells vs other novel treatments or allogeneic CAR T cells. However, it also highlights the need to incorporate monocyte depletion strategies for better CAR T production., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Introduction: The synthesis of the periprosthetic capsule during implant-based breast reconstruction is the result of a coordinate cascade of inflammatory events ending in a fibrous tissue deposition around the expander or implant. Although the development of small volumes of fluid is one of the complications of prosthetic-based breast reconstruction, the characterization of the periprosthetic effusions coupled with the micro-textured devices, that have been recently introduced after the recall of macro-textured ones, is still lacking. The investigation of these periprosthetic effusions and paired capsules in terms of immunological content were the primary and secondary aims of the present study, respectively., Methods: For this, 68 women, 41 of whom had periprosthetic effusions at the time of expander replacement with implant, were recruited. For each case, capsule and healthy dermal tissues were taken and for women with periprosthetic effusion, peripheral blood was also collected. Periprosthetic effusions and peripheral blood were characterized by cytometry while capsules and dermal tissues by immunohistochemistry and Nanostring analysis., Results: The results showed an increase of Th1, Th2 lymphocytes and a HLA-DR+ bright CD16+ cells (likely representing monocytes-derived macrophages) in periprosthetic effusions in respect to peripheral blood. These pro-inflammatory cells were counterbalanced by the gain of suppressive CD4 Treg cells. In the corresponding capsules, immunohistochemistry revealed the absence of Th1 cells and the presence of tissutal FOXP3 Treg. No significant difference in expression of inflammatory-related genes between capsules and dermal tissues was present., Conclusions: These results suggest the presence of a Treg-controlled inflammation in both periprosthetic effusions and capsules., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Association with hypomethylating agents is a promising strategy to improve the efficacy of immune checkpoint inhibitors-based therapy. The NIBIT-M4 was a phase Ib, dose-escalation trial in patients with advanced melanoma of the hypomethylating agent guadecitabine combined with the anti-CTLA-4 antibody ipilimumab that followed a traditional 3 + 3 design (NCT02608437). Patients received guadecitabine 30, 45 or 60 mg/m 2 /day subcutaneously on days 1 to 5 every 3 weeks starting on week 0 for a total of four cycles, and ipilimumab 3 mg/kg intravenously starting on day 1 of week 1 every 3 weeks for a total of four cycles. Primary outcomes of safety, tolerability, and maximum tolerated dose of treatment were previously reported. Here we report the 5-year clinical outcome for the secondary endpoints of overall survival, progression free survival, and duration of response, and an exploratory integrated multi-omics analysis on pre- and on-treatment tumor biopsies. With a minimum follow-up of 45 months, the 5-year overall survival rate was 28.9% and the median duration of response was 20.6 months. Re-expression of immuno-modulatory endogenous retroviruses and of other repetitive elements, and a mechanistic signature of guadecitabine are associated with response. Integration of a genetic immunoediting index with an adaptive immunity signature stratifies patients/lesions into four distinct subsets and discriminates 5-year overall survival and progression free survival. These results suggest that coupling genetic immunoediting with activation of adaptive immunity is a relevant requisite for achieving long term clinical benefit by epigenetic immunomodulation in advanced melanoma patients., (© 2023. Springer Nature Limited.)

Background: Gene expression profiling (GEP)-based prognostic signatures are being rapidly integrated into clinical decision making for systemic management of breast cancer patients. However, GEP remains relatively underdeveloped for locoregional risk assessment. Yet, locoregional recurrence (LRR), especially early after surgery, is associated with poor survival., Patients and Methods: GEP was carried out on two independent luminal-like breast cancer cohorts of patients developing early (≤5 years after surgery) or late (>5 years) LRR and used, by a training and testing approach, to build a gene signature able to intercept women at risk of developing early LRR. The GEP data of two in silico datasets and of a third independent cohort were used to explore its prognostic value., Results: Analysis of the first two cohorts led to the identification of three genes, CSTB, CCDC91 and ITGB1, whose expression, derived by principal component analysis, generated a three-gene signature significantly associated with early LRR in both cohorts (P value <0.001 and 0.005, respectively), overcoming the discriminatory capability of age, hormone receptor status and therapy. Remarkably, the integration of the signature with these clinical variables led to an area under the curve of 0.878 [95% confidence interval (CI) 0.810-0.945]. In in silico datasets we found that the three-gene signature retained its association, showing higher values in the early relapsed patients. Moreover, in the third additional cohort, the signature significantly associated with relapse-free survival (hazard ratio 1.56, 95% CI 1.04-2.35)., Conclusions: Our three-gene signature represents a new exploitable tool to aid treatment choice in patients with luminal-like breast cancer at risk of developing early recurrence., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Peritoneal metastases (PM) are common routes of dissemination for colorectal cancer (CRC) and remain a lethal disease with a poor prognosis. The properties of the extracellular matrix (ECM) are important in cancer development; studying their changes is crucial to understand CRC-PM development. We studied the elastic properties of ECMs derived from human samples of normal and neoplastic PM by atomic force microscopy (AFM); results were correlated with patient clinical data and expression of ECM components related to metastatic spread. We show that PM progression is accompanied by stiffening of the ECM, increased cancer associated fibroblasts (CAF) activity and increased deposition and crosslinking in neoplastic matrices; on the other hand, softer regions are also found in neoplastic ECMs on the same scales. Our results support the hypothesis that local changes in the normal ECM can create the ground for growth and spread from the tumour of invading metastatic cells. We have found correlations between the mechanical properties (relative stiffening between normal and neoplastic ECM) of the ECM and patients' clinical data, like age, sex, presence of protein activating mutations in BRAF and KRAS genes and tumour grade. Our findings suggest that the mechanical phenotyping of PM-ECM has the potential to predict tumour development., (© 2023. The Author(s).)

In pseudomyxoma peritonei (PMP), KRAS and GNAS mutations are frequent. We hypothesized that these mutations may contribute to the suppression of antitumor immunity: KRAS may induce GMCSF expression, while GNAS may enhance the expression of cyclic adenosine monophosphate and A2AR signaling. This study aimed to explore possible mechanisms facilitated by KRAS and GNAS mutations for escaping immune surveillance. Additionally, we looked for new potential therapeutic and prognostic targets in this rare disease which is poorly characterized at the molecular level. GM-CSF, A2AR, CD73, CD39, and PD-L1 expression was investigated by immunohistochemistry in 40 PMPs characterized for GNAS and KRAS mutational status. Immune cell populations were studied by immunohistochemistry and nanostring nCounter ® . Following the criteria of a prognostic nomogram reported for PMP, we stratified the patients into two different risk groups, with 28 "low-risk" and 12 "high-risk" patients. We observed the expression of GM-CSF (74%); CD39 (37%); CD73 (53%); A2AR (74%); and PD-L1 (16%) which was unrelated to GNAS or KRAS status. The tumor microenvironment showed the presence of CD4+ T cells (86%); CD8+ T cells (27%); CD20+ B (67%); CD15+ cells (86%); and CD163+ M2 macrophages (67%), while CD56+ NK cells were absent. CD163 expression (27%) in PMP tumor cells was associated with poor prognosis. GNAS mutation and A2AR expression were not associated with a specific immune transcriptional signature. However, the expression assay revealed 21 genes associated with prognosis. The "high-risk" patients exhibited worse progression-free survival (HR = 2.3, CI 95%: 1.1-5.1, p = 0.034) and significant downregulation of MET , IL8 , PPARG , DTX4 , HMGA1, ZIC2 , WNT5B, and CCRL2 . In conclusion, we documented the presence of immunosuppressive factors such as GM-CSF, A2AR, and PD-L1 in PMP. These factors were not associated with GNAS and KRAS status and could be explored as therapeutic molecular targets. Additionally, a set of potential prognostic biomarkers, including CD163 expression in tumor cells, deserve further investigation.

Purpose: Trastuzumab is an HER2-specific agent approved as the gold-standard therapy for advanced HER2-positive (HER2+) gastric cancer (GC), but the high rate and rapid appearance of resistance limit its clinical efficacy, resulting in the need to identify new vulnerabilities. Defining the drivers influencing HER2+ cancer stem cell (CSC) maintenance/survival could represent a clinically useful strategy to counteract tumor growth and therapy resistance. Accumulating evidence show that targeting crucial metabolic hubs, as the fatty acid synthase (FASN), may be clinically relevant., Methods: FASN protein and transcript expression were examined by WB and FACS and by qRT-PCR and GEP analyses, respectively, in trastuzumab-sensitive and trastuzumab-resistant HER2+ GC cell lines cultured in adherent (2D) or gastrosphere promoting (3D) conditions. Molecular data were analyzed in silico in public HER2+ GC datasets. The effectiveness of the FASN inhibitor TVB3166 to overcome anti-HER2 therapy resistance was tested in vitro in gastrospheres forming efficiency bioassays and in vivo in mice bearing trastuzumab-resistant GC cells., Results: We compared the transcriptome profiles of HER2+ GC cells cultured in 2D versus 3D conditions finding a significant enrichment of FASN in 3D cultures. FASN upregulation significantly correlated with high stemness score and poor prognosis in HER2+ GC cases. TVB3166 treatment significantly decreased GCSCs in all cell targets. HER2 and FASN cotargeting significantly decreased the capability to form gastrospheres versus monotherapy and reduced the in vivo growth of trastuzumab-resistant GC cells., Conclusion: Our findings indicate that cotargeting HER2 and FASN increase the benefit of anti-HER2 therapy representing a new opportunity for metabolically combating trastuzumab-resistant HER2+ GC., (© 2023. The Author(s).)

Background: Chemoimmunotherapy represents the standard of care for patients with advanced non-small cell lung cancer (NSCLC) and programmed death-ligand 1 (PD-L1) <50%. Although single-agent pembrolizumab has also demonstrated some activity in this setting, no reliable biomarkers yet exist for selecting patients likely to respond to single-agent immunotherapy. The main purpose of the study was to identify potential new biomarkers associated with progression-free-survival (PFS) within a multiomics analysis., Methods: PEOPLE (NTC03447678) was a prospective phase II trial evaluating first-line pembrolizumab in patients with advanced EGFR and ALK wild type treatment-naïve NSCLC with PD-L1 <50%. Circulating immune profiling was performed by determination of absolute cell counts with multiparametric flow cytometry on freshly isolated whole blood samples at baseline and at first radiological evaluation. Gene expression profiling was performed using nCounter PanCancer IO 360 Panel (NanoString) on baseline tissue. Gut bacterial taxonomic abundance was obtained by shotgun metagenomic sequencing of stool samples at baseline. Omics data were analyzed with sequential univariate Cox proportional hazards regression predicting PFS, with Benjamini-Hochberg multiple comparisons correction. Biological features significant with univariate analysis were analyzed with multivariate least absolute shrinkage and selection operator (LASSO)., Results: From May 2018 to October 2020, 65 patients were enrolled. Median follow-up and PFS were 26.4 and 2.9 months, respectively. LASSO integration analysis, with an optimal lambda of 0.28, showed that peripheral blood natural killer cells/CD56dimCD16+ (HR 0.56, 0.41-0.76, p=0.006) abundance at baseline and non-classical CD14dimCD16+monocytes (HR 0.52, 0.36-0.75, p=0.004), eosinophils (CD15+CD16-) (HR 0.62, 0.44-0.89, p=0.03) and lymphocytes (HR 0.32, 0.19-0.56, p=0.001) after first radiologic evaluation correlated with favorable PFS as well as high baseline expression levels of CD244 (HR 0.74, 0.62-0.87, p=0.05) protein tyrosine phosphatase receptor type C (HR 0.55, 0.38-0.81, p=0.098) and killer cell lectin like receptor B1 (HR 0.76, 0.66-0.89, p=0.05). Interferon-responsive factor 9 and cartilage oligomeric matrix protein genes correlated with unfavorable PFS (HR 3.03, 1.52-6.02, p 0.08 and HR 1.22, 1.08-1.37, p=0.06, corrected). No microbiome features were selected., Conclusions: This multiomics approach was able to identify immune cell subsets and expression levels of genes associated to PFS in patients with PD-L1 <50% NSCLC treated with first-line pembrolizumab. These preliminary data will be confirmed in the larger multicentric international I3LUNG trial (NCT05537922)., Trial Registration Number: 2017-002841-31., Competing Interests: Competing interests: GLR provided consultation, attended advisory boards and/or provided lectures for the following organizations, from whom received honoraria or education grants: Merck Sharp and Dohme, Takeda, Amgen, Eli Lilly, BMS, Roche, Italfarmaco, Novartis, Sanofi, Pfizer and AstraZeneca. AP declares personal fees from AstraZeneca, Italfarmaco, Roche, BMS. RF declares advisory role from Merck Sharp and Dohme. FDB provided consultation, attended advisory boards and/or provided lectures for the following organizations, from whom received honoraria or education grants: Amgen, AstraZeneca, Boehringer-Ingelheim, BMS, Eli Lilly, F. Hoffmann-La Roche, Ignyta, Merck Sharp and Dohme, Merck Serono, Novartis, Pfizer.CP declares personal fees from Italfarmaco, AstraZeneca, BMS and Merck Sharp and Dohme.MCG declares personal financial interests with the following organizations: AstraZeneca, MSD International GmbH, BMS, Boehringer Ingelheim Italia S.p.A, Celgene, Eli Lilly, Ignyta, Incyte, Inivata, MedImmune, Novartis, Pfizer, Roche, Takeda, Seattle Genetics, Mirati, Daiichi Sankyo, Regeneron, Merck, Ose Immuno Therapeutics, Blueprint, Jansenn, Sanofi; she also declares Institutional financial interests with the following organizations: Eli Lilly, MSD, Pfizer (MISP); AstraZeneca, MSD International GmbH, BMS, Boehringer Ingelheim Italia S.p.A, Celgene, Eli Lilly, Ignyta, Incyte, MedImmune, Novartis, Pfizer, Roche, Takeda, Tiziana, Foundation Medicine, Glaxo Smith Kline GSK, Spectrum pharmaceuticals., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

This study exploited a novel patient-derived xenograft (PDX) of desmoplastic small round cell tumor (DSRCT), which reproduces histomorphological and molecular characteristics of the clinical tumor, to assess the activity of cytotoxic and targeted anticancer agents. Antitumor effect was moderate for doxorubicin, pazopanib and larotrectenib [maximum tumor volume inhibition (max TVI), 55-66%], while trabectedin had higher activity (max TVI, 82%). Vinorelbine, irinotecan and eribulin achieved nearly complete tumor growth inhibition (max TVI, 96-98%), although tumors regrew after the end of treatment. The combination of irinotecan with either eribulin or trabectedin resulted in complete responses, which were maintained until the end of the experiment for irinotecan plus trabectedin. Irinotecan-based combinations nearly abrogated the expression of proteins of the G2/M checkpoint, preventing cell entrance in mitosis, and induced apoptotic and necroptotic cell death. Consistently, irinotecan plus trabectedin resulted in reprogramming of DSCRT transcriptome, with downregulation of E2F targets, G2/M checkpoint and mitotic spindle gene sets. This study emphasizes the importance of patient-derived preclinical models to explore new treatments for DSRCT and fosters clinical investigation into the activity of irinotecan plus trabectedin., Competing Interests: Competing interests V.Z., S. Pasquali, M.T., S. Percio, V.D., M.B., P.C., G.P.D., S.B., P.G., M.F., A.M.F., A.G., A.F. and N.Z. have no competing or financial interests. M.C. is on Advisory Boards for Pfizer, Bayer, Astra Zeneca, Servier and BMS. S.S. receives institutional research funding from Amgen Dompe, Advenchen, Bayer, Blueprint Medicines, Deciphera, Eli Lilly, Epizyme, Daiichi Sankyo Pharma, GSK, Karyopharm, Novartis, Pfizer, PharmaMar, SpringWorks and Hutchinson MediPharma International Inc.; has received honoraria from Aadi, Bayer, Deciphera, Daiichi, Eli Lilly, Maxivax, Novartis, GSK and PharmaMar; and participates in a Data Safety Monitoring Board or Advisory Board for Bayer, Bavarian Nordic, Deciphera, Eli Lilly, Daiichi, GSK, Ikena, Maxivax, Novartis and Pharmamar., (© 2023. Published by The Company of Biologists Ltd.)

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments., (© The Author(s) (2022). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Background: Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents., Methods: Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets., Results: Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset., Conclusions: The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches., (© 2022. The Author(s).)

Purpose: This is a multicenter, single-arm phase II trial evaluating the efficacy and safety of an immune-sensitizing strategy with temozolomide priming followed by a combination of low-dose ipilimumab and nivolumab in patients with microsatellite-stable (MSS) and O 6 -methylguanine-DNA methyltransferase (MGMT)-silenced metastatic colorectal cancer (mCRC)., Patients and Methods: Patients with pretreated mCRC were centrally prescreened for MSS status and MGMT silencing (ie, lack of MGMT expression by immunohistochemistry plus MGMT methylation by pyrosequencing). Eligible patients received two priming cycles of oral temozolomide 150 mg/sqm once daily, days 1-5, once every 4 weeks (first treatment part) followed, in absence of progression, by its combination with ipilimumab 1 mg/kg once every 8 weeks and nivolumab 480 mg once every 4 weeks (second treatment part). The primary end point was the 8-month progression-free survival (PFS) rate calculated from enrollment in patients who started the second treatment part, with ≥ 4 out of 27 subjects progression-free by the 8-month time point as decision rule., Results: Among 716 prescreened patients, 204 (29%) were molecularly eligible and 135 started the first treatment part. Among these, 102 (76%) were discontinued because of death or disease progression on temozolomide priming, whereas 33 patients (24%) who achieved disease control started the second treatment part and represented the final study population. After a median follow-up of 23.1 months (interquartile range, 14.9-24.6 months), 8-month PFS rate was 36%. Median PFS and overall survival were 7.0 and 18.4 months, respectively, and overall response rate was 45%. Grade 3-4 immune-related adverse events were skin rash (6%), colitis (3%), and hypophysitis (3%). No unexpected adverse events or treatment-related deaths were reported., Conclusion: The MAYA study provided proof-of-concept that a sequence of temozolomide priming followed by a combination of low-dose ipilimumab and nivolumab may induce durable clinical benefit in MSS and MGMT-silenced mCRC., Competing Interests: Federica MoranoHonoraria: ServierTravel, Accommodations, Expenses: Sanofi, Servier Sara LonardiConsulting or Advisory Role: Amgen, Merck Serono, Lilly, Servier, AstraZeneca, Incyte, Daiichi Sankyo, Bristol Myers Squibb, MSDSpeakers' Bureau: Roche, Lilly, Bristol Myers Squibb, Servier, Merck Serono, Pierre Fabre, GlaxoSmithKline, AmgenResearch Funding: Amgen, Merck Serono, Bayer (Inst), Roche (Inst), Lilly (Inst), AstraZeneca (Inst), Bristol Myers Squibb (Inst) Lisa SalvatoreHonoraria: Roche, Merck Serono, Servier, Bayer, Amgen, Sanofi, AstraZeneca, Pierre Fabre, MSDConsulting or Advisory Role: Merck Serono, Servier, Bayer, Roche, Amgen, AstraZeneca, Sanofi, Pierre Fabre, MSDTravel, Accommodations, Expenses: Sanofi, Merck Serono, Bayer, Roche, Servier, Celgene Chiara CremoliniHonoraria: Roche, Amgen, Bayer, Servier, MSD, MerckConsulting or Advisory Role: Roche, Bayer, Amgen, MSDSpeakers' Bureau: ServierResearch Funding: Merck, Bayer, Roche, Servier Patrizia RaccaHonoraria: Merck Serono, Roche, Amgen, Servier Monica NigerConsulting or Advisory Role: Incyte, Basilea Pharmaceutical, EMD Serono, MSD/AstraZenecaTravel, Accommodations, Expenses: Celgene Francesca CortiTravel, Accommodations, Expenses: Advanced Accelerator Applications Alberto ZaniboniConsulting or Advisory Role: Amgen, Servier, Bayer, Merck Serono, MerckSpeakers' Bureau: servier, Astellas Pharma (Inst) Celeste CagnazzoSpeakers' Bureau: Boehringer Ingelheim, Novartis Massimo Di MaioHonoraria: Pfizer, Takeda, AstraZeneca, Janssen, Eisai, Novartis, Roche, Astellas Pharma, MSD OncologyConsulting or Advisory Role: AstraZeneca, Pfizer, Takeda, Janssen, Eisai, Novartis, Roche, MSD Oncology, AmgenResearch Funding: Tesaro (Inst), GlaxoSmithKline (Inst) Maria Di BartolomeoHonoraria: Lilly, MSD Oncology, ServierConsulting or Advisory Role: Lilly, MSD OncologyResearch Funding: LillyTravel, Accommodations, Expenses: Roche, Sanofi Filippo De BraudHonoraria: Roche, Pfizer, BMS, Merck, MSD, Servier, Sanofi, Amgen Astellas BioPharma, IncyteConsulting or Advisory Role: Roche, Incyte, EMD SERONO, Bristol Myers Squibb, Nerviano Medical Sciences, Sanofi, Novartis Italy, nms medical science, MenariniResearch Funding: Novartis (Inst), Roche (Inst), Merck Serono (Inst), Pfizer (Inst), Servier (Inst), Philogen (Inst), Loxo (Inst), Tesaro (Inst), Nerviano Medical Sciences (Inst), Kymab (Inst), Bristol Myers Squibb/Medarex, Merck KGaA, Ignyta, MedImmune, Exelixis, Bayer Health, Daiichi Sankyo Europe GmbH, Incyte, Basilea Pharmaceutical, Janssen Oncology Filippo PietrantonioHonoraria: Servier, Bayer, AstraZeneca/MedImmune, Lilly, Sanofi, MSD Oncology, AmgenConsulting or Advisory Role: Amgen, Servier, MSD Oncology, MerckResearch Funding: Bristol Myers Squibb (Inst), Astrazeneca (Inst)No other potential conflicts of interest were reported.

Aim: To unveil genomic and immunohistochemical expression profiles associated with primary resistance to EGFR/BRAF targeted therapy in patients with BRAF-mutated and microsatellite stable (MSS) metastatic colorectal cancer., Experimental Design: In this multicenter case-control study on patients treated with EGFR/BRAF ± MEK blockade, we compared a primary resistance cohort (N = 20; RECISTv1.1 PD/SD, and progression-free survival [PFS] <16 weeks) versus a sensitive one (N = 19; RECISTv1.1 PR/CR, and PFS ≥16 weeks) in terms of clinical and genomic/expression data by means of comprehensive genomic profiling, tumour mutational burden (TMB), BRAF-mutant transcriptional subtypes (BM) classification and PTEN expression., Results: Left-sided tumours (28% of the total) were enriched in the sensitive versus resistant cohort (53% versus 10%, P = 0.010). Genomic alterations in the PIK3CA/MTOR pathway, BM1 status and PTEN loss were similarly distributed among patients with resistant and sensitive tumours. Amplification of CCND1-3 genes were found only in patients with primary resistance (20% versus 0%, P = 0.106). TMB and prevalence of intermediate TMB (TMB-I 6-20 mutations/Mb) were higher in the resistant versus sensitive cohort (median TMB: 6 [IQR, 3-7.29] versus 3 [IQR, 1.26-3.5]; P = 0.013; TMB-I/H: 60% versus 11%; P = 0.001). Patients with TMB-I had shorter PFS (3.3 versus 5.9 months; HR = 2.19, 95%CI, 1.07-4.47, P = 0.031) and overall survival (6.3 versus 10.5 months; HR = 2.22, 95%CI, 1.02-4.81, P = 0.044)., Conclusion: Despite the small sample size, the association of a relatively higher TMB with limited benefit from EGFR/BRAF blockade in patients with MSS and BRAF-mutated mCRC deserves prospective validation., Competing Interests: Conflict of interest statement Filippo Pietrantonio received honoraria for speakers bureau from Servier, research grants from BMS and honoraria for advisory boards/speakers bureau from Roche, Amgen, Merck-Serono, Lilly, Sanofi, Bayer and Servier. Elena Élez reports personal fees from Hoffman La - Roche, Bristol Myers Squibb, Servier, Amgen, Merck Serono, ArrayBiopharma, Sanofi. Chiara Cremolini reports personal fees from Roche, Amgen, Bayer, and Servier; research funding from Merck Serono; and a consulting or advisory role with Roche, Bayer, Amgen. Matteo Fassan reports a consulting or advisory role with Astellas Pharma, GlaxoSmithKline, Diaceutics, and Roche; research funding from Astellas Pharma, QED Therapeutics, and Macrophage Pharma. Sara Lonardi received honoraria for Speakers Bureau, Consulting or Advisory Role from Amgen, Merck Serono, Lilly, Astra Zeneca, Incyte, Daiichi-Sankyo, Bristol-Myers Squibb, Servier, MSD, Roche, Pierre-Fabre, GSK and Research Funding (to Institution) from Amgen, Merck Serono, Bayer, Roche, Lilly, Astra Zeneca, Bristol-Myers Squibb. All remaining authors have declared no conflicts of interest. This Study was partially funded by a grant from Ministero della Salute, Bando della Ricerca Finalizzata 2019, Project Number GR-2019-12368903, CUP code J99C21000260001., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We previously published the results of a pilot study showing that vaccination with tumor-loaded dendritic cells (DCs) induced both T and B cell response and produced clinical benefit in the absence of toxicity in patients with relapsed, indolent non-Hodgkin lymphoma (iNHL). The purpose of the present short report is to provide a 15-year follow-up of our study and to expand the biomarker analysis previously performed. The long-term follow-up highlighted the absence of particular or delayed toxicity and the benefit of active immunization with DCs loaded with autologous, heat-shocked and UV-C treated tumor cells in relapsed iNHL (5-year and 10-year progression-free survival (PFS) rates: 55.6% and 33.3%, respectively; 10-year overall survival (OS) rate: 83.3%). Female patients experienced a better PFS (p=0.016) and a trend towards a better OS (p=0.185) compared with male patients. Of note, we observed a non-negligible fraction of patients (22%) who experienced a long-lasting complete response. In a targeted gene expression profiling of pre-treatment tumor biopsies in 11 patients with available formalin-fixed, paraffin-embedded tissue, we observed that KIT , ATG12 , TNFRSF10C , PBK , ITGA2 , GATA3 , CLU , NCAM1 , SYT17 and LTK were differentially expressed in patients with responder versus non-responder tumors. The characterization of peripheral monocytic cells in a subgroup of 14 patients with available baseline blood samples showed a higher frequency of the subset of CD14 ++ CD16 + cells (intermediate monocytes) in patients with responding tumors. Since in patients with relapsed iNHL the available therapeutic options are often incapable of inducing a long-lasting complete remission and can be sometimes characterized by intolerable toxicity, we think that the encouraging results of our long-term follow-up analysis represent a stimulus to further investigate the role of active vaccination in this specific setting and in earlier lines of therapy and to explore novel combinatorial strategies encompassing other innovative immunotherapy agents, such as immune-checkpoint inhibitors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The aim of this work was to assess the antimicrobial efficacy on Pseudomonas aeruginosa of nanomicelles loaded with curcumin (CUR) alone and activated by blue laser light in an antimicrobial photodynamic therapy (APDT) approach. First, free CUR in liquid suspension and loaded in three amphiphilic nanomicelles (CUR-DAPMA, CUR-SPD and CUR-SPM) were tested both on bacteria and keratinocytes. While free CUR exerted limited efficacy showing moderate cytotoxicity, a strong inhibition of bacterial growth was obtained using all three nanosystems without toxicity on eukaryotic cells. CUR-SPM emerged as the most effective, and was therefore employed in APDT experiments. Among the three sublethal blue laser (λ 445 nm) protocols tested, the ones characterized by a fluence of 18 and 30 J/cm 2 further decreased the antimicrobial concentration to 50 nM. The combination of blue laser APDT with CUR-SPM nanomicelles results in an effective synergistic activity that represents a promising novel therapeutic approach on resistant species., (© 2020 Wiley-VCH GmbH.)

Background: Dedifferentiated liposarcoma (DDLPS), a tumor that lacks effective treatment strategies and is associated with poor outcomes, expresses amplified MDM2 in the presence of wild-type p53. MDM2 ubiquitination of p53 facilitates its XPO1-mediated nuclear export, thus limiting p53 tumor suppressor functions. Consequently, nuclear export is a rational target in DDLPS. We directly compared the antitumor activity of the first-in class XPO1 inhibitor selinexor and doxorubicin, the standard front-line therapy in sarcomas, in DDLPS patient-derived xenografts (PDXs) and primary cell lines., Methods: Drug activity was assessed in three PDXs (and two corresponding cell lines) established from the dedifferentiated component of primary untreated retroperitoneal DDLPS with myogenic (N = 2) and rhabdomyoblastic (N = 1) differentiation from patients who underwent surgery. These models were marked by amplification of MDM2, CDK4 and HMGA2 genes., Results: Selinexor was moderately active in the three PDXs but achieved greater tumor response compared to doxorubicin (maximum tumor volume inhibition: 46-80 % vs. 37-60 %). The PDX harboring rhabdomyoblastic dedifferentiation showed the highest sensitivity to both agents. PDX response to selinexor and doxorubicin was not associated with the extent of MDM2 and CDK4 gene amplification. Interestingly, the most chemosensitive PDX model showed the lowest extent of HMGA2 amplification. Selinexor was also more efficient than doxorubicinin in inducing an apoptotic response in PDXs and cell lines. Consistently, an increased nuclear accumulation of p53 was seen in all selinexor-treated models. In addition, a time-dependent decrease of survivin expression, with an almost complete abrogation of the cytoplasmic anti-apoptotic pool of this protein, was observed as a consequence of the decreased acetylation/activation of STAT3 and the increased ubiquitination of nuclear survivin., Conclusions: Selinexor showed a moderate antitumor activity in three DDLPS PDXs, which was, however, consistently higher than doxorubicin across all different models regardless the extent of MDM2 amplification and the histological differentiation. The depletion of survivin protein seems to significantly contribute to the induction of apoptosis through which selinexor exerts its antitumor activity.

Background: Immune-contexture of tumour microenvironment (TME) influences prognosis of colorectal cancer (CRC) patients and can be altered by cytotoxic and targeted agents. Limited data are available regarding the immune-TME of CRC after treatment., Methods: An extensive immunohistochemistry evaluation of immunological parameters on tumour cells and TME of colorectal liver metastases from 106 patients who underwent secondary resection, after receiving triplets FOLFOXIRI (5-fluorouracil, oxaliplatin and irinotecan) or COI (capecitabine, oxaliplatin and irinotecan) plus bevacizumab (N = 59) or cetuximab (N = 47) in five first-line no-profit clinical trials was performed., Results: No substantial differences were reported in immunological parameters according to administered targeted agent, RAS/BRAF mutational status and histopathological or Response Evaluation Criteria in Solid Tumours response. Stromal expression of Cyclooxygenase-2 (COX-2) (p = 0.002), Human leukocyte antigen (HLA) (p = 0.003) and Programmed cell death protein 1 (PD1) (p = 0.002) were independent prognostic factors for longer relapse-free survival (RFS) at multivariate analysis with a positive trend for post-resection overall survival (OS). Patients whose metastases expressed stromal COX-2, HLA and PD1 (inflamed-score positive) reported longer RFS (25.5 versus 9.8 months; p < 0.001) and post-resection OS (64.3 versus 37.7 months; p = 0.003) as compared with others. In addition, patients with higher expression of CD4 and CD8 T-cells in tumour core and invasive margin (CD4/CD8-score) showed a better post-resection OS (not-reached versus 41.6 months; p = 0.032). A combined score of inflamed-score and CD4/CD8-score (combo-score) showed a clear prognostic role., Conclusions: The present study emphasises the role of immune-TME as independent predictor of survival in patients resected after triplets plus biologic. Inflamed-, CD4/C8- and combo-scores should be confirmed as prognostic factors in further studies., Competing Interests: Conflict of interest statement F.M. declared Honoraria/speaker's bureau from Servier. S. L. receiving advisory board fees from Amgen, Eli Lilly, and Merck. F. L. receiving lecture fees from Amgen and F. Hoffmann–La Roche and advisory fees from Bayer.F.d.B. provided consultation, attended advisory boards, and/or provided lectures for the following organisations from whom honoraria or education grants were received: Amgen, AstraZeneca, Boehringer-Ingelheim, BMS, Eli Lilly, F. Hoffmann-La Roche, Ignyta, Merck Sharp and Dohme, Merck Serono, Novartis, Pfizer. A.F. is a consultant/advisory board member for Bayer, Roche, Amgen, Eli-Lilly, Merck Serono, Sanofi and Servier. F. P. declared Honoraria/speaker's bureau from Roche, Amgen, Merck-Serono, Lilly, Sanofi, Bayer andServier. Research Grants from BMS. C. C. is a consultant/advisory board member for Roche, Amgen, Bayer, Merck Serono and Servier. All other authors have declared no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of this study was to assess the performance of fluorescence in situ hybridization (FISH) in identifying the copy number profiles of the three key peritoneal mesothelioma tumor suppressor genes BAP1, CDKN2A, and NF2, with particular emphasis on minute homozygous deletions, a copy number abnormality recently unveiled at the 3p21 (BAP1) chromosomal region using high-throughput methods. FISH was performed on 75 formalin-fixed-paraffin-embedded peritoneal mesotheliomas and recognized two types of monoallelic loss (monosomy, and hemizygous deletion) and two types of biallelic loss (canonical homozygous deletion with a complete loss of FISH signal and homozygous deletion with diminished signal). Diminished FISH signals revealed deletions occurring within the genomic region covered by the gene-specific probe and affected all three tumor suppressors. BAP1 homozygous deletions with diminished signal outnumbered canonical homozygous deletions (13 vs 3): conversely, canonical homozygous deletions were prevalent for CDKN2A (2 vs 14). Diminished signal homozygous deletion was the only pattern of biallelic loss observed for NF2 (2 cases). Hemizygous deletion mainly affected BAP1 (21 vs 6), while monosomy was prevalent for CDKN2A (14 vs 7) and particularly for NF2 where it accounts for all monoallelic losses. FISH/immunohistochemistry (BAP1, CDKN2A, and MTAP) correlation showed that all homozygous deletions, including those with diminished signals, resulted in a null BAP1 and CDKN2A immunophenotype but only canonical CDKN2A homozygous deletions resulted in MTAP loss of expression. BAP1 hemizygous deletion, but not monosomy, was also invariably associated with loss of protein expression whereas neither type of CDKN2A monoallelic loss correlated with p16 or MTAP immunohistochemistry. Array comparative genomic hybridization performed on a spontaneously emerging peritoneal mesothelioma cell line provided support for the interpretation of the FISH patterns and allowed us to extend the number of chromatin remodeling factors involved in mesothelioma to SETD7 and PCGF5, two previously unreported genes.

Thyroid carcinoma (TC) comprises several histotypes with different aggressiveness, from well (papillary carcinoma, PTC) to less differentiated forms (poorly differentiated and anaplastic thyroid carcinoma, PDTC and ATC, respectively). Previous reports have suggested a functional role for cancer-associated fibroblasts (CAFs) or senescent TC cells in the progression of PTC. In this study, we investigated the presence of CAFs and senescent cells in proprietary human TCs including PTC, PDTC, and ATC. Screening for the driving lesions BRAFV600E and N/H/KRAS mutations, and gene fusions was also performed to correlate results with tumor genotype. In samples with unidentified drivers, transcriptomic profiles were used to establish a BRAF- or RAS-like molecular subtype based on a gene signature derived from The Cancer Genome Atlas. By using immunohistochemistry, we found co-occurrence of stromal CAFs and senescent TC cells at the tumor invasive front, where deposition of collagen (COL1A1) and expression of lysyl oxidase (LOX) enzyme were also detected, in association with features of local invasion. Concurrent high expression of CAFs and of the senescent TC cells markers, COL1A1 and LOX was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in BRAF mutated or BRAF-like tumors. In this study, we show that CAFs and senescent TC cells co-occur in various histotypes of BRAF-driven thyroid tumors and localize at the tumor invasive front., Competing Interests: The authors declare no conflict of interest.

Purpose: This study was aimed at investigating whether the PPARγ agonist pioglitazone-given in combination with trabectedin-is able to reactivate adipocytic differentiation in myxoid liposarcoma (MLS) patient-derived xenografts, overcoming resistance to trabectedin., Experimental Design: The antitumor and biological effects of trabectedin, pioglitazone, and the combination of the two drugs were investigated in nude mice bearing well-characterized MLS xenografts representative of innate or acquired resistance against trabectedin. Pioglitazone and trabectedin were given by daily oral and weekly i.v. administrations, respectively. Molecular studies were performed by using microarrays approach, real-time PCR, and Western blotting., Results: We found that the resistance of MLS against trabectedin is associated with the lack of activation of adipogenesis. The PPARγ agonist pioglitazone reactivated adipogenesis, assessed by histologic and gene pathway analyses. Pioglitazone was well tolerated and did not increase the toxicity of trabectedin. The ability of pioglitazone to reactivate adipocytic differentiation was observed by morphologic examination, and it is consistent with the increased expression of genes such as ADIPOQ implicated in the adipogenesis process. The determination of adiponectin by Western blotting constitutes a good and reliable biomarker related to MLS adipocytic differentiation., Conclusions: The finding that the combination of pioglitazone and trabectedin induces terminal adipocytic differentiation of some MLSs with the complete pathologic response and cure of tumor-bearing mice provides a strong rationale to test the combination of trabectedin and pioglitazone in patients with MLS., (©2019 American Association for Cancer Research.)

Background-There are currently no effective therapies for diffuse malignant peritoneal mesothelioma (DMPM) patients with disease recurrence. In this study, we investigated the biology of DMPM by analyzing the EGFR family, Axl, and MET, in order to assess the presence of cross-talk between these receptors, suggesting the effectiveness of combined targeted treatments in DMPM. Method-We analyzed a series of 22 naïve epithelioid DMPM samples from a single institute, two of which showed higher-grade malignancy ("progressed"). EGFR, HER2, HER3, Axl, and MET activation and expression were investigated by biochemical analysis, real-time PCR immunofluorescence, immunohistochemistry, next-generation sequencing, miRNA, and mRNA in situ hybridization. Results-In most DMPMs, a strong EGFR activation was associated with HER2, HER3, Axl, and MET co-activation, mediated mainly by receptor heterodimerization and autocrine-paracrine loops induced by the expression of their cognate ligands. Axl expression was downregulated by miRNA34a. Mutations in MET Sema domain were exclusively found in two "progressed" DMPMs, and the combined Axl and MET inhibition reduced cellular motility in a DMPM cell line obtained from a "progressed" DMPM. Conclusion-The results indicate that the coordinated activity of multiple cross-talks between RTKs is directly involved in the biology of DMPM, suggesting the combined inhibition of PIK3 and mTOR as an effective strategy that may be easily implemented in clinical practice, and indicating that the combined inhibition of EGFR/HER2 and HER3 and of Axl and MET deserves further investigation.

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC., (© 2019 UICC.)

Background: Myxoid liposarcoma is a histological subtype of liposarcoma particularly sensitive to trabectedin. In clinical use this drug does not cause cumulative toxicity, allowing prolonged treatment, generally until disease progression. No other effective therapies are available for trabectedin-resistant patients., Methods: Through repeated in vivo treatment in athymic nude mice, we have obtained a patient-derived xenograft with acquired resistance to trabectedin., Results: At basal level, the morphology of the resistant and sensitive models did not differ, in keeping with the finding that the transcriptional profiles of the resistant and sensitive tumours were very similar. After trabectedin treatment adipogenesis was induced in the parental xenograft but not in the resistant one, as assessed by pathological and molecular analysis. A defective transcription-coupled-nucleotide excision repair in the resistant tumour due to mutation of the UVSSA gene may be implicated in the mechanism of resistance., Conclusions: This is the first in vivo model of myxoid liposarcoma with acquired resistance to trabectedin. Although further studies are necessary to characterise the resistance mechanisms, this is a useful tool for studying new therapeutic strategies to overcome trabectedin resistance in patients.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma histotype with uncertain differentiation. EMC is hallmarked by the rearrangement of the NR4A3 gene, which in most cases fuses with EWSR1 or TAF15. TAF15-translocated EMC seem to feature a more aggressive course compared to EWSR1-positive EMCs, but whether the type of NR4A3 chimera impinges upon EMC biology is still largely undefined. To gain insights on this issue, a series of EMC samples (7 EWSR1-NR4A3 and 5 TAF15-NR4A3) were transcriptionally profiled. Our study unveiled that the two EMC variants display a distinct transcriptional profile and that the axon guidance pathway is a major discriminant. In particular, class 4-6 semaphorins and axonal guidance cues endowed with pro-tumorigenic activity were more expressed in TAF15-NR4A3 tumors; vice versa, class 3 semaphorins, considered to convey growth inhibitory signals, were more abundant in EWSR1-NR4A3 EMC. Intriguingly, the dichotomy in axon guidance signaling observed in the two tumor variants was recapitulated in in vitro cell models engineered to ectopically express EWSR1-NR4A3 or TAF15-NR4A3. Moreover, TAF15-NR4A3 cells displayed a more pronounced tumorigenic potential, as assessed by anchorage-independent growth. Overall, our results indicate that the type of NR4A3 chimera dictates an axon guidance switch and impacts on tumor cell biology. These findings may provide a framework for interpretation of the different clinical-pathological features of the two EMC variants and lay down the bases for the development of novel patient stratification criteria and therapeutic approaches. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Background: Extraskeletal myxoid chondrosarcoma is a rare sarcoma with low sensitivity to cytotoxic chemotherapy. Retrospective evidence suggests that antiangiogenic drugs could be a treatment option. We aimed to investigate the activity of pazopanib, an antiangiogenic drug, in patients with advanced extraskeletal myxoid chondrosarcoma., Methods: In this single-arm, open-label phase 2 trial, three parallel independent cohorts of different histotypes of advanced sarcomas were recruited (extraskeletal myxoid chondrosarcoma, typical solitary fibrous tumour, and malignant-dedifferentiated solitary fibrous tumour). In each cohort, patients received pazopanib. In this Article, we report the results of the cohort of patients with advanced extraskeletal myxoid chondrosarcoma. Separate reporting of the three cohorts was prespecified in the study protocol. In this cohort, adult patients (aged ≥18 years) with a diagnosis of NR4A3-translocated, metastatic, or unresectable extraskeletal myxoid chondrosarcoma, who had Response Evaluation Criteria in Solid Tumors (RECIST) progression in the previous 6 months, and had an Eastern Cooperative Oncology Group performance status of 0-2, were enrolled at 11 study sites of the Spanish, Italian, and French sarcoma groups. Patients received oral pazopanib (800 mg/day) continuously, until disease progression, unacceptable toxicity, death, non-compliance, patient refusal, or investigator's decision. The primary endpoint was the proportion of patients achieving an objective response according to RECIST 1·1 in the modified intention-to-treat population (patients who provided consent and had a central molecularly confirmed diagnosis of extraskeletal myxoid chondrosarcoma). The safety analysis included all patients who received at least one dose of pazopanib. This study is registered with ClinicalTrials.gov, number NCT02066285., Findings: Between June 24, 2014, and Jan 17, 2017, 26 patients entered the study and started pazopanib. Of these, 23 met the eligibility criteria for the modified intention-to-treat analysis. Median follow-up was 27 months (IQR 18-30). 22 patients (one patient died before the primary analysis) were evaluable for the primary endpoint: four (18% [95% CI 1-36]) had a RECIST objective response. No deaths or grade 4 adverse events occurred. The most frequent grade 3 adverse events were hypertension (nine [35%] of 26 patients), increased concentration of alanine aminotransferase (six [23%]), and increased aspartate aminotransferase (five [19%])., Interpretation: Pazopanib had clinically meaningful antitumour activity in patients with progressive and advanced extraskeletal myxoid chondrosarcoma, and could be considered a suitable option after failure to respond to first-line anthracycline-based chemotherapy in these patients., Funding: Spanish Group for Research on Sarcomas, Italian Sarcoma Group, French Sarcoma Group, GlaxoSmithKline, and Novartis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Epithelioid sarcoma (ES) is a rare mesenchymal malignancy marked by SMARCB1/INI1 deficiency. Retrospective clinical data report on the activity of anthracycline- and gemcitabine-based regimens. EZH2 inhibitors are currently being tested in clinical trials. Since comparisons of these agents are unlikely to be prospectively evaluated in the clinics, we took advantage of an INI1-deficient proximal-type ES patient-derived xenograft (PDX ES-1) to comparatively assess its preclinical antitumor activity. Mice were treated with doxorubicin and ifosfamide, singly or in combination, gemcitabine, and the EZH2 inhibitor EPZ-011989. Comparable antitumor activity (max tumor volume inhibition: ~90%) was caused by gemcitabine, EPZ-011989, and the doxorubicin-ifosfamide combination. The integration of RNAseq data, generated on tumors obtained from untreated and EPZ-011989-treated mice, and results from functional studies, carried out on the PDX-derived ES-1 cell line, revealed autophagy induction as a possible survival mechanism in residual tumor cells following EPZ-011989 treatment and identified HMGA2 as a main player in this process. Our data support the clinical use of gemcitabine and the doxorubicin-ifosfamide combination, confirm EZH2 as a therapeutic target in proximal-type ES, and suggest autophagy as a cytoprotective mechanism against EZH2 inhibition.

Even if thyroid tumors are generally curable, a fraction will develop resistance to therapy and progress towards undifferentiated forms, whose treatment remains a demanding challenge. To identify potential novel targets for treatment of thyroid cancer, in a previous study using siRNA-mediated functional screening, we identified several genes that are essential for the growth of thyroid tumor, but not normal cells. Among the top-ranking hits, we found microtubule associated serine/threonine kinase-like (MASTL), which is known to play an essential role in mitosis regulation, and is also involved in the DNA damage response. Herein, we examine the effects of MASTL depletion on growth and viability of thyroid tumor cells. MASTL depletion impaired cell proliferation and increased the percentage of cells presenting nuclear anomalies, which are indicative of mitotic catastrophe. Furthermore, MASTL depletion was associated with enhanced DNA damage. All these effects eventually led to cell death, characterized by the presence of apoptotic markers. Moreover, MASTL depletion sensitized thyroid tumor cells to cisplatin. Our results demonstrate that MASTL represents vulnerability for thyroid tumor cells, which could be explored as a therapeutic target for thyroid cancer., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive, and poorly investigated simple sarcoma with a low frequency of genetic deregulation other than an Ewing sarcoma RNA binding protein 1 (EWSR1)-Wilm's tumor suppressor (WT1) translocation. We used whole-exome sequencing to interrogate six consecutive pre-treated DSRCTs whose gene expression was previously investigated., Methods: DNA libraries were prepared from formalin-fixed, paraffin-embedded archival tissue specimens following the Agilent SureSelectXT2 target enrichment protocol and sequenced on Illumina NextSeq 500. Raw sequence data were aligned to the reference genome with Burrows-Wheeler Aligner algorithm. Somatic mutations and copy number alterations (CNAs) were identified using MuTect2 and EXCAVATOR2, respectively. Biological functions associated with altered genes were investigated through Ingenuity Pathway Analysis (IPA) software., Results: A total of 137 unique somatic mutations were identified: 133 mutated genes were case-specific, and 2 were mutated in two cases but in different positions. Among the 135 mutated genes, 27% were related to two biological categories: DNA damage-response (DDR) network that was also identified through IPA and mesenchymal-epithelial reverse transition (MErT)/epithelial-mesenchymal transition (EMT) already demonstrated to be relevant in DSRCT. The mutated genes in the DDR network were involved in various steps of transcription and particularly affected pre-mRNA. Half of these genes encoded RNA-binding proteins or DNA/RNA-binding proteins, which were recently recognized as a new class of DDR players. CNAs in genes/gene families, involved in MErT/EMT and DDR, were recurrent across patients and mostly segregated in the MErT/EMT category. In addition, recurrent gains of regions in chromosome 1 involving many MErT/EMT gene families and loss of one arm or the entire chromosome 6 affecting relevant immune-regulatory genes were recorded., Conclusions: The emerging picture is an extreme inter-tumor heterogeneity, characterized by the concurrent deregulation of the DDR and MErT/EMT dynamic and plastic programs that could favour genomic instability and explain the refractory DSRCT profile.

Malignant peritoneal mesothelioma (MpM), arising in the setting of local inflammation, is a rare aggressive tumour with a poor prognosis and limited therapeutic options. The three major MpM histological variants, epithelioid (E-MpMs), biphasic, and sarcomatoid MpMs (S-MpMs), are characterised by an increased aggressiveness and enhanced levels of EZH2 expression. To investigate the MpM immune contexture along the spectrum of MpM histotypes, an extended in situ analysis was performed on a series of 14 cases. Tumour-infiltrating immune cells and their functionality were assessed by immunohistochemistry, immunofluorescence, qRT-PCR, and flow cytometry analysis. MpMs are featured by a complex immune landscape modulated along the spectrum of MpM variants. Tumour-infiltrating T cells and evidence for pre-existing antitumour immunity are mainly confined to E-MpMs. However, Th1-related immunological features are progressively impaired in the more aggressive forms of E-MpMs and completely lost in S-MpM. Concomitantly, E-MpMs show also signs of active immune suppression, such as the occurrence of Tregs and Bregs and the expression of the immune checkpoint inhibitory molecules PD1 and PDL1. This study enriches the rising rationale for immunotherapy in MpM and points to the E-MpMs as the most immune-sensitive MpM histotypes, but it also suggests that synergistic interventions aimed at modifying the tumour microenvironment (TME) should be considered to make immunotherapy beneficial for these patients.

Background: We present the results of an academic phase 2 study on imatinib plus everolimus in patients who have progressive advanced chordoma., Methods: In January 2011, 43 adult chordoma patients were enrolled in the study and received imatinib 400 mg/day and everolimus 2.5 mg/day until progression or limiting toxicity. Eligible patients had progressed in the 6 months before study entry. PDGFRB, S6, and 4EBP1 expression and phosphorylation were evaluated by way of immunohistochemistry and/or western blotting. The primary endpoint was the overall response rate (ORR) according to Choi criteria. Secondary endpoints were RECIST 1.1 response, progression-free survival (PFS), overall survival (OS), correlation between S6/4EBP1 phosphorylation and response., Results: Thirteen of 43 patients were pretreated with imatinib. Among 40 of the 43 patients who were evaluable by Choi criteria, the best responses were 9 with partial response (ORR, 20.9%), 24 with stable disease (SD) (ORR, 55.8%), and 7 with progressive disease (ORR, 16.3%). Forty-two patients were evaluable by RECIST criteria, with 1 partial response (ORR, 2.3%), 37 stable disease (ORR, 86%), and 4 progressive disease (ORR, 9.3%). The median PFS according to Choi criteria was 11.5 months (range, 4.6-17.6 months), and 58.8% and 48.1% of patients were progression-free at 9 and 12 months, respectively. The median PFS by RECIST criteria was 14 months; the median OS was 47.1 months. When assessable, S6/4EBP1 was phosphorylated in a high and moderate/low proportion of tumor cells in responsive and nonresponsive patients, respectively. Toxicity caused a temporary and definitive treatment discontinuation in 60.5% and 30.2% of patients, respectively., Conclusions: Imatinib plus everolimus showed a limited activity in progressing advanced chordoma. Interestingly, the amount of tumor cells activated for mammalian target of rapamycin effectors correlated with the response. Toxicity was not negligible., (© 2018 American Cancer Society.)

Solitary fibrous tumors (SFTs) are rare soft tissue sarcomas that rely on several epithelial-mesenchymal transition (EMT) protein regulators for invasion/metastatic progression. Curcumin (CUR) has several pharmacological activities, including anticancer activity and the ability to suppress the EMT process. However, poor absorption, rapid metabolism, and side effects at high doses limit the clinical applications of CUR. Here we present the results obtained by treating SFT cells with free CUR and three different CUR-loaded nanomicelles (NMs), each of which has its surface decorated with different ligands. All CUR-loaded NMs were more efficient in suppressing SFT cell viability and expression of EMT markers than CUR alone. Combined treatments with the pan-histone deacetylase dual inhibitor SAHA revealed a differential ability in inhibiting EMT markers expression and SFT cell invasiveness, depending on the NM-ligand type. Finally, combinations of photodynamic therapy and CUR-loaded NM administrations resulted in almost complete SFT cell viability abrogation 24 h after laser irradiation.

To gain new insights into desmoplastic small round cell tumors (DSRCTs) by means of gene expression profiling (GEP). Formalin-fixed, paraffin-embedded surgical specimens obtained from seven pretreated DSRCT patients were interrogated using GEP complemented by immunohistochemistry, a cancer stem cell array, and miRNA in situ hybridisation, including the combined chimera modules miRNA-200/ZEB1 and miRNA-34/SLUG. The chimera modules divided the cases into three classes that respectively recapitulated the traits of mesenchymal epithelial reverse transition (MErT), epithelial mesenchymal transition (EMT), and hybrid/partial EMT. This indicates a close correlation between the reprogramming governed by EMT regulators and DSRCT biology, which was further confirmed by miRNA-21 and is consistent with the broad morphological spectrum of DSRCTs. Starting from the miRNA-200/ZEB1 axis, we also found that DSRCTs carry a signature of immunological ignorance that is not responsive to PD--L1 blockade. Evidence that the up-regulation of miRNA-200 and E-cadherin, and quite a high level of miRNA-21 expression segregate with the MErT supports the idea that, in addition to the hybrid/partial state, MErT is also enriched in stemness: the androgen-positive cases, whose stemness traits were confirmed by stem cell arrays, all fell into these two classes. Our findings also confirmed that tumoral cell PDGFRA expression correlates with desmoplasia, and demonstrated the co-expression of PDGFRA and ISLR/Meflin, another marker of pluripotency. Despite the limited number of cases, these findings provide unexpectedly relevant information concerning the pathogenesis of DSRCTs, and prove the validity of miRNA-based chimera circuit modelling in the clinico-pathological setting.

The aim of this study was to reconsider the biological characteristics of epithelioid malignant peritoneal mesothelioma (E-MpM) in the light of new concepts about epithelial mesenchymal transition and mesenchymal epithelial reverse transition (EMT/MErT) and the role of epigenetic reprogramming in this context. To this end we profiled surgical specimens and derived cells cultures by a number of complementary approaches i.e. immunohistochemistry, immunofluorescence, in situ hybridization, biochemistry, pluripotent stem cell arrays, treatments with cytokines, growth factors and specific inhibitors.The analyses of the surgical specimens showed that i) EZH2 is expressed throughout the spectrum of MpM, ii) that E-MpM (including the high-grade undifferentiated form) are characterised by c-MYC and miRNA 17-5p expression, and iii) that progression to sarcomatoid MpM is dictated by EMT regulators. They also showed that E-MpM expressed c-MET and are enriched in E- and P-cadherins- and VEGFR2-expressing CSCs, thus strongly supporting a role for MErT reprogramming in endowing E-MpM tumour cells with stemness and plasticity, and hence with a drug resistant phenotype. The cell culture-based experiments confirmed the stemness traits and plasticity of E-MpM, and support the view that EZH2 is a druggable target in this tumor.

Background: The aim of this study was to report on sirolimus activity in a series of patients with hemangioendothelioma (HE) treated at the National Cancer Institute, Milan (Istituto Nazionale Tumori; INT) and within the Italian Rare Cancer Network ("Rete Tumori Rari"; RTR)., Methods: We retrospectively reviewed patients with advanced and progressing epithelioid hemangioendothelioma (EHE) treated with sirolimus at the INT and/or within the RTR. Pathologic review and molecular analysis for WWTR1 rearrangement were performed. Sirolimus was administered until unacceptable toxicity or progression, with the dose being adjusted to reach target plasma levels of 15-20 ng/dL. Responses were assessed using the Response Evaluation Criteria In Solid Tumors (RECIST) criteria., Results: Since 2005, 18 patients (17 EHE, 1 retiform HE; 1 locally advanced, 17 metastatic; WWTR1 rearrangement: 16) have been identified, with 17/18 patients being evaluable for response. Mean sirolimus daily dose was 4.5 mg. According to RECIST, best responses in EHE were 1 partial response (PR), 12 stable disease (SD), and 3 progressive disease (PD); the patient with retiform HE also achieved a PR, lasting >2 years. Four patients with a reversed interval progression on interruption were observed. Median overall survival was 16 months, and median progression-free survival was 12 months (range 1-45), with four patients progression-free at 24 months. The clinical benefit (complete response [CR] + PR + SD >6 months) was 56 %. Seven patients receiving sirolimus experienced an increase in pleural/peritoneal effusion plus worsening of tumor-related symptoms; six of these patients died within 1-8 months from evidence of effusion progression, while a RECIST PD was assessed in two of seven patients., Conclusions: A clinical benefit was achieved in 56 % of patients receiving sirolimus, which lasted >24 months in four patients. Most patients with pleural effusion did not benefit from sirolimus and had a poor outcome.

Unlabelled: Dermatofibrosarcoma protuberans (DFSP) is a rare and indolent cutaneous sarcoma. At times, a fibrosarcomatous transformation marked by a more aggressive clinical behavior may be present. We investigated the natural history and the molecular bases of progression from classic DFSP to the fibrosarcomatous form (FS-DFSP), looking, retrospectively, at the outcome of all patients affected by primary DFSP treated at our institution from 1993 to 2012 and analyzing the molecular profile of 5 DFSPs and 5 FS-DFSPs by an integrated genomics approach (whole transcriptome sequencing, copy number analysis, FISH, qRT-PCR, IHC). The presence of fibrosarcomatous features was identified in 20 (7.6%) patients out of 263 DFSP. All cases were treated with macroscopic complete surgery. A local relapse occurred in 4 of 23 patients who received a microscopic marginal surgery (2 classic DFSP, 2 FS-DFSP), while metastasis affected 2 patients, both FS-DFSP (10% of FS-DFSP), being the first event. DFSP evolution to FS-DFSP was paralleled by a transcriptional reprogramming. The recurrent loss of chromosome 22q appeared to contribute to this phenomenon by promoting the expression of epigenetic regulators, such as EZH2. Loss of the p16/CDKN2A/INK4A locus at 9p was also observed in two FS-DFSP metastatic cases., Implications: FS-DFSP is a rare subgroup among DFSP, with a 10% metastatic risk, that was independent from local recurrence and that was not observed in DFSP, that were all cured by wide surgery. Chromosome 22q deletion might play a role in FS-DFSP, and p16 loss may convey a poor outcome. EZH2 dysregulation was also found and represents a druggable target. Mol Cancer Res; 14(9); 820-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

Sunitinib improves the outcomes of patients with solitary fibrous tumours (SFTs). The aim of this study was to investigate and contextualise sunitinib-induced morpho-functional changes in order to gain insights into the drug's mechanism of action.To this end, four surgical specimens obtained from two sunitinib-responsive patients with malignant SFT, and one primary cell culture obtained from fresh tumoral tissue and its stabilised cell line, were studied by means of immunohistochemistry, bright field in situ hybridisation, immunofluorescence/confocal microscopy, and biochemistry.The post-sunitinib surgical samples were characterised by two biologically relevant morpho-functional changes: clear areas and necrotic foci. The first were associated with the attenuation/loss of PDGFRB expression and decreased mTOR signalling, and corresponded to a pathological response. The second were associated with the over-expression of PDGFRB and VEGFA, strong mTOR signalling activation, and the appearance of HIF1α expression, hallmarks of pathological progression. The analysis clearly showed that sunitinib reduces the vascular supply network and inhibits tumoral cells. It also either induces autophagy, thus favouring drug response, or impairs autophagy as a result of lysosome sequestration, thus favouring disease progression. These distinct autophagic events were associated with different myeloid immune contextures. Finally, we also found that PDGFRB is one of the components of a complex that includes Beclin 1 and VPS34.The results of these tissue-based analyses provide new insights into sunitinib's mechanism of action in SFT patients., Competing Interests: SS declares research funding from Pfizer. All of the other authors declare that they have no conflicts of interest.