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

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.

Solitary fibrous tumors: loss of chimeric protein expression and genomic instability mark dedifferentiation.

Solitary fibrous tumors, which are characterized by their broad morphological spectrum and unpredictable behavior, are rare mesenchymal neoplasias that are currently divided into three main variants that have the NAB2-STAT6 gene fusion as their unifying molecular lesion: usual, malignant and dedifferentiated solitary fibrous tumors. The aims of this study were to validate molecular and immunohistochemical/biochemical approaches to diagnose the range of solitary fibrous tumors by focusing on the dedifferentiated variant, and to reveal the genetic events associated with dedifferentiation by integrating the findings of array comparative genomic hybridization. We studied 29 usual, malignant and dedifferentiated solitary fibrous tumors from 24 patients (including paired samples from five patients whose tumors progressed to the dedifferentiated form) by means of STAT6 immunohistochemistry and (when frozen material was available) reverse-transcriptase polymerase chain reaction and biochemistry. In addition, the array comparative genomic hybridization findings were used to profile 12 tumors from nine patients. The NAB2/STAT6 fusion was detected in all of the tumors, but immunohistochemistry and western blotting indicated that chimeric protein expression was atypical or absent in 9 out of 11 dedifferentiated tumors. The comparative genomic hybridization results revealed that the usual and malignant solitary fibrous tumors had a simple profile, whereas the genome of the dedifferentiated tumors was complex and unstable, and suggested that 13q and 17p deletions and TP53 mutations may be present in malignant lesions before the full expression of a dedifferentiated phenotype. Solitary fibrous tumor dedifferentiation is associated with the loss of chimeric oncoprotein expression, genomic instability, and cell decommitment and reprogramming. The assessment of dedifferentiated solitary fibrous tumors is based on the presence of the fusion transcripts and, in principle, negative STAT6 immunohistochemistry should not rule out a diagnosis of solitary fibrous tumor.

Melan-A/MART-1 immunity in a EWS-ATF1 translocated clear cell sarcoma patient treated with sunitinib: a case report.

BACKGROUND: Clear cell sarcoma (CCS), initially named malignant melanoma of soft parts, is an aggressive soft tissue sarcoma (STS) that, due to MITF activation, shares with melanoma the expression of melanocyte differentiation antigens. CCS is poorly sensitive to chemotherapy. Multi-kinase inhibitors have been used as therapeutic agents. In the case we report here, treatment with sunitinib induced a long-lasting clinical response that was associated with an immune activation directed against Melan-A/MART-1 antigen. CASE PRESENTATION: A 28 years old female patient with an advanced molecularly confirmed CCS resistant to conventional chemotherapy was started in January 2012 on sunitinib, 37.5 mg/day, with evidence of radiologic and metabolic response at the primary and metastatic sites of disease. Pathologic response and loss of the Melan-A/MART-1 antigen were evidenced on residual tumor removed in April 2012. Immunological monitoring performed on patient's blood during pharmacological treatment revealed a systemic, Melan-A/MART-1 specific immunity and a low frequency of immunosuppressive cells. Sunitinib was restarted in May 2012, with a new response, and continued for 11 months although with repeatedly interruptions due to toxicity. Disease progression and new responses were documented at each treatment interruption and restart. Sunitinib was definitively interrupted in April 2013 for disease progression. CONCLUSION: The analysis of this case proves that antigens expressed by CCS, as for melanoma, can be immunogenic in vivo and that tumor-antigen specific T cells may exert anti-tumor activity in CCS patient. Thus, manipulation of the immune response may have therapeutic potential for this STS subtype and immunotherapy approaches, can be promising therapeutic options for these patients.

Structured myeloid cells and anti-angiogenic therapy in alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma and the clinical management of patients with unresectable, metastatic disease is still challenging. ASPS expresses an array of potentially therapeutically targetable, angiogenesis-related molecules and, importantly, it has a distinctive angiogenic phenotype marked by a peculiar tumor-associated vasculature. Several studies, conducted in transgenic mouse models and in a large variety of human tumors of different histotype, clearly proved the substantial contribution of tumor-infiltrating myeloid cells, such as myeloid derived suppressor cells, monocytes and macrophages, in the formation and maintenance of abnormal blood vessels in tumors. By immunohistochemistry we thus explored the presence and the distribution of cells expressing myeloid markers in the inflammatory infiltrate of surgical treated metastatic ASPS. Indeed, we found that myeloid cells expressing CD14 and CD163 markers constitute the prominent cells in the inflammatory infiltrate of ASPS. These macrophage-like cells form a network surrounding the endothelial cells, or, interspersed in the tumor nest, they keep deep contact with tumor cells. In this commentary, we discussed our findings in relation to the recently published paper by Kummar and colleagues reporting the clinical and molecular results of a phase II clinical trial in patients with unresectable, metastatic ASPS treated with the anti-angiogenic drug cediranib, targeting the VEGFR-1,-2,-3 tyrosine kinases.

Gastrointestinal stromal tumor of the rectum: results of surgical and multimodality therapy in the era of imatinib.

BACKGROUND: The rectum is a rare site of gastrointestinal stromal tumor (GIST), and factors determining long-term outcome remain unclear. In a population study, we assessed the outcome of rectal GIST patients treated at two referral centers. METHODS: A total of 39 patients diagnosed with rectal GIST between January 2002 and December 2010 were identified in prospective databases. Tumor and patient characteristics, treatment details, and outcome were evaluated. Median follow-up was 41 (3-110) months. RESULTS: A male predominance was noticed (M/F = 29/10). Median age was 53 years (range, 32-80 years). The cohort included, of 39 patients, 12 low-risk, 26 high-risk, and 1 with M1 disease. Of 38 patients with nonmetastatic disease, 36 underwent surgery as transabdominal (15 of 36) or local (21 of 36) resection. There were 21 patients who received preoperative and/or postoperative imatinib treatment. Patients with preoperative imatinib (16 of 36) had a significantly higher rate of R0 resections (p = .02). Five patients developed local recurrences. All of them had undergone local tumor excision with positive margins and without perioperative imatinib. Also, five patients suffered from distant metastases. All belonged to the high-risk group and underwent tumor surgery (3 R0, 2 R1) without receiving perioperative imatinib. A total of three patients died of disease. Perioperative imatinib was associated with improved local disease-free, disease-free, and overall survival (p < .01, p < .01, and p = .03, respectively). Local disease-free survival was significantly improved by negative resection margins (p < .01). CONCLUSIONS: Complete resection is recommended to achieve local disease control. Preoperative imatinib was associated with improved surgical margins. Perioperative imatinib was associated with improved local disease-free, disease-free, and overall survival.

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

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.

Imatinib response in two GIST patients carrying two hitherto functionally uncharacterized PDGFRA mutations: an imaging, biochemical and molecular modeling study.

Beside the well known "in vivo" and "in vitro" Imatinib resistant D842V mutation in PDGFRA receptor, very few are the information concerning the "in vivo" Imatinib activity with respect to the other PDGFRA mutations for which only "in vitro" data are available. Two patients carrying PDGFRA mutations in exons 18 (involving residues DIMH842-845) and 12 (V561D), respectively, were treated with Imatinib at a dose of 400 mg/day. According to Response Evaluation Criteria in Solid Tumors criteria, after a median treatment of 7 months both patients showed clinical partial response, and underwent surgery of the minimal residual disease. Tumor response was confirmed pathologically. In both patients, analyses of PDGFRA performed on pre- and/or post-treatment material were compared to affinity data of the mutated receptor towards the inhibitor. Molecular modeling evidence was found to be consistent with sensitivity of mutated PDGFRA receptors to Imatinib. Thus, the "in vivo" evidence that these two mutations of PDGFRA are sensitive to Imatinib was confirmed by a multidimensional approach comprising "in silico" experiments that, in association to molecular and biochemical analyses, constitutes a powerful tool to predict Imatinib sensitivity, clinically beneficial in the treatment of these tumors with molecularly targeted therapies.

Dermatofibrosarcoma protuberans-derived fibrosarcoma: clinical history, biological profile and sensitivity to imatinib.

Dermatofibrosarcoma Protuberans (DFSP) carries a translocation resulting in the COL1A1/PDGFB fusion-gene, responsible for platelet derived growth factor beta receptor (PDGFRB) activation. Fibrosarcomatous (FS) transformation in DFSP rarely occur. The fusion-gene and PDGFRB expression/activation pattern and imatinib role in DFSP-derived FS is less defined. We reviewed all consecutive patients operated for localized DFSP at our institution from 1994 to 2009, selecting cases with FS component. We also reviewed patients treated with imatinib for advanced FS-DFSP over the same period. When cryopreserved material was available, biochemical/molecular analyses were performed. Of 275 DFSPs, 13 (4.7%) showed a FS component. Fifteen percent of these patients developed metastases, one to the brain. Four patients with DFSP-derived FS received imatinib, with a Response Evaluation Criteria in Solid Tumor Partial Response. Response was followed by early secondary progression in two. One died for brain metastases. Three patients underwent surgery after imatinib. The fusion-gene was detected in all cases in both the classical and FS component, before and after imatinib. PDGFRB expression/activation was confirmed in all cases. mTOR was switched-off, despite the phosphorylation of its effectors. However, a strong phosphorylation of S6 and 4EBP1 was restricted to the FS component. In conclusion, DFSP-derived FS maintains the fusion-gene, being sensitive to imatinib. However, responses are short-lasting. Secondary resistance to imatinib is not related to PDGFRB.

Chromosome band 17q21 in breast cancer: significant association between beclin 1 loss and HER2/NEU amplification.

Treatment success of breast cancer patients with trastuzumab alone or in combination depends not only on HER2/NEU amplification but also on PTEN and PI3K status and efficient cell death programs. In this pilot study, we found a significant association between loss of beclin 1 and HER2/NEU amplification (both on 17q21) in breast cancers. This finding was confirmed in two public copy number microarray datasets. Furthermore, there is a trend associating beclin 1 loss with TP53 mutations, PI3KCA gene gain, and PTEN mutations. Finally, the observation that beclin 1 gene loss predicted a response to trastuzumab alone or in combination with other drugs is worthy of further confirmation in larger cohorts. Our results suggest that, beclin 1 loss may contribute to genome instability and to a defective autophagy that may lead to tumoral cell death in presence of competent apoptosis or senescence pathways.

c-Kit/PDGFRA gene status alterations possibly related to primary imatinib resistance in gastrointestinal stromal tumors.

PURPOSE: To correlate morphologic changes with molecular, biochemical, and cytogenetic profiles in gastrointestinal stromal tumor (GIST) patients before and after imatinib treatment. EXPERIMENTAL DESIGN: We investigated 132 tumor samples obtained from 35 patients with advanced disease who underwent resective surgery after imatinib treatment according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group protocol. On the basis of imaging findings, 27 patients were responders and 8 progressors, and retaining this radiological subdivision, we analyzed posttreatment morphologic changes correlating them with molecular, biochemical, and cytogenetic analyses. RESULTS: On the basis of morphology (residual viable cellularity/proliferation markers), three subgroups were identified showing high, moderate, or low response. All of the progressing cases clustered in the low-response subgroup, whereas the responding cases were distributed in all three subgroups. The correlation between morphology and the molecular findings showed that secondary mutations segregated with the low-response subgroup, whereas c-Kit primary resistance mutations were randomly distributed in the three subgroups. Fluorescence in situ hybridization analysis of c-Kit/PDGFRA genes showed that all of the progressing cases were disomic. Referring to morphology, among the responding cases, a disomic pattern was mainly restricted to the high responders, whereas the moderate and low responders were aneusomic. Comparison of post-imatinib genomic profiles with the 23 available primary tumors showed that 17 cases carried the same cytogenetic pattern. Overall, 12 of the 27 primary tumors presented a gain/loss of c-Kit/PDGFRA gene copy number. CONCLUSIONS: Our findings show that c-Kit/PDGFRA genomic alterations were present at disease onset in 1/3 of the examined cases. They therefore represent an early event possibly related to primary imatinib resistance in GISTs.

Cyclooxygenase-2 and platelet-derived growth factor receptors as potential targets in treating aggressive fibromatosis.

PURPOSE: To explore the molecular bases of potential new pharmacologic targets in aggressive fibromatosis (desmoid tumor). EXPERIMENTAL DESIGN: Tumor specimens from 14 patients surgically treated for aggressive fibromatosis (6 familial adenomatous polyposis and 8 sporadic cases), analyzed for adenomatous polyposis coli (APC) and CTNNB1 (beta-catenin) mutations, were further investigated for beta-catenin, cyclooxygenase-2 (COX-2), platelet-derived growth factor (PDGF) receptor alpha (PDGFRA)/PDGF receptor beta (PDGFRB), their cognate ligands (PDGFA and PDGFB), and KIT using a comprehensive immunohistochemical, biochemical, molecular, and cytogenetic approach. RESULTS: No CTNNB1 (beta-catenin) mutations were found in the familial adenomatous polyposis patients, but previously reported activating mutations were found in six of the eight sporadic patients. All of the cases carrying an altered WNT pathway showed nuclear and cytoplasmic immunoreactivity for beta-catenin, whereas beta-catenin expression was restricted to the cytoplasm in the sporadic patients lacking CTNNB1 mutations. COX-2 protein and mRNA overexpression was detected in all 14 cases, together with the expression and phosphorylation of PDGFRA and PDGFRB, which in turn paralleled the presence of their cognate ligands. No PDGFRB mutations were found. The results are consistent with PDGFRA and PDGFRB activation sustained by an autocrine/paracrine loop. CONCLUSIONS: Aggressive fibromatosis is characterized by WNT/oncogene pathway alterations triggering COX-2-mediated constitutive coactivation of PDGFRA and PDGFRB, and may therefore benefit from combined nonsteroidal anti-inflammatory drug + tyrosine kinase inhibitor treatment.

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

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.

Molecular and biochemical analyses of platelet-derived growth factor receptor (PDGFR) B, PDGFRA, and KIT receptors in chordomas.

PURPOSE: We have previously shown the presence of an activated platelet-derived growth factor (PDGF) receptor (PDGFR) B and its ligand PDGFB in a limited number of patients with clinical and radiological responses to imatinib mesylate treatment. This article describes the results of comprehensive molecular/biochemical analyses of the three receptors targeted by the drug (PDGFRB, PDGFRA, and KIT) in a series of 31 chordoma patients. EXPERIMENTAL DESIGN: The presence and activation status of PDGFRB, PDGFRA, and KIT receptors were investigated by means of immunoprecipitation and Western blot analyses complemented by immunohistochemistry, their expression level was analyzed by means of real-time PCR, and the occurrence of activating point mutations was investigated by means of cDNA sequencing. The PDGFB, PDGFA, and stem cell factor cognate ligands were investigated by reverse transcription-PCR, and gene status was assessed by fluorescence in situ hybridization. RESULTS: The results show that PDGFRB was highly expressed and phosphorylated, whereas PDGFRA and KIT were less expressed but phosphorylated and thus activated. These findings, together with the absence of gain-of-function mutations and the presence of the cognate ligands, strongly support the hypothesis that the activation mechanism is the autocrine/paracrine loop. No role seems to be played by gene amplification. CONCLUSIONS: In the light of our findings, the clinical benefit observed in chordoma patients treated with imatinib seems to be attributable to the switching off of all three receptors.

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

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.

Identification of SRF-E2F1 fusion transcript in EWSR-negative myoepithelioma of the soft tissue.

Myoepithelial neoplasms (MN) are rare and not well-circumstanced entities displaying a heterogeneous spectrum of genetic abnormalities, including EWSR1, FUS and PLAG1 rearrangements. However, in the remaining MN no other fusion gene has been described and knowledge concerning secondary acquired molecular alterations is still poor. Therefore, we screened 5 cases of MN of the soft tissue by RNA sequencing with the aim of identifying novel fusion transcripts. A novel SRF-E2F1 fusion was detected in two cases: one was negative for other fusions while the other showed also the presence of FUS-KLF17. The fusion was validated through independent techniques and, in both cases, SRF-E2F1 was detected only in a subclone of the tumoral mass. SRF-E2F1 maintained the coding frame, thus leading to the translation of a chimeric protein containing the DNA-binding domain of SRF and the trans-activation domain of E2F1. Moreover, ectopical expression of SRF-E2F1 demonstrated that the chimeric transcript is functionally active and could affect tumor growth. Occurrence in two cases and biological relevance of the two genes involved suggest that the SRF-E2F1 fusion might become a helpful diagnostic tool. Further biologic studies are needed to better assess its role in MN biology.

Adaptive Immunity in Fibrosarcomatous Dermatofibrosarcoma Protuberans and Response to Imatinib Treatment.

Dermatofibrosarcoma protuberans (DFSP), although rare, is the most frequent skin sarcoma. Here, we focus on DFSP carrying the fibrosarcomatous transformation (FS-DFSP). FS-DFSP responds to imatinib (IM); however, tumor relapse often occurs. In a series of 21 pre- and post-treatment FS-DFSP samples, the present study explored the events that occur at the tumor site during IM therapy. Gene expression profile and immunohistochemistry analyses documented the occurrence of IM-induced senescence phenotype in the tumor cells and showed the accumulation of activated CD3+ T cells and CD163+CD14+ myeloid cells expressing the CD209 marker in post-therapy lesions. In post-IM specimens, the pathological response and tumor apoptosis were tightly associated with T-cell infiltration, thus suggesting the presence of an ongoing anti-tumor response, which was further confirmed by in vitro functional assays with CD3+ T cells isolated from an IM-responding FS-DFSP lesion. The integration of targeted therapies with immune therapies is currently under investigation to achieve longer tumor control. Our data outline the in situ immunological effects of IM and classify IM-treated FS-DFSP as potentially sensitive to immunotherapy, thus providing the rationale for further investigations of combination treatment for this soft-tissue sarcoma.

Decorin and biglycan expression is differentially altered in several muscular dystrophies.

Biglycan and decorin are small extracellular proteoglycans that interact with cytokines, whose activity they may modulate, and with matrix proteins, particularly collagens. To better understand their role in muscle fibrosis, we investigated expression of decorin and biglycan transcripts and protein in muscle of several forms of muscular dystrophy, and also expression of perlecan, an extracellular proteoglycan unrelated to collagen deposition. In Duchenne muscular dystrophy (DMD) and LAMA2-mutated congenital muscular dystrophy (MDC1A) we also quantitated transcript levels of the profibrotic cytokine TGF-beta1. We examined muscle biopsies from nine DMD patients, aged 2-8 years; 14 BMD (Becker muscular dystrophy) patients (nine aged 1-5 years; five aged 30-37 years); four MDC1A patients (aged 2-7 years); six dysferlin-deficient patients (aged 19-53 years) with mutation ascertained in two, and normal expression of proteins related to limb girdle muscular dystrophies in the others; 10 sarcoglycan-deficient patients: seven with alpha-sarcoglycan mutation, two with beta-sarcoglycan mutation and one with gamma-sarcoglycan mutation (five aged 8-15 years; five aged 26-43 years); and nine children (aged 1-6 years) and 12 adults (aged 16-61 years) suspected of neuromuscular disease, but who had normal muscle on biopsy. Biglycan mRNA levels varied in DMD and MDC1A depending on the quantitation method, but were upregulated in BMD, sarcoglycanopathies and dysferlinopathy. Decorin mRNA was significantly downregulated in DMD and MDC1A, whereas TGF-beta1 was significantly upregulated. Decorin mRNA was normal in paediatric BMD, but upregulated in adult BMD, sarcoglycanopathies and dysferlinopathy. Perlecan transcript levels were similar to those of age-matched controls in all disease groups. By immunohistochemistry, decorin and biglycan were mainly localized in muscle connective tissue; their presence increased in relation to increased fibrosis in all dystrophic muscle. By visual inspection, decorin bands on immunoblot did not differ from those of age-matched controls in all patient groups. However, when the intensity of the bands was quantitated against vimentin and normalized against sarcomeric actin, in DMD and MDC1A the ratio of band intensities was significantly lower than in age-matched controls. Variations in the transcript and protein levels of these proteoglycans in different muscular dystrophies probably reflect the variable disruption of extracellular matrix organization that occurs in these diseases. The significantly lowered decorin levels in DMD and MDC1A may be related to the increased TGF-beta1 levels, suggesting a therapeutic role of decorin in these severe dystrophies.

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

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.

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

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.

Efficacy and Biological Activity of Imatinib in Metastatic Dermatofibrosarcoma Protuberans (DFSP).

PURPOSE: To report on imatinib mesylate (IM) in patients with metastatic dermatofibrosarcoma protuberans (DFSP)/fibrosarcomatous (FS)-DFSP and on the impact of the treatment on tumor biology. EXPERIMENTAL DESIGN: Ten consecutive patients treated with IM from 2007 to 2015 for a metastatic relapse from DFSP/FS-DFSP were identified. FISH analysis for COL1A1-PDGFB was performed. Two IM-treated and 4 naïve FS-DFSP were transcriptionally profiled by RNAseq on HiScanSQ platform. Differential gene expression was analyzed with edgeR (Bioconductor), followed by hierarchical clustering and Principal Component Analysis. RESULTS: All cases featured fibrosarcomatous in the metastasis and retained the COL1A1-PDGFB. Best RECIST response was: 8 partial response, 1 stable disease, and 1 progressive disease. Median progression-free survival was 11 months. Five patients received surgery after IM and all relapsed. IM was restored in 4 patients with a new response. After IM, the most upregulated genes included those encoding for immunoglobulins and those affecting functions and differentiation of endothelial cells. Pathway enrichment analysis revealed upregulation in genes involved in antigen processing and presentation, natural killer-mediated cytotoxicity, and drug and xenobiotics metabolism. Conversely, a significant down-regulation of kinase signaling pathways was detected. CONCLUSIONS: All metastatic cases were fibrosarcomatous. Most patients responded to IM, but PFS was shorter than reported in published series which included both DFSP and FS-DFSP. All patients operated after IM had a relapse, suggesting that IM cannot eradicate metastatic cases and that the role of surgery is limited. Transcriptional profile of naïve and posttreatment samples pointed the contribution of immune infiltrates in sustaining the response to IM.