Publications by authors named "Silvia Brich"

34 Publications

Selinexor versus doxorubicin in dedifferentiated liposarcoma PDXs: evidence of greater activity and apoptotic response dependent on p53 nuclear accumulation and survivin down-regulation.

J Exp Clin Cancer Res 2021 Mar 1;40(1):83. Epub 2021 Mar 1.

Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale Tumori, Via Amadeo 42, 20133, Milan, Italy.

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.
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http://dx.doi.org/10.1186/s13046-021-01886-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923610PMC
March 2021

Antimicrobial activity of amphiphilic nanomicelles loaded with curcumin against Pseudomonas aeruginosa alone and activated by blue laser light.

J Biophotonics 2021 Mar 18;14(3):e202000350. Epub 2020 Nov 18.

Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy.

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 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.
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http://dx.doi.org/10.1002/jbio.202000350DOI Listing
March 2021

Prognostic impact of immune-microenvironment in colorectal liver metastases resected after triplets plus a biologic agent: A pooled analysis of five prospective trials.

Eur J Cancer 2020 08 15;135:78-88. Epub 2020 Jun 15.

Unit of Medical Oncology 2, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy; Department of Translational Research and New Technology in Medicine and Surgery, University of Pisa, Pisa, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejca.2020.04.045DOI Listing
August 2020

Correction: Establishment and characterisation of a new patient-derived model of myxoid liposarcoma with acquired resistance to trabectedin.

Br J Cancer 2020 Mar;122(7):1120

Laboratory of Cancer Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156, Milan, Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41416-020-0746-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109093PMC
March 2020

Cancer Associated Fibroblasts and Senescent Thyroid Cells in the Invasive Front of Thyroid Carcinoma.

Cancers (Basel) 2020 Jan 1;12(1). Epub 2020 Jan 1.

Molecular Mechanisms Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan 20133, Italy.

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 and 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, and was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in 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.
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http://dx.doi.org/10.3390/cancers12010112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016563PMC
January 2020

Molecular Signatures for Combined Targeted Treatments in Diffuse Malignant Peritoneal Mesothelioma.

Int J Mol Sci 2019 Nov 19;20(22). Epub 2019 Nov 19.

Laboratory of Molecular Pathology, Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, via Venezian 1, 20133 Milan, Italy.

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.
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http://dx.doi.org/10.3390/ijms20225817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888071PMC
November 2019

Fluorescence in situ hybridization (FISH) provides estimates of minute and interstitial BAP1, CDKN2A, and NF2 gene deletions in peritoneal mesothelioma.

Mod Pathol 2020 02 30;33(2):217-227. Epub 2019 Sep 30.

Department of Pathology, Laboratory of Experimental Molecular Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

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.
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http://dx.doi.org/10.1038/s41379-019-0371-0DOI Listing
February 2020

Combination of PPARγ Agonist Pioglitazone and Trabectedin Induce Adipocyte Differentiation to Overcome Trabectedin Resistance in Myxoid Liposarcomas.

Clin Cancer Res 2019 12 3;25(24):7565-7575. Epub 2019 Sep 3.

Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy.

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 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.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0976DOI Listing
December 2019

Establishment and characterisation of a new patient-derived model of myxoid liposarcoma with acquired resistance to trabectedin.

Br J Cancer 2019 09 14;121(6):464-473. Epub 2019 Aug 14.

Laboratory of Cancer Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156, Milan, Italy.

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.
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http://dx.doi.org/10.1038/s41416-019-0550-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738121PMC
September 2019

Pazopanib for treatment of advanced extraskeletal myxoid chondrosarcoma: a multicentre, single-arm, phase 2 trial.

Lancet Oncol 2019 09 19;20(9):1252-1262. Epub 2019 Jul 19.

Department of Medical Oncology, University Hospital Virgen del Rocio, Seville, Spain; Institute of Biomedicine of Sevilla, Universidad de Sevilla, Seville, Spain.

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.
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http://dx.doi.org/10.1016/S1470-2045(19)30319-5DOI Listing
September 2019

Comparative Assessment of Antitumor Effects and Autophagy Induction as a Resistance Mechanism by Cytotoxics and EZH2 Inhibition in INI1-Negative Epithelioid Sarcoma Patient-Derived Xenograft.

Cancers (Basel) 2019 Jul 19;11(7). Epub 2019 Jul 19.

Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale Tumori, Via Amadeo 42, 20133 Milan, Italy.

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.
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http://dx.doi.org/10.3390/cancers11071015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678245PMC
July 2019

NR4A3 fusion proteins trigger an axon guidance switch that marks the difference between EWSR1 and TAF15 translocated extraskeletal myxoid chondrosarcomas.

J Pathol 2019 09 14;249(1):90-101. Epub 2019 May 14.

Unit of Oncogenetics and Functional Oncogenomics, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, National Cancer Institute, Aviano, Italy.

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.
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http://dx.doi.org/10.1002/path.5284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766969PMC
September 2019

Identification of mutations associated with acquired resistance to sunitinib in renal cell cancer.

Int J Cancer 2019 10 30;145(7):1991-2001. Epub 2019 Mar 30.

Department of Oncology, Clinica Universidad de Navarra, Pamplona, Spain.

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.
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http://dx.doi.org/10.1002/ijc.32256DOI Listing
October 2019

Complex Immune Contextures Characterise Malignant Peritoneal Mesothelioma: Loss of Adaptive Immunological Signature in the More Aggressive Histological Types.

J Immunol Res 2018 29;2018:5804230. Epub 2018 Oct 29.

Unit of Immunotherapy of Human Tumours, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

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.
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http://dx.doi.org/10.1155/2018/5804230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231377PMC
January 2019

The genomics of desmoplastic small round cell tumor reveals the deregulation of genes related to DNA damage response, epithelial-mesenchymal transition, and immune response.

Cancer Commun (Lond) 2018 11 28;38(1):70. Epub 2018 Nov 28.

Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, 20133, Milan, Italy.

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.
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http://dx.doi.org/10.1186/s40880-018-0339-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260689PMC
November 2018

Mitosis perturbation by MASTL depletion impairs the viability of thyroid tumor cells.

Cancer Lett 2019 02 14;442:362-372. Epub 2018 Nov 14.

Molecular Mechanisms Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.canlet.2018.11.010DOI Listing
February 2019

Imatinib and everolimus in patients with progressing advanced chordoma: A phase 2 clinical study.

Cancer 2018 10 14;124(20):4056-4063. Epub 2018 Sep 14.

Medical Oncology Unit 2, Medical Oncology Department, Fondazione IRCCS Istituto Nazione dei Tumori, Milan, Italy.

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.
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http://dx.doi.org/10.1002/cncr.31685DOI Listing
October 2018

Self-Assembled Nanomicelles as Curcumin Drug Delivery Vehicles: Impact on Solitary Fibrous Tumor Cell Protein Expression and Viability.

Mol Pharm 2018 10 17;15(10):4689-4701. Epub 2018 Sep 17.

Molecular Biology and Nanotechnology Laboratory (MolBNL@Units), DEA , University of Trieste , 34127 Trieste , Italy.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00655DOI Listing
October 2018

New transcriptional-based insights into the pathogenesis of desmoplastic small round cell tumors (DSRCTs).

Oncotarget 2017 May;8(20):32492-32504

Department of Diagnostic Pathology and Laboratory Medicine, Laboratory of Experimental Molecular Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

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.
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http://dx.doi.org/10.18632/oncotarget.16477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464804PMC
May 2017

Epithelioid peritoneal mesothelioma: a hybrid phenotype within a mesenchymal-epithelial/epithelial-mesenchymal transition framework.

Oncotarget 2016 11;7(46):75503-75517

Laboratory of Experimental Molecular Pathology, Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

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.
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http://dx.doi.org/10.18632/oncotarget.12262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342756PMC
November 2016

Sirolimus in Advanced Epithelioid Hemangioendothelioma: A Retrospective Case-Series Analysis from the Italian Rare Cancer Network Database.

Ann Surg Oncol 2016 09 22;23(9):2735-44. Epub 2016 Jun 22.

Medical Oncology Unit 2 - Adult Mesenchymal Tumours and Rare Cancers, Department of Cancer Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.

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.
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http://dx.doi.org/10.1245/s10434-016-5331-zDOI Listing
September 2016

Sunitinib-induced morpho-functional changes and drug effectiveness in malignant solitary fibrous tumours.

Oncotarget 2016 Jul;7(29):45015-45026

Laboratory of Experimental Molecular Pathology, Department of Diagnostic Pathology and Laboratory, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

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.
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http://dx.doi.org/10.18632/oncotarget.7523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216702PMC
July 2016

Evolution of Dermatofibrosarcoma Protuberans to DFSP-Derived Fibrosarcoma: An Event Marked by Epithelial-Mesenchymal Transition-like Process and 22q Loss.

Mol Cancer Res 2016 09 2;14(9):820-9. Epub 2016 Jun 2.

Unit of Experimental Oncology 1, CRO Aviano National Cancer Institute, Aviano, Italy.

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.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0068DOI Listing
September 2016

Identification of a gene expression driven progression pathway in myxoid liposarcoma.

Oncotarget 2014 Aug;5(15):5965-77

Laboratory of Experimental Molecular Pathology, Department of Diagnostic Pathology and Laboratory, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan Italy.

Aim: to investigate the events involved in the progression of myxoid liposarcoma (MLS). Gene expression profiling and immunohistochemical/biochemical analyses were applied to specimens representative of the opposite ends of the MLS spectrum: pure myxoid (ML) and pure round cell (RC) liposarcomas. The analyses revealed the involvement of both coding and non coding RNAs (SNORDs located in DLK1-DIO3 region) and support a model of stepwise progression mainly driven by epigenetic changes involving tumour vascular supply and tumoral cellular component. In this model, a switch in the vascular landscape from a normal to a pro-angiogenic signature and the silencing of DLK1-DIO3 region mark the progression from ML to RC in concert with the acquisition by the latter of the over-expression of YYI/C-MYC/HDAC2, together with over-expression of genes involved in cell proliferation and stemness: MKNK2, MSX1 and TRIM71. Taken together, these findings strongly suggest that to progress from ML to RC liposarcoma the cells have to overcome the epigenetic silencing restriction point in order to reset their new stem-like differentiation signature. Our findings provide a first attempt at identifying the missing links between ML and RC liposarcomas, that may also have broader applications in other clinico-pathological settings characterised by a spectrum of progression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4171605PMC
http://dx.doi.org/10.18632/oncotarget.2023DOI Listing
August 2014

Antiangiogenic activity of trabectedin in myxoid liposarcoma: involvement of host TIMP-1 and TIMP-2 and tumor thrombospondin-1.

Int J Cancer 2015 Feb 19;136(3):721-9. Epub 2014 Jun 19.

Tumor Angiogenesis Unit, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy.

Trabectedin is a marine natural product, approved in Europe for the treatment of soft tissue sarcoma and relapsed ovarian cancer. Clinical and experimental evidence indicates that trabectedin is particularly effective against myxoid liposarcomas where response is associated to regression of capillary networks. Here, we investigated the mechanism of the antiangiogenic activity of trabectedin in myxoid liposarcomas. Trabectedin directly targeted endothelial cells, impairing functions relying on extracellular matrix remodeling (invasion and branching morphogenesis) through the upregulation of the inhibitors of matrix metalloproteinases TIMP-1 and TIMP-2. Increased TIMPs synthesis by the tumor microenvironment following trabectedin treatment was confirmed in xenograft models of myxoid liposarcoma. In addition, trabectedin upregulated tumor cell expression of the endogenous inhibitor thrombospondin-1 (TSP-1, a key regulator of angiogenesis-dependent dormancy in sarcoma), in in vivo models of myxoid liposarcomas, in vitro cell lines and primary cell cultures from patients' myxoid liposarcomas. Chromatin Immunoprecipitation analysis showed that trabectedin displaced the master regulator of adipogenesis C/EBPβ from the TSP-1 promoter, indicating an association between the up-regulation of TSP-1 and induction of adipocytic differentiation program by trabectedin. We conclude that trabectedin inhibits angiogenesis through multiple mechanisms, including directly affecting endothelial cells in the tumor microenvironment--with a potentially widespread activity--and targeting tumor cells' angiogenic activity, linked to a tumor-specific molecular alteration.
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http://dx.doi.org/10.1002/ijc.29023DOI Listing
February 2015

Receptor tyrosine kinase pathway analysis sheds light on similarities between clear-cell sarcoma and metastatic melanoma.

Genes Chromosomes Cancer 2012 Feb 1;51(2):111-26. Epub 2011 Nov 1.

Laboratory of Molecular Pathology, Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

To highlight possible similarities and differences in receptor tyrosine kinase (RTK) and downstream signalling activation profiles between clear-cell sarcomas (CCS) and metastatic melanomas (MM), frozen, and paired-matched fixed samples of six CCS with EWSR1 rearrangement (EWSR1+), five CCS without EWSR1 rearrangement (EWSR1-), and seven MM were investigated by means of biochemical, immunohistochemical, FISH, molecular analyses, and immunofluorescence confocal microscopy. Fixed samples of a further 10 CCS and 14 MM were investigated by means of sequencing for BRAF, NRAS, and KRAS mutations and FISH analyses for the gain of chromosomes 22 and 8. RTK analysis of all CCS/MM samples showed activation of short-form (sf) recepteur d'origine nantais (RON) RTK and of PDGFRB, MET, and HER3. Analysis of downstream signaling revealed consistent phosphorylation patterns of PI3K/AKT, RSK, and the mTOR targets S6 and 4EBP1. Analysis of frozen and fixed material from 21 CCS and 21 MM showed the presence of the V600E BRAF mutation in 2/12 EWSR1+ and 3/9 EWSR1- CCS and 9/21 MM and demonstrated a significant (P < 0.001) correlation between the gain of chromosomes 22 and 8 and EWSR1- CCS. Our results show that BRAF mutation can also be present in CCS and support the proposed aberration of chromosomes 22 and 8 as a possibly useful nonrandom hallmark of EWSR1- CCS. Besides, they broaden the spectrum of the similarities of RTK pathway activation between CCS and MM, thus suggesting that new drugs found to be active in melanoma and RON inhibitors could have a role in CCS treatment. © 2011 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/gcc.20933DOI Listing
February 2012

High CD133 expression levels in gastrointestinal stromal tumors.

Cytometry B Clin Cytom 2011 Jul-Aug;80(4):238-47. Epub 2011 Apr 1.

Laboratory of Experimental Molecular Pathology, Department of Pathology, Fondazione IRCCS, Istituto Nazionale Tumori, Milan, Italy.

Background: Gastrointestinal stromal tumours (GISTs) have activating KIT or PDGFRA gene mutations. Imatinib mesylate, which targets KIT and PDGFRA, is effective in treating GISTs, but 90% of GIST patients become imatinib-resistant as a result of acquiring secondary KIT mutations. Recent findings suggest that tumour growth can be driven by mutated self-renewing progenitors known as cancer stem cells (CSCs), which are believed to be present in all neoplastic proliferations and are thought to accumulate mutations. It is therefore possible that the acquisition of secondary KIT mutations during imatinib treatment may occur in putative GIST CSCs.

Methods: Using flow cytometry, in vivo murine xenografts and molecular characterization, we tried to identify putative GIST CSCs by looking for the occurrence of common CSC markers such as KIT, CD133, CD90, CD44, and CD34 in 18 surgical samples obtained from nine untreated and nine imatinib-treated KIT-mutated GIST patients.

Results: The results indicated the homogeneous and previously unreported expression of CD133 (18/18), CD90 (15/16), and CD44 (12/14), together with KIT (18/18) and CD34 (13/18). This profile is similar to that identified in bone marrow mesenchymal progenitors and does not seem to be significantly modified by imatinib as only marginal changes in KIT and CD133 expression (P ≤ 0.05, Mann-Whitney test) were found in the treated samples.

Conclusions: These findings suggest that GISTs are a clonal expansion of quite primitive cells that strictly depend on KIT oncogenic addiction, and have no cancer/stem cell component that can be detected by means of the antigens used in this study.
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http://dx.doi.org/10.1002/cyto.b.20589DOI Listing
October 2011

Fluorescence in situ hybridization analysis and immunophenotyping of c-Kit/PDGFRA and Bcl-2 expression in gastrointestinal stromal tumors.

Anal Quant Cytol Histol 2010 Aug;32(4):225-33

Unit of Experimental Molecular Pathology, Department of Pathology, Fondazione Istituto di Ricerca e di Cura a Carattere Scientifico (IRCCS), Istituto Nazionale dei Tumori, Milan, Italy.

Objective: To investigate c-Kit/PDGFRA genomic alterations, KIT-PDGFRA coexpression in gastrointestinal stromal tumors (GISTs) and the role of Bcl-2.

Study Design: We analyzed 70 primary tumors, 6 recurrences, 4 metastases and 1 recurrence plus metastasis, all molecularly characterized. Alterations in gene copy number were detected by fluorescence in situ hybridization (FISH) and expression of KIT, PDGFRA and Bcl-2 by immunohistochemistry.

Results: c-Kit/PDGFRA gene alterations affected 38% of all cases and 39% of primary tumors, with major changes accounting for 15% in both all the cases and primary tumors. Cytoplasmic KIT/PDGFRA coexpression was present in 96.5% of the c-Kit-mutated cases, 100% of the wt c-Kit/PDGFRA cases and 66.6% of the PDGFRA-mutated cases. Bcl-2 immunoreactivity was present in 70% of cases, with expression levels of +++ in 29%, ++ in 38% and + in 33%.

Conclusion: FISH confirmed cytogenetic alterations in about 40% of primary GISTs at the onset. The high rate of c-Kit/PDGFRA coexpression suggests that both receptors are involved in oncogenicity and may affect imatinib efficacy. The assumption that Bcl-2 expression is supported by the KIT pathway and that its imatinib-mediated down-regulation contributes to autophagic cell death, although attractive, needs to be further confirmed.
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August 2010

Receptor tyrosine kinase and downstream signalling analysis in diffuse malignant peritoneal mesothelioma.

Eur J Cancer 2010 Oct 6;46(15):2837-48. Epub 2010 Aug 6.

Experimental Molecular Pathology, Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Via G. Venezian 1, 20133 Milano, Italy.

Our aim was to assess the activation profile of EGFR, PDGFRB and PDGFRA receptor tyrosine kinases (RTK) and their downstream effectors in a series of cryopreserved diffuse malignant peritoneal mesothelioma (DMPM) surgical specimens to discover the targets for drug inhibition. We also made a complementary analysis of the cytotoxic effects of some kinase inhibitors on the proliferation of the human peritoneal mesothelioma STO cell line. We found the expression/phosphorylation of EGFR and PDGFRB in most of the tumours, and PDGFRA activation in half. The expression of the cognate ligands TGF-α, PDGFB and PDGFA in the absence of RTK mutation and amplification suggested the presence of an autocrine/paracrine loop. There was also evidence of EGFR and PDGFRB co-activation. RTK downstream signalling analysis demonstrated the activation/expression of ERK1/2, AKT and mTOR, together with S6 and 4EBP1, in almost all the DMPMs. No KRAS/BRAF mutations, PI3KCA mutations/amplifications or PTEN inactivation were observed. Real-time polymerase chain reaction revealed the decreased expression of TSC1 c-DNA in half of the tumours. In vitro cytotoxicity studies showed the STO cell line to be resistant to gefitinib and sensitive to sequential treatment with RAD001 and sorafenib; these findings were consistent with the presence of the KRAS mutation G12D in these cells although it was not detectable in the original tumour. Our results highlight the ligand-dependent activation and co-activation of EGFR and PDGFRB, as well as a connection between these activated RTKs and the downstream mTOR pathway, thus supporting the role of combined treatment with RTK and mTOR inhibitors in DMPM.
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http://dx.doi.org/10.1016/j.ejca.2010.06.130DOI Listing
October 2010