Search

Heatmap of key genes in adipogenesis process.

Supplementary data, material and results. Figure S1.1. Overall analysis schema Table S1.1 REMARK form. Figure S1.2 a) Kaplan Meier curves of patients divided by tumor grades (OS and PFS model). The table below summarizes the models used. Figure S1.2 b) Kaplan Meier curves of patients divided in patients who received chemotherapy (Yes) and patients who did not undergo chemotherapy (No). Figure S1.2 c) Kaplan Meier curves of patients divided in FIGO substages. The table below summarizes the models used.

Purpose: Stage I epithelial ovarian cancer (EOC) represents about 10% of all EOCs and is characterized by good prognosis with fewer than 20% of patients relapsing. As it occurs less frequently than advanced-stage EOC, its molecular features have not been thoroughly investigated. We have demonstrated that in stage I EOC miR-200c-3p can predict patients' outcome. In the present study, we analyzed the expression of long non-coding RNAs (lncRNA) to enable potential definition of a non-coding transcriptional signature with prognostic relevance for stage I EOC.Experimental Design: 202 snap-frozen stage I EOC tumor biopsies, 47 of which relapsed, were gathered together from three independent tumor tissue collections and subdivided into a training set (n = 73) and a validation set (n = 129). Median follow up was 9 years. LncRNAs' expression profiles were correlated in univariate and multivariate analysis with overall survival (OS) and progression-free survival (PFS).Results: The expression of lnc-SERTAD2-3, lnc-SOX4-1, lnc-HRCT1-1, and PVT1 was associated in univariate and multivariate analyses with relapse and poor outcome in both training and validation sets (P < 0.001). Using the expression profiles of PVT1, lnc-SERTAD2-3, and miR-200c-3p simultaneously, it was possible to stratify patients into high and low risk. The OS for high- and low-risk individuals are 36 and 123 months, respectively (OR, 15.55; 95% confidence interval, 3.81–63.36).Conclusions: We have identified a non-coding transcriptional signature predictor of survival and biomarker of relapse for stage I EOC. Clin Cancer Res; 23(9); 2356–66. ©2016 AACR.

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.

Myxoid liposarcoma (MLPS) is a rare soft-tissue sarcoma characterised by the expression of FUS-DDIT3 chimera. Trabectedin has shown significant clinical anti-tumour activity against MLPS. To characterise the molecular mechanism of trabectedin sensitivity and of resistance against it, we integrated genomic and transcriptomic data from treated mice bearing ML017 or ML017/ET, two patient-derived MLPS xenograft models, sensitive to and resistant against trabectedin, respectively. Longitudinal RNA-Seq analysis of ML017 showed that trabectedin acts mainly as a transcriptional regulator: 15 days after the third dose trabectedin modulates the transcription of 4883 genes involved in processes that sustain adipocyte differentiation. No such differences were observed in ML017/ET. Genomic analysis showed that prolonged treatment causes losses in 4p15.2, 4p16.3 and 17q21.3 cytobands leading to acquired-resistance against the drug. The results dissect the complex mechanism of action of trabectedin and provide the basis for novel combinatorial approaches for the treatment of MLPS that could overcome drug-resistance.

Glioblastoma (GBM) is the most aggressive type of brain tumor, and existing treatments are inadequate at preventing recurrence. Tumor Treating Fields (TTFields) is a novel treatment that has been proven to extend patients' lives during the previous two decades. The antitumoral impact of TTFields involve different mechanism and is an optional add-on to standard temozolomide maintenance. Although TTFields therapy slows tumor progression initially, it does not prevent tumor relapse in most patients. We subjected patient-derived GBM primary cultures to continuous TTFields stimulation to mimic the GBM standard of care and examine the molecular pathways behind GBM sensitivity to TTFields treatment. TTFields initially limit cellular growth well. However, even under constant TTFields administration, the remaining cells regain their ability to proliferate at their own rate in long-term trials. Prolonged treatment with TTFields increases the expression of many stemness markers as well as the overexpression of different genes involved in cell cycle progression. There is also a rise in membrane ionic conductance activity. After sustained exposure to TTFields, sodium, potassium, and chloride channels are functionally increased. This influences the properties of tumor cells. Increased sodium channel functional activity, for example, promotes membrane potential depolarization. Furthermore, earlier research has shown that the membrane form of the chloride intracellular channel 1 (tmCLIC1) is important in the development of GBM. In patient-derived GBM cells exposed to TTFields, tmCLIC1 is significantly concentrated, and membrane depolarization improves its function. The evaluation of GBM tissues obtained from many patients who were treated with TTFields validates the in vitro tumor relapse model.Our findings imply that the process by which GBM cells develop a decrease in sensitivity to TTFields correlates with an increase in stemness markers and an increase in ionic channel functional expression. Citation Format: Stefania Castiglione, Gaetano Cannavale, Francesca Cianci, Ivan Verduci, Chiara Biella, Chiara Mercurio, Emanuela Pastorelli, Monica Lupi, Laura Paracchini, Laura Mannarino, Dietmar Krex, Federico Brandalise, Michele Mazzanti. TTFields reduce sensitivity in glioblastoma is associated with the functional expression of the chloride intracellular channel 1 and with voltage dependent sodium channel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1425.

Pleural mesothelioma (PM) is an aggressive tumor with few therapeutic options. Although patients with epithelioid PM (ePM) survive longer than non-epithelioid PM (non-ePM), heterogeneity of tumor response in ePM is observed. The role of the tumor immune microenvironment (TIME) in the development and progression of PM is currently considered a promising biomarker. A few studies have used high-throughput technologies correlated with TIME evaluation and morphologic and clinical data. This study aimed to identify different morphological, immunohistochemical, and transcriptional profiles that could potentially predict the outcome. A retrospective multicenter cohort of 129 chemonaive PM patients was recruited. Tissue slides were reviewed by dedicated pathologists for histotype classification and immunophenotype of tumor-infiltrating lymphocytes (TILs) and lymphoid aggregates or tertiary lymphoid structures (TLS). ePM (n = 99) survivors were further classified into long (>36 months) or short (

We have previously demonstrated that longitudinal untargeted analysis of plasma samples withdrawn from patients with high-grade serous ovarian cancer (HGS-EOC) can intercept the presence of molecular recurrence (TRm) earlier than the diagnosis of clinical recurrence (TRc). This finding opens a clinical important temporal window to acquire through plasma sample analysis a real-time picture of those emerging molecular lesions that will drive and sustain the growth of relapsed disease and ultimately will confer resistance. In this proof of principle study, the same genomic libraries obtained at the diagnosis (T0), TRm and TRc were further analyzed by targeted resequencing approach to sequence the coding region of a panel of 65 genes to provide longitudinal analysis of clonal evolution as a novel strategy to support clinical decisions for the second-line treatment. Experiments were performed on plasma and tumor tissues withdrawn on a selection of previously analyzed cohorts of cases (i.e., 33 matched primary and synchronous lesions and 43 plasma samples from 18 patients). At T0, the median concordance of mutations shared by each tumor tissue biopsy and its matched plasma sample was 2.27%. This finding confirms the limit of a single tumor biopsy to be representative of the entire disease, while plasma analysis can recapitulate most of the main molecular lesions of the disease. A comparable scenario was observed during longitudinal analysis, where, with the exception of the TP53 gene and germline mutations in BRCA1/2 genes, no other gene shared the same locus specific gene mutation across T0, TRm and TRc time points. This high level of temporal heterogeneity has important implications for planning second-line treatment. For example, in three out of 13 cases, plasma ctDNA analysis at TRm or TRc reported acquired novel variants in the TP53BP1 gene not present at T0. In particular, patient 21564, potentially eligible for PARP-inhibitor (PARPi) treatment at the time of diagnosis (BRCA1 c.5182delA mutation), would unlikely respond to these drugs in second-line therapy due to the presence of eight distinct TP53BP1 variants in plasma samples collected TRc. This study demonstrates that liquid biopsy provides a real-time molecular picture to intercept those actionable genetic vulnerabilities or drug resistance mechanisms that could be used to plan a more rational second-line treatment.

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.

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.

High-grade serous epithelial ovarian cancer (HGS-EOC) is a systemic disease, with marked intra and interpatient tumor heterogeneity. The issue of spatial and temporal heterogeneity has long been overlooked, hampering the possibility to identify those genomic alterations that persist, before and after therapy, in the genome of all tumor cells across the different anatomical districts. This knowledge is the first step to clarify those molecular determinants that characterize the tumor biology of HGS-EOC and their route toward malignancy. In our study, -omics data were generated from 79 snap frozen matched tumor biopsies, withdrawn before and after chemotherapy from 24 HGS-EOC patients, gathered together from independent cohorts. The landscape of somatic copy number alterations depicts a more homogenous and stable genomic portrait than the single nucleotide variant profile. Genomic identification of significant targets in cancer analysis identified two focal and minimal common regions (FMCRs) of amplification in the cytoband 3q26.2 (region α, 193 kb long) and 8q24.3 (region β, 495 kb long). Analysis in two external databases confirmed regions α and β are features of HGS-EOC. The MECOM gene is located in region α, and 15 genes are in region β. No functional data are yet available for the genes in the β region. In conclusion, we have identified for the first time two FMCRs of amplification in HGS-EOC, opening up a potential biological role in its etiopathogenesis.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with a 5-year survival rate of less than 10% and it remains a cancer lacking effective therapy options when tumors progress. The development of novel drug regimens for MPM treatment have been limited during the last two decades, with the exception of combinations including immune checkpoint inhibitors. In 2019, Tumor Treating Fields (TTFields) in combination with pemetrexed plus platinum-based chemotherapy were approved by the FDA for the first-line treatment of unresectable, locally advanced or metastatic, MPM. However, the mechanism of action of TTFields and their interaction with chemotherapeutic agents are not fully elucidated yet, thus a better knowledge may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients’ thoracic cavity, before administration of therapeutic treatment, obtaining cell lines from the 3 subtypes of MPM. Epithelioid and sarcomatoid subtypes revealed different sensitivities to TTFields, with a stronger impact of the treatment on cell proliferation in the epithelioid subtype. This was associated with a higher modulation of transcription at different times of TTFields exposure, as demonstrated by RNA-seq analysis. Some of the genes modulated by the treatment encode membrane transport proteins, especially of the solute carrier family (SLC), in both sarcomatoid and epithelioid cells. This suggested a possible role of TTFields in cellular drug uptake and efflux. To test this hypothesis two cell lines, the sarcomatoid CD60 and the epithelioid CD473, have been treated with 150 kHz TTFields in combination with doxorubicin (DXR) following different schedules of treatment and the intracellular drug concentrations were assessed by HPLC measurements. TTFields exposure increased cellular uptake of DXR in both MPM models, but the effect was higher in CD473 epithelioid cells. After 24h of treatment with 1 µM DXR the amount of intracellular drug in sarcomatoid cells exposed to TTFields was almost twice that of the samples treated with DXR alone, while in epithelioid cells intracellular DXR concentration was almost four times higher than that found in TTFields untreated samples. This behavior might be ascribed to a different modulation of the gene coding for P-glycoprotein (ABCB1), which was markedly downregulated by TTFields treatment in epithelioid cells only. Similar experiments are in progress to test the impact of TTFields on cellular drug uptake in different combination schedules with other anticancer compounds. These data suggest that TTFields may increase the efficacy of drug treatments by enhancing the intracellular drug concentration and their possible confirmation by further experiments may represent an important rationale for the design of novel MPM therapies. Citation Format: Monica Lupi, Rosy Amodeo, Lavinia Morosi, Laura Mannarino, Lara Paracchini, Sergio Marchini, Federica Grosso, Roberta Libener, Giovanni L. Ceresoli, Maurizio D'Incalci. Tumor treating fields enhance cellular drug uptake in mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 380.

// Lara Paracchini 1, * , Laura Mannarino 1, * , Ilaria Craparotta 1 , Chiara Romualdi 2 , Robert Fruscio 3 , Tommaso Grassi 3 , Vittoria Fotia 4 , Giulia Caratti 1 , Patrizia Perego 5 , Enrica Calura 2 , Luca Clivio 1 , Maurizio D’Incalci 1 , Luca Beltrame 1, § , Sergio Marchini 1, § 1 Department of Oncology, IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy 2 Department of Biology, University of Padova, Padova, Italy 3 Clinic of Obstetrics and Gynaecology, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy 4 PhD program in Experimental Medicine, University of Pavia, Pavia, Italy 5 Pathology Unit University of Milan-Bicocca, San Gerardo Hospital, Monza, Italy * both are first authors § both are last co-authors Correspondence to: Maurizio D’Incalci, email: maurizio.dincalci@marionegri.it Keywords: ovarian cancer, temporal heterogeneity, spatial heterogeneity, targeted next generation sequencing, drug resistance Received: March 01, 2016 Accepted: May 23, 2016 Published: July 09, 2016 ABSTRACT Stage III/IV epithelial ovarian cancer (EOC) is a systemic disease. The clonal relationship among different tumor lesions at diagnosis (spatial heterogeneity) and how tumor clonal architecture evolves over time (temporal heterogeneity) have not yet been defined. Such knowledge would help to develop new target-based strategies, as biomarkers which can adjudge the success of therapeutic intervention should be independent of spatial and temporal heterogeneity. The work described in this paper addresses spatial and temporal heterogeneity in a cohort of 71 tumor biopsies using targeted NGS technology. These samples were taken from twelve high grade serous (HGS) and seven non HSG-EOC, both at the time of primary surgery when the tumor was naive to chemotherapy and after chemotherapy. Matched tumor lesions growing in the ovary or at other anatomical sites show very different mutational landscapes with branched tumor evolution. Mutations in ATM, ATR, TGFB3, VCAM1 and COL3A1 genes were shared across all lesions. BRCA1 and BRCA2 genes were frequently mutated in synchronous lesions of non HGS-EOC. Relapsed disease seems to originate from resistant clones originally present at the time of primary surgery rather than from resistance acquired de novo during platinum based therapy. Overall the work suggests that EOC continues to evolve. More detailed mapping of genetic lesions is necessary to improve therapeutic strategies.

In cancer, myeloid cells have tumor-supporting roles. We reported that the protein GPNMB (glycoprotein nonmetastatic B) was profoundly upregulated in macrophages interacting with tumor cells. Here, using mouse tumor models, we show that macrophage-derived soluble GPNMB increases tumor growth and metastasis in Gpnmb-mutant mice (DBA/2J). GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. Through the CD44 receptor, GPNMB mechanistically activates tumor cells to express the cytokine IL-33 and its receptor IL-1R1L. We also determined that recombinant IL-33 binding to IL-1R1L is sufficient to induce tumor spheroid formation with features of cancer stem cells. Overall, our results reveal a new paracrine axis, GPNMB and IL-33, which is activated during the cross talk of macrophages with tumor cells and eventually promotes cancer cell survival, the expansion of cancer stem cells and the acquisition of a metastatic phenotype.

Summary About one-third of endometrial carcinomas (ECs), mainly of endometrioid histology, harbor the mismatch repair (MMR) defects and microsatellite instability (MSI). Among these, ECs arising in women with Lynch syndrome (LS) account for a large proportion. To date, no somatic genetic analyses have been published comparing LS-ECs with sporadic ECs. In this work, we examined the mutational profiles of a well-characterized series of sporadic and LS-related ECs, performing exonic targeted sequencing of 16 genes mainly involved in MSI ECs. Next-generation sequencing analysis was performed in 35 ECs on the MiSeq platform (Illumina, San Diego, CA), and the mutational profile was analyzed integrating molecular and immunohistochemical data. PTEN, ARID1A, and ARID2 were the most frequently mutated genes regardless of MSI status or family history. MSI ECs showed a higher mutational load than MMR-proficient cases, exhibiting an MMR-deficient mutational signature. Among MSI tumors, LS-related and sporadic ECs exhibited similar mutational profiles, with MSH2 as the most commonly mutated gene. KRAS mutations seemed to be more common in sporadic MSI ECs than in LS-related ECs even if further studies are needed to confirm this finding. MMR-deficient ECs carried a higher mutational load and an excess of C>T transitions compared with MMR-proficient ECs, suggesting that the use of a small gene panel may be adequate to highlight significant differences between these 2 groups. An integrated analysis of genetic and epigenetic features of LS-related and sporadic ECs provides useful insights into disease biology and diagnostic classification of these tumors.

Background: Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells. Methods: In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice. Results: Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes. Conclusions: The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.

Despite the increasing knowledge of the biology of malignant pleural mesothelioma (MPM), patients with this diagnosis still have a poor prognosis with a 5-year survival rate of lower than 10%. The effectiveness demonstrated by Tumor Treating Fields (TTFields) in combination with chemotherapy in preclinical experiments and in MPM patients (NCT02397928) affirms TTFields as a promising therapeutic tool. Knowledge of the mechanism of action of TTFields and its interaction with chemotherapeutic agents may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients' thoracic cavity, before administration of therapeutic treatment, obtaining six cell lines from the 3 types of MPM cells: epithelioid CD473 and CD484, sarcomatoid CD60 and CD432, and biphasic CD487 and CD491. RNAseq analysis revealed that the transcriptional profiles of CD473 and CD60 cells were comparable with those derived from patient biopsies of the same histotype, representing clinically relevant MPM models. Antiproliferative effects induced by TTFields alone (1.12 V/cm; 150 KHz; for 96h duration) were studied in these MPM cell lines. Cell survival and cell cycle perturbations analyses during and after treatment were performed. Consistent with clinical observations, epithelioid MPM cells demonstrated greater sensitivity to TTFields than biphasic and sarcomatoid MPM cells. Cell cycle analysis revealed that the former cell type was blocked in G2M phase and was followed by formation of polyploid cells; while the latter cell types were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptotic cells were present in all treated samples, but while epithelioid and biphasic cell death was already observed during the first 24h of treatment, sarcomatoid cells needed to be treated for at least 24h before starting apoptotic pathways. The antiproliferative effect observed in epithelioid cells was persistent even after treatment cessation, whereas biphasic and sarcomatoid cells were able to reinitiate growth upon TTFields cessation. RNAseq experiments were performed in CD473 and CD60 cells at different times during TTFields application to explore transcriptional modifications that may explicate these varied MPM cell responses. Preliminary data suggested that TTFields induced a transcriptional response already detectable at earlier time points of treatment (8h; maximum effect at 24h). The number of differentially expressed genes was higher in CD473 relative to CD60 cells (1951 in CD473 and 336 in CD60 at 24h), involving several pathways, that need to be further investigated. These data demonstrate that TTFields induce specific effects on cell proliferation in varied subsets of mesothelioma cells and provide a mechanistic rationale for future therapies and combinations with other anticancer drugs for MPM treatment. Citation Format: Monica Lupi, Federica Mirimao, Nicolò Panini, Greta Piazza, Lara Paracchini, Laura Mannarino, Sergio Marchini, Federica Grosso, Serena Penpa, Antonio Maconi, Giovanni L. Ceresoli, Maurizio D'Incalci. Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1064.

This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.

Malignant pleural mesothelioma (MPM) is a rare malignancy mainly caused by asbestos exposure. Germinal and acquired mutations in genes of DNA repair pathways, in particular of homologous recombination repair, are frequent in MPM. Here we overview the available experimental data suggesting that an impaired DNA repair system affects MPM pathogenesis by leaving lesions through the genome unresolved. DNA repair defects represent a vulnerability of MPM, and it seems plausible to propose that leveraging these deficiencies could have therapeutic potential for patients with MPM, for whom there is an urgent need of more effective therapies.

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.Through repeated in vivo treatment in athymic nude mice, we have obtained a patient-derived xenograft with acquired resistance to trabectedin.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.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.

Combined surgical-orthodontic treatment of impacted maxillary canines has developed significantly in the last few years, regarding management of both hard and soft tissues and forces of traction. The aim of this report is to describe a combined surgical-orthodontic approach used to treat an impacted maxillary canine and to value the functional and esthetic results after 5 years of followup.A 13-year-old boy had been seen by surgeons in the Operative Unit of Orthodontics of Policlinico Tor Vergata in Rome. Radiographic images showed intraosseous impaction of teeth 1.3 and 2.3 in a late mixed dentition, and the patient was scheduled for the combined surgical-orthodontic treatment.After the five-year follow-up, the patient had a good occlusal stability. The maxillary canine that had been orthodontically repositioned showed an adequate width of attached gingiva, which was well keratinized, and the margin of free gingiva that followed the course of the cement-enamel junction. Bleeding was absent on probing, the periodontal pocket depth was4 mm, and there was no radiographically evident bone loss.The combined surgical-orthodontic technique used in this case (closed eruption towards the center of the alveolar ridge associated with conservative periodontal surgery, the adhesive technique, and controlled orthodontic traction) simulates physiological tooth eruption and results in proper alignment with good periodontal results. It should be considered as the treatment of choice for impacted teeth whose eruption is not precluded by the position of the tooth and/or the presence of ankyloses.Mucoperiosteal flap, Surgical tecnique.Il trattamento combinato chirurgico-ortodontico per il recupero dei canini mascellari inclusi si è sviluppato notevolmente negli ultimi anni, considerando non solo il management dei tessuti duri e molli, ma anche delle forze di trazione. Lo scopo del presente lavoro è descrivere una tecnica combinata chirurgico-ortodontica per il trattamento di un canino incluso mascellare, con una tecnica chirurgica minimamente invasiva, valutandone i risultati funzionali ed estetici nel follow-up a medio termine (5 anni).P.B., di 13 anni, alle indagini radiografi che si evidenzia la malposizione intraossea degli elementi 13 e 2.3 in una dentizione mista tardiva e si è deciso pertanto. Per il suo recupero in arcata attraverso un approccio combinato ortodontico-chirurgico.Nel follow-up a 5 anni il paziente si è presentato con una buona stabilità occlusale. Gli elementi riposizionati ortodonticamente in arcata presentano adeguata banda di gengiva aderente ben cheratinizzata porzione che da un ottimo spessore ed il margine della gengiva libera che segue l’andamento della giunzione amelo- cementizia, assenza di sanguinamento al sondaggio, una profondità di tasca4 mm e assenza di perdita ossea visibile radiografi camente.Il trattamento combinato chirurgico ortodontico (eruzione “chiusa” verso il centro della cresta alveolare con l’applicazione dei principi di una chirurgia conservativa, della tecnica adesiva e della trazione ortodontica controllata) simula l’eruzione fisiologica dentale, ottenendo un corretto allineamento in arcata associato a condizioni di salute parodontale. Dovrebbe quindi essere considerato come trattamento di scelta per i denti inclusi, almeno quando la loro posizione e l’assenza di anchilosi permetta la loro eruzione.

The reported clinical activity of lurbinectedin in Small Cell Lung Cancer (SCLC) prompted us to investigate the selective mechanism of this drug in SCLC cell lines. Since lurbinectedin binds the DNA minor groove modifying DNA structure, we hypothesized that it can interfere with transcription regulation. Using SHP-77 SCLC cell line, we investigated the effect of lurbinectedin on transcriptomic profile by microarray approach, at drug concentration that reduced the cell growth rate by 50%. We found only 89 differently expressed genes (DEG), 31 up- regulated and 58 down-regulated, after 6 hours of treatment whereas there was a dramatic change in gene expression profiles after 24 hours (4529 DEGs of which 1982 up-regulated and 2547 down-regulated) and 48 hours (5457 DEGs of which 2368 up-regulated and 3089 down-regulated) of exposure. Among the genes that were clearly down-regulated at both 24 and 48 hours, there was ASCL1, a gene that encodes a transcription factor required for proper development of neuronal and pulmonary neuroendocrine cells. ASCL1 regulates stemness and cell cycle progression and it is increased in high grade neuroendocrine tumours including SCLC. Western blot analysis confirmed strong reduction of the ASCL1 protein level in SHP-77 cell line. From literature data we retrieved the ChIP-Seq target genes of ASCL1 derived from different SCLC cell lines and compared them to the list of genes at basal condition in SHP-77 cell line. Then we computed Pearson correlation between the expression of ASCL1 and these targets and filtered only strong positive correlations. Through this approach, we finally selected 213 target genes of ASCL1 many of which were coherently inhibited after 24 and 48 hours of treatment. These genes were mainly involved in MAPK and Wnt signalling pathways, both crucial for SCLC. The mechanism of transcription modulation driven by ASCL1 after lurbinectedin treatment is under investigation by ChIP-Seq technology. Moreover, work is in progress to extend our findings on others SCLC cell lines and in sub-lines in which ASCL1 was silenced. We tested whether lurbinectedin shared a similar effect on transcriptomic profile as other known drugs: by dividing up and down regulated genes and testing their correlation to the drug-related gene profiles provided by the tool Enrichr, we found that lurbinectedin has a high correlation to the topoisomerase I inhibitor class. This finding deserves further experimental verifications also in view of potential clinical interest in the combination of lurbinectedin and topoisomerase I inhibitors. Citation Format: Laura Mannarino, Ilaria Craparotta, Federica Mirimao, Nicolò Panini, Monica Lupi, Giulia Protti, Roberta Frapolli, Sergio Marchini, Maurizio D'Incalci. Lurbinectedin down-regulates ASCL1 transcription factor in Small Cell Lung Cancer (SCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B13. doi:10.1158/1535-7163.TARG-19-LB-B13

Purpose: Stage I epithelial ovarian cancer (EOC) represents about 10% of all EOCs and is characterized by good prognosis with fewer than 20% of patients relapsing. As it occurs less frequently than advanced-stage EOC, its molecular features have not been thoroughly investigated. We have demonstrated that in stage I EOC miR-200c-3p can predict patients' outcome. In the present study, we analyzed the expression of long non-coding RNAs (lncRNA) to enable potential definition of a non-coding transcriptional signature with prognostic relevance for stage I EOC. Experimental Design: 202 snap-frozen stage I EOC tumor biopsies, 47 of which relapsed, were gathered together from three independent tumor tissue collections and subdivided into a training set (n = 73) and a validation set (n = 129). Median follow up was 9 years. LncRNAs' expression profiles were correlated in univariate and multivariate analysis with overall survival (OS) and progression-free survival (PFS). Results: The expression of lnc-SERTAD2-3, lnc-SOX4-1, lnc-HRCT1-1, and PVT1 was associated in univariate and multivariate analyses with relapse and poor outcome in both training and validation sets (P < 0.001). Using the expression profiles of PVT1, lnc-SERTAD2-3, and miR-200c-3p simultaneously, it was possible to stratify patients into high and low risk. The OS for high- and low-risk individuals are 36 and 123 months, respectively (OR, 15.55; 95% confidence interval, 3.81–63.36). Conclusions: We have identified a non-coding transcriptional signature predictor of survival and biomarker of relapse for stage I EOC. Clin Cancer Res; 23(9); 2356–66. ©2016 AACR.

Background: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN. Methods: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines. Results: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models. Conclusions: Trabectedin could be good candidate for clinical studies in JMML/CMML patients.

Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen.One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis.Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-β activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P0.001).This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.