Patient-derived solitary fibrous tumour xenografts predict high sensitivity to doxorubicin/dacarbazine combination confirmed in the clinic and highlight the potential effectiveness of trabectedin or eribulin against this tumour.

BACKGROUND: Preclinical models that mimic pathological and molecular features of solitary fibrous tumour (SFT) represent an important tool to select effective regimes and novel compounds to be tested in the clinic. This study was aimed at developing two preclinical models of SFT, assessing their predictive value in the clinic and selecting potential novel effective treatments. MATERIAL AND METHODS: Two dedifferentiated-SFT (D-SFT) models obtained from patients' biopsies were grown in immunodeficient mice. The antitumour activity on these models of doxorubicin, dacarbazine (DTIC), ifosfamide (monotherapy or combination), trabectedin and eribulin was tested. Twelve SFT patients were treated with doxorubicin and DTIC. Response by RECIST, progression-free survival and overall survival were retrospectively evaluated, distinguishing malignant-SFT (M-SFT) and D-SFT. RESULTS: Two D-SFT patient-derived xenografts (PDXs) that represent the first available preclinical in vivo models of SFT were developed and characterised. Doxorubicin/DTIC, DTIC/ifosfamide, doxorubicin/ifosfamide combinations consistently induced better antitumour activity than the single-agents. Particularly, doxorubicin/DTIC combination caused a max tumour volume inhibition >80% in both models. Doxorubicin/DTIC combo showed activity also in the case-series. Best RECIST responses were: 6 responses (M-SFT = 2 of 7, D-SFT = 4 of 5), 1 stable disease, 5 progressions, with a 6-month median progression-free survival (M-SFT = 6, D-SFT = 10 months). The PDXs were very sensitive to trabectedin and eribulin. CONCLUSION: Doxorubicin plus DTIC combination was effective in our two D-SFT mice models and appeared to be active also in the clinic, especially in high-grade D-SFT patients. Among additional drugs tested in the PDXs, trabectedin and eribulin were highly effective, providing a rational to test these drugs in D-SFT patients.

Preclinical and clinical evidence of activity of pazopanib in solitary fibrous tumour.

BACKGROUND: To explore the activity of pazopanib in solitary fibrous tumour (SFT). PATIENTS AND METHODS: In a preclinical study, we compared the activity of pazopanib, sorafenib, sunitinib, regorafenib, axitinib and bevacizumab in a dedifferentiated-SFT (DSFT) xenotransplanted into Severe Combined Immunodeficiency (SCID) mice. Antiangiogenics were administered at their reported optimal doses when mean tumour volume (TV) was 80 mm(3). Drug activity was assessed as TV inhibition percentage (TVI%). From May 2012, six consecutive patients with advanced SFT received pazopanib, on a national name-based programme. In one case sunitinib was administered after pazopanib failure. RESULTS: In the xenograft model, pazopanib showed the lowest antitumour activity (21%TVI), while regorafenib was the most active (95%TVI). Sorafenib, bevacizumab, sunitinib were markedly active (78/70/65%TVI). Axitinib was marginally active (51%TVI). In the retrospective case-series, three patients carried malignant-SFT (MSFT), three DSFT. Best Response Evaluation Criteria in Solid Tumour (RECIST) responses were: three stable disease (SD), all MSFT, three progressive disease (PD), all DSFT, corresponding to one partial response (PR), two SD, three PD by Choi criteria. Median-progression-free survival was 3 months (range 1-15). In one patient, sunitinib was started after pazopanib failure, with a response. CONCLUSIONS: In dedifferentiated-SFT xenograft pazopanib induced a marginal antitumour activity, while regorafenib appeared the most active and promising agent. When administered in patients, pazopanib showed a modest activity in terms of tumour growth stabilisation, observed only in non-dedifferentiated cases.

Adaptive immune contexture at the tumour site and downmodulation of circulating myeloid-derived suppressor cells in the response of solitary fibrous tumour patients to anti-angiogenic therapy.

BACKGROUND: Host immunity is emerging as a key player in the prognosis and response to treatment of cancer patients. However, the impact of the immune system and its modulation by therapies are unknown in rare soft tissue sarcomas such as solitary fibrous tumours (SFTs), whose management in the advanced forms includes anti-angiogenic therapy. Here, we studied the in situ and systemic immune status of advanced SFT patients and the effects of sunitinib malate (SM) in association with the clinical efficacy. METHODS: Immune contexture of SFTs was assessed by immunohistochemistry in lesions from untreated or SM-treated patients. Frequency of circulating myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and T-cell functions was assessed ex vivo in SFT patients prior and during anti-angiogenic therapy. Patients with long-term tumour control were included to correlate immune profiles and clinical responses. RESULTS: Anti-angiogenic naïve SFT lesions were heavily infiltrated by CD163(+)CD14(+)CD68(-) and CD163(+)CD14(-)CD68(-) myeloid cells but devoid of T cells. Conversely, post-SM tumours acquired a new subset of CD68(+)CD14(+) myeloid cells and displayed traits of an on-going adaptive immunity, strongly enriched in activated CD8(+) and CD4(+) T cells. These changes at the tumour site paralleled the alleviation of systemic immunosuppression and the drop in the frequency of circulating monocytic MDSCs (mMDSCs) and granulocytic MDSCs (gMDSCs). Rebound in the number of mMDSCs, but not of gMDSCs occurred at disease progression, and a reduced percentages of mMDSCs, comparable to those found in healthy donors (HDs), endured only in the SM-responsive patients. CONCLUSIONS: The immune contexture of SFT patients is heavily involved in anti-angiogenic therapy and it could be exploited to achieve more durable disease control through immune-based combination strategies.

Activity of sunitinib in extraskeletal myxoid chondrosarcoma.

BACKGROUND: Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue sarcoma, marked by NR4A3 rearrangement. Herein we report on the activity of sunitinib in a series of 10 patients, strengthening what initially observed in two cases. PATIENTS AND METHODS: From July 2011, 10 patients with progressive metastatic translocated EMC have been consecutively treated with sunitinib 37.5mg/day, on a named-use basis. In an attempt to interpret the activity of sunitinib in EMC, genotype/phenotype correlations were carried out by fluorescence in situ hybridization (FISH) analyses. Moreover, transcriptome, immunohistochemical and biochemical analyses of a limited set of samples were performed focusing on some putative targets of sunitinib. RESULTS: Eight of 10 patients are still on therapy. Six patients had a Response Evaluation Criteria in Solid Tumours (RECIST) partial response (PR), two were stable, two progressed. Positron emission tomography (PET) was consistent in 6/6 evaluable cases. One patient underwent surgery after sunitinib, with evidence of a pathologic response. At a median follow-up of 8.5 months (range 2-28), no secondary resistance was detected. Median progression free survival (PFS) has not been reached. Interestingly, all responsive cases turned out to express the typical EWSR1-NR4A3 fusion, while refractory cases carried the alternative TAF15-NR4A3 fusion. Among putative sunitinib targets, only RET was expressed and activated in analysed samples. CONCLUSIONS: This report confirms the therapeutic activity of sunitinib in EMC. Genotype/phenotype analyses support a correlation between response and EWSR1-NR4A3 fusion. Involvement of RET deserves further investigation.

Dacarbazine in solitary fibrous tumor: a case series analysis and preclinical evidence vis-a-vis temozolomide and antiangiogenics.

PURPOSE: To explore the value of triazines in solitary fibrous tumor (SFT). EXPERIMENTAL DESIGN: We retrospectively reviewed 8 cases of patients with SFT treated with dacarbazine (1,200 mg/m(2) every 3 weeks) as from January 2012. Then, we studied a dedifferentiated-SFT subcutaneously xenotransplanted into severe combined immunodeficient (SCID) mice. Dacarbazine, temozolomide, sunitinib, bevacizumab, and pazopanib were administered at their reported optimal doses for the mouse model when mean tumor volume (TV) was about 80 mm(3); each experimental groups included 6 mice. Drug activity was assessed as tumor volume inhibition percentage (TVI%). Dacarbazine was tested according to two different schedules of administration. One hunded twenty days after treatment interruption, mouse tumor samples were analyzed. RESULTS: Among the eight patients treated with dacarbazine, best response evaluation criteria in solid tumors responses (RECIST) were three partial responses, 4 stable disease, 1 progression. Two responsive patients had paraneoplastic hypoglycemia that disappeared after 10 days from starting dacarbazine. In the dedifferentiated-SFT xenograft model, dacarbazine and temozolomide showed the highest antitumor activity (about 95% TVI), confirmed pathologically. Sunitinib and pazopanib were only marginally active (52% and 41% TVI, respectively), whereas bevacizumab caused a 78% TVI. No tumor regrowth was observed up to 100 days from end of treatment with temozolomide and dacarbazine, whereas secondary progression followed sunitinib, pazopanib, and bevacizumab interruption. CONCLUSIONS: Dacarbazine as single agent has antitumor activity in SFT. Our preclinical results suggest a cytotoxic effect of temozolomide and dacarbazine, as compared with a cytostatic role for sunitinib, pazopanib, and bevacizumab. A phase II study on dacarbazine in advanced SFT is planned.

Phase II study on lapatinib in advanced EGFR-positive chordoma.

BACKGROUND: To report on a prospective, investigator-driven, phase II study on lapatinib in epidermal growth factor receptor (EGFR)-positive advanced chordoma patients. PATIENTS AND METHODS: From December 2009 to January 2012, 18 advanced progressing chordoma patients entered this study (median age: 61 years; disease extent: metastatic 72% and locally advanced 28%). Epidermal growth factor receptor (EGFR) expression and activation were evaluated by immunohistochemistry and/or phospho-arrays, real-time polimerase chain reaction, fluorescence immunostaining. Fluorescence in situ hybridization analysis was also carried out. Patients received lapatinib 1500 mg/day (mean dose intensity = 1282 mg/day), until progression or toxicity. The primary study end point was response rate (RR) as per Choi criteria. Secondary end points were RR by Response Evaluation Criteria in Solid Tumor (RECIST), overall survival, progression-free survival (PFS) and clinical benefit rate (CBR; RECIST complete response + partial response (PR) + stable disease (SD) ≥ 6 months). RESULTS: All patients were evaluable for response. Six (33.3%) patients had PR and 7 (38.9%) SD, as their best Choi responses, corresponding to RECIST SD in all cases. Median PFS by Choi was 6 [interquartile (IQ) range 3-8] months. Median PFS by RECIST was 8 (IQ range 4-12) months, with a 22% CBR. CONCLUSIONS: This phase II study showed a modest antitumor activity of lapatinib in chordoma. The clinical exploitation of EGFR targeting in chordoma needs to be further investigated, both clinically and preclinically. Clinical trial Registration No: EU Clinical Trials Register trial no. 2009-014456-29.

Response to chemotherapy of solitary fibrous tumour: a retrospective study.

BACKGROUND: To report on anthracycline-based chemotherapy in a retrospective case-series analysis of solitary fibrous tumour (SFT) patients treated within the Italian Rare Cancer Network. PATIENTS AND METHODS: We reviewed a set of SFT treated with chemotherapy since 2002, focusing on anthracycline, administered alone or in combination with ifosfamide. Responses to ifosfamide as a single agent were also evaluated. Pathologic diagnosis was centrally reviewed, distinguishing typical, malignant (MSFT) and dedifferentiated (DSFT) subtypes. RESULTS: Among 42 SFT patients treated with chemotherapy, we selected 31 cases (mean age: 62 years; locally advanced/metastatic: 13/18; front-line/further line: 25/6; typical/MSFT/DSFT/not assessable: 1/17/12/1) who received anthracycline-based chemotherapy (anthracycline monotherapy: eight; anthracycline+ifosfamide: 23). 30 patients are evaluable for response. Best response by Response Evaluation Criteria in Solid Tumours (RECIST) was: partial response (PR): 6 (20%), stable disease (SD): eight (27%), progressive disease (PD): 16 (53%) cases. Responses were confirmed after 3 months. Median progression-free survival (PFS) was 4 (range 2-15) months, with 20% of patients being progression-free at 6 months. PR was found in 2/18 (11%) MSFT and 4/12 (30%) DSFT, with a median PFS of 3.5 and 5 months in MSFT and DSFT, respectively. 19 patients received high-dose prolonged-infusion ifosfamide (front-line/further line: 11/8; typical/MSFT/DSFT: 0/15/4) with two (10%) PR, five (26%) SD, 12 (63%) PD. CONCLUSIONS: This retrospective series suggests that in SFT anthracyclines have a degree of antitumour activity in the range of soft tissue sarcoma chemotherapy. Ifosfamide monotherapy seemed to have lower activity. A higher response rate was observed in DSFT in comparison to MSFT. Studies on targeted therapies are ongoing.

Targeting metabolism for cancer treatment and prevention: metformin, an old drug with multi-faceted effects.

Understanding the complexity of cancer and of the underlying regulatory networks provides a new paradigm that tackles cancer development and treatment through a system biology approach, contemporarily acting on various intersecting pathways. Cancer cell metabolism is an old pathogenetic issue that has recently gained new interest as target for therapeutic approaches. More than 70 years ago, Warburg discovered that malignant cells generally have altered metabolism with high rates of glucose uptake and increased glycolysis, even under aerobic condition. Observational studies have provided evidence that impaired metabolism, obesity, hyperglycemia and hyperinsulinemia may have a role in cancer development, progression and prognosis, and actually diabetic and obese patients have increased cancer risk. On the other hand, caloric restriction has been shown to prolong life span and reduce cancer incidence in several animal models, having an impact on different metabolic pathways. Metformin, an antidiabetic drug widely used for over 40 years, mimics caloric restriction acting on cell metabolism at multiple levels, reducing all energy-consuming processes in the cells, including cell proliferation. By overviewing molecular mechanisms of action, epidemiological evidences, experimental data in tumor models and early clinical study results, this review provides information supporting the promising use of metformin in cancer prevention and treatment.

Sunitinib malate in solitary fibrous tumor (SFT).

BACKGROUND: To report on sunitinib activity in a retrospective series of 35 solitary fibrous tumor (SFT) treated at a single institution. PATIENTS AND METHODS: From April 2008, 35 patients with progressive advanced SFT (male/female: 20/15; mean age: 58 years; meningeal/extrameningeal: 6/29; locally advanced/metastatic: 15/20; prior chemotherapy: 25) were treated, on an individual use basis, with continuous-dosing sunitinib 37.5 mg/day. Platelet-derived growth factor receptor beta (PDGFRB) and vascular endothelial growth factor receptor 2 (VEGFR2) status were assessed by immunohistochemistry and, in a subgroup of patients, by real time PCR. RESULTS: Thirty-one patients were assessable for response by RECIST (one early death; three early interruptions). Best responses were 2 partial response (PR), 16 stable disease, 13 progressive disease. A <30% decrease in size was observed in three patients. Fourteen of 29 patients assessable by Choi criteria had a PR. Median progression-free survival by RECIST was 6 months (range 1-22). In two of six patients, resistance to sunitinib was overcome by increasing sunitinib to 50 mg/day. PDGFRB and/or VEGFR2 were positive in all cases and not predictive of response; a less aggressive morphology corresponded to an increased response rate (53% PR by Choi in the malignant SFT, 20% PR in the pleomorphic/dedifferentiated SFT). CONCLUSIONS: Sunitinib is active in SFT. Response can be long-lasting.

DHM simulation in virtual environments: a case-study on control room design.

This paper will present the workflow developed for the application of serious games in the design of complex cooperative work settings. The project was based on ergonomic studies and development of a control room among participative design process. Our main concerns were the 3D human virtual representation acquired from 3D scanning, human interaction, workspace layout and equipment designed considering ergonomics standards. Using Unity3D platform to design the virtual environment, the virtual human model can be controlled by users on dynamic scenario in order to evaluate the new work settings and simulate work activities. The results obtained showed that this virtual technology can drastically change the design process by improving the level of interaction between final users and, managers and human factors team.

Role of EGFR family receptors in proliferation of squamous carcinoma cells induced by wound healing fluids of head and neck cancer patients.

BACKGROUND: Mounting evidence suggests that recurrence of resected head and neck squamous cell carcinomas (HNSCCs) is due to the outgrowth of unrecognized residual tumor cells as well as to the premalignant and/or precursor-field epithelial cells. We studied the impact of processes triggered by HNSCC surgery in stimulating both residual tumor cells [demonstrated to overexpress epidermal growth factor receptor (EGFR)], and premalignant cells surrounding the resected lesion. PATIENTS AND METHODS: EGFR expression/activation by immunohistochemistry/biochemistry and gene status by FISH were investigated in 23 primary HNSCCs and surrounding tissues. The ability to induce cell proliferation of wound healing drainages collected from 12 relapsed and 11 not relapsed patients was evaluated by a colorimetric assay in squamous cell carcinoma cell lines A431 (carrying EGFR amplification) and CAL27 (carrying three EGFR copies) in the presence/absence of EGFR therapeutic inhibitors. RESULTS: Primary tumors showed intermediate/high EGFR expression (91%), EGFR phosphorylation and EGFR-positive FISH (35%). Normal, metaplastic and dysplastic epithelium surrounding the resected tumor displayed EGFR overexpression. EGFR activation and gene amplification were observed in normal and dysplastic epithelium, respectively. Each tested wound healing drainage induced the cells to proliferate and the proliferation was significantly higher in relapsed compared with not relapsed HNSCC patients (P = 0.02 and P = 0.03). Anti-EGFR treatments inhibited the drainage-induced proliferation, with the highest inhibitory efficiency by cetuximab on A431 cells, while CAL27 cell growth was more efficiently inhibited by tyrosine kinase inhibitors. CONCLUSIONS: Surgery could favor the proliferation of cells showing EGFR overexpression/activation/amplification such as residual tumor cells and/or precursor-field epithelial cells already present after surgery. Treatment with anti-EGFR reagents inhibits wound-induced stimulation, according to the EGFR family status.

Sunitinib in advanced alveolar soft part sarcoma: evidence of a direct antitumor effect.

BACKGROUND: The purpose of this study was to confirm sunitinib activity in alveolar soft part sarcoma (ASPS) and to report on new insights into the molecular bases thereof. PATIENTS AND METHODS: From July 2007, nine patients with progressive metastatic ASPS received sunitinib 37.5 mg/day, within a named use program. Cryopreserved material was available for five naive patients, among whom three received sunitinib. Immunofluorescence (IF)/confocal microscopy, biochemical, and molecular/cytogenetic analyses were carried out, complemented by antiproliferative and activation assays in a short-term culture derived from one case. RESULTS: All patients were eligible for response. Best RECIST response was partial response in five cases, stable disease in three, and progression in one. The median progression-free survival was 17 months. Positron emission tomography results were consistent. Two cases of interval progressions were recorded. Antiproliferative assays and biochemistry on short-term culture showed that sunitinib is able to markedly impair ASPS cells growth and switch-off PDGFRB. IF/confocal microscopy demonstrated coexpression and physical association between PDGFRB/vascular endothelial growth factor receptor 2 (VEGFR2) and RET/VEGFR2 in ASPS cells, which was validated by biochemistry. PDGFRB, RET, and MET ligand-dependent activation was confirmed. CONCLUSIONS: We confirm the clinical efficacy of sunitinib in ASPS, mediated by PDGFRB, VEGFR2, and RET, which are all expressed in tumor cells. A direct antitumor effect was shown in a short-term cell culture.

Oncogenic and ligand-dependent activation of KIT/PDGFRA in surgical samples of imatinib-treated gastrointestinal stromal tumours (GISTs).

As the range of receptor tyrosine kinase (RTK) inhibitors widens, a detailed understanding of the activating mechanisms of KIT/platelet-derived growth factor receptor (PDGFR)A and the related downstream pathways involved in the development and maintenance of GISTs is becoming increasingly important. We analysed areas with different histological response ratios in surgical specimens taken from imatinib-treated and untreated GIST patients in order to investigate KIT and PDGFRA expression/activation, the presence of their cognate ligands and the activation of downstream signalling, by means of biochemistry, immunohistochemistry and flow cytometry. All of the cases showed KIT and PDGFRA co-expression. In addition to the oncogenic activation of mutated receptors, activation of wild-type KIT and wild-type PDGFRA, sustained by heterodimerization and an autocrine-paracrine loop, was demonstrated by the presence of their specific ligands, stem cell factor (SCF) and PDGFA. To confirm RTK activation further, all of the samples (including those with the highest regression ratios) were investigated for downstream effectors, and all proved to have activated downstream signalling. The results show that after the mutated receptors are switched off, heterologous wild-type receptors become important in imatinib-treated GISTs as a means of maintaining signalling activation. Taken together, our findings suggest that drugs targeting wild-type receptors should be tested in imatinib-treated GIST patients.

Analysis of potential receptor tyrosine kinase targets in intimal and mural sarcomas.

Primary sarcomas of the great vessels are very rare neoplasms and only a few cases have been reported. They are divided into the two broad categories of intimal or luminal and mural sarcomas. We analysed eight advanced high-grade sarcomas originating from major vessels (seven intimal and one mural sarcoma) by means of immunohistochemistry and FISH analysis for PDGFRA, PDGFRB, EGFR and KIT receptor tyrosine kinases (RTKs), together with immunoprecipitation/western blotting, sequencing of the corresponding genes, and the search for cognate ligands. The intimal sarcomas showed a wide spectrum of morphologies and immunophenotypes, whereas the mural sarcoma had common leiomyosarcomatous features. Regardless of their category, all of the cases had a PDGFRA-deregulated cytogenetic profile mainly consisting of an amplification cluster; five were also polysomic for PDGFRB, whereas three showed disomy. Six cases had a deregulated EGFR gene, and c-Kit gene status was similar to that of PDGFRA. In one case, biochemical analysis revealed the presence of activated and highly expressed PDGFRA, PDGFRB and EGFR, whereas KIT was expressed at reference level. Sequencing of the corresponding genes revealed no activating mutations in any of the analysed receptors. The cognate ligands were detected in all cases. In predictive terms, the evidence of gene amplification/high polysomy of several RTKs, together with PDGFRA, PDGFRB and EGFR expression and phosphorylation, suggests that these tumours may be sensitive to RTK-inhibiting treatments.

Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients.

Imatinib-acquired resistance related to the presence of secondary point mutations has become a frequent event in gastrointestinal stromal tumors. Here, transient transfection experiments with plasmids carrying two different KIT-acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived from imatinib-treated and untreated cells. The molecular mechanics/Poisson Boltzmann surface area computational techniques were applied to study the interactions of the wild-type and mutated receptors with imatinib at the molecular level. Biochemical analyses showed KIT phosphorylation in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that T670I was insensitive to the drug at all the applied concentrations, whereas V654A was inhibited by 6 microM of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib-binding site, but to a different extent: T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined structural changes. We demonstrated that T670I and V654A cause indeed imatinib-acquired resistance and that the former is more resistant to imatinib than the latter. The application of molecular simulations allowed us to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance.

PDGFRalpha, PDGFRbeta and KIT expression/activation in conventional chondrosarcoma.

Chondrosarcomas represent 20% of all primary bone sarcomas, and many studies have attempted to unravel molecular targets for future development of new therapies. The aim of this study was to investigate the expression/activation of PDGFRalpha, PDGFRbeta and KIT receptor tyrosine kinases (RTKs) as potential therapeutic targets in conventional central primary chondrosarcomas (CCS). The expression of PDGFRalpha, PDGFRbeta and KIT RTKs was detected in 16 CCSs using immunohistochemistry (IHC), and their level of expression and activation status were analysed by immunoprecipitation and western blot experiments. PDGFRalpha, PDGFRbeta and KIT cDNAs were screened to verify the presence of activating mutations and the presence of the cognate ligands was analysed by means of RT-PCR. RTK gene amplification was further studied by means of fluorescence in situ hybridization (FISH) analysis. The immunophenotyping and biochemical analyses showed that the CCSs co-expressed PDGFRalpha and PDGFRbeta, with the latter showing definitively greater protein expression and phosphorylation levels. PDGFRbeta was expressed but not activated in control healthy joint cartilage, in line with no PDGFB detection. Conversely, the KIT gene product did not seem to play a relevant role. These findings, in the absence of activating mutations or an abnormal genomic profile and the presence of PDGFA and PDGFB expression, are consistent with an autocrine/paracrine loop activation of the corresponding receptors. The CCS gene profile described here offers a rationale for the use of RTK inhibitors alone or in combination with chemotherapy, and supports further investigation of RTKs and their downstream signals.

Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.

Utrophin is highly homologous and structurally similar to dystrophin, and in gene delivery experiments in mdx mice was able to functionally replace dystrophin. We performed mini-utrophin gene transfer in Golden Retriever dogs with canine muscular dystrophy (CXMD). Unlike the mouse model, the clinicopathological phenotype of CXMD is similar to that of Duchenne muscular dystrophy (DMD). We injected an adenoviral vector expressing a synthetic utrophin into tibialis anterior muscles of newborn dogs affected with CXMD and examined transgene expression by RNA and protein analysis at 10, 30 and 60 days postinjection in cyclosporin-treated and -untreated animals. Immunosuppression by cyclosporin was required to mitigate the immune response to viral and transgene antigens. RT-PCR analysis showed the presence of the exogenous transcript in the muscle of cyclosporin-treated and -untreated animals. The transgenic utrophin was efficiently expressed at the extrajunctional membrane in immunosuppressed dogs and this expression was stable for at least 60 days. We found reduced fibrosis and increased expression of dystrophin-associated proteins (DAPs) in association with muscle areas expressing the utrophin minigene, indicating that mini-utrophin can functionally compensate for lack of dystrophin in injected muscles. For this reason, utrophin transfer to dystrophin-deficient muscle appears as a promising therapeutic approach to DMD.