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PDF file, 227K, SFT xenotransplanted in SCID mice pathologic evaluation before and after treatment. A and B Pre-treatment samples: high-grade sarcoma aspects mixed with cartilaginous (A) and osseous component (B). C and D Post-sunitinib, post-bevacizumab and post-pazopanib samples. 120 days after the end of treatment with sunitinib (C), bevacizumab (D) xenografts were sacrificed. The tumor samples were all made of hypercellular viable, mainly spindle tumor cells, intermingled with osteoid matrix. Pazopanib (E) xenograft was instead sacrificed soon after treatment end, Even in this case tumor sample appeared to be completely vital.

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/m2 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 mm3; 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. Clin Cancer Res; 19(18); 5192–201. ©2013 AACR.

PDF file, 256K, A Human primary tumor: morphology (i.e., patternless growth, collagen deposition, moderate cellularity, mitotic index (>4X10 High Power Field (HPF), as shown in panel 1) and immunophenotype (i.e., diffuse positivity for CD34, as shown in panel 1/a) were consistent with a malignant SFT. Immunohistochemistry was positive for PDGFRB expression (panel 1/b). B Human relapsed tumor: morphology was consistent with a dedifferentiated SFT (DSFT), being marked by the abrupt transition from areas with MSFT aspects similar to what observed in the primary tumor (panel 2) to areas with high-grade sarcoma features (panel 3). High-grade areas showed hypercellularity (panel 3/a), chondroid (panel 3/b) and osteoid differentiation (panel 3/c). C Xenograft tumor: morphology was consistent with a high-grade DSFT (panel 4), superimposable to what observed in panel B, 3a/3b/3c, again with presence of mineralized trabeculaes and osteoid (panel 4/a). Immunohistochemistry was positive for PDGFRB expression (panel 4/b). Phospho receptor tyrosine kinase (pRTK) array (ARY001 Proteome ProfilerTM Array, R&D Systems) showed similar profiles for human relapsed tumor (panel B, 3/d) and xenograft (panel C, 4/c) with the activation of PDGFRA (green boxes), PDGFRB (red boxes) and VEGFR1 (yellow boxes). White boxes are the proteome profiler negative controls.

PDF file, 110K, The xenografts were sacrificed five days after starting sunitinib, bevacizumab and pazopanib. One sample was obtained also 4 weeks after the end of treatment with pazopanib. PDGFRB and VEGFR2 activation was analyzed trough immunoprecipitation (IP) and western blotting (WB). PDGFRB activation was reduced by sunitinib and pazopanib, while VEGFR2 activation was decreased by bevacizumab and pazopanib. A re-activation of both PDGFRB and VEGFR2 was detected after 4 weeks of treatment with pazopanib.

BackgroundRheumatoid arthritis (RA) patients usually present extra-articular manifestations (1,2), which affect muscle mass and, consequently, physical function (3). Among the various methods to assess muscle mass we tested muscle morphology by ultrasound (MU) to verify the associations of the quadriceps muscle with clinical features, muscle strength, functional capacity and physical function.ObjectivesTo assess the MU of the quadriceps muscle and verify the muscle quality assessed by the pennation angle and its associations with clinical features, muscle strength, functional capacity and physical function in RA patients.MethodsRA women, age ≥18years and who met 2010 American College of Rheumatology (ACR) criteria were included. Morphological parameters in quadriceps muscle consisted of the pennation angle of rectus femoris (RF), vastus intermedius (VI) and vastus lateralis (VL). RA activity was measured by 28-joint disease activity score (DAS28), muscle strength by handgrip and chair stand tests, functional capacity by health assessment questionnaire (HAQ), and physical function by timed-up-and-go (TUG) test and short physical performance battery (SPPB). Pearson’s or Spearman’s correlation coefficients were explored. The significance level was set at p ≤ 0.05 for all analyzes.ResultsEighty-one patients were included (age: 58.64±9.52 years old; DAS28: 3.24±1.34). Smaller pennation angle in rectus femoris (RF) were associated with lower handgrip strength (r= 0.224, p=0.044), chair stand test (r= -0.372, p=0.004), HAQ (r= -0.404, p=0.001), SPPB (r= 0.262, p=0.047), as well as higher disease activity by DAS-28 (r= -0.415, pConclusionThe pennation angles of the quadriceps muscle evaluated by ultrasound (RF, VI and VL muscles) were associated with chair stand test and DAS-28. In addition, the level of disease activity assessed by DAS-28 also appears to be affecting the quadriceps muscle. Finally, MU may be a useful method to evaluate the impact of the disease on skeletal muscle.References[1]Summers GD, Deighton CM, Rennie MJ, Booth AH. Rheumatoid cachexia : a clinical perspective. 2008;(April):1124–31.[2]da Rocha OM, Batista A de AP, Maestá N, Burini RC, Laurindo IMM, Kayser C. Sarcopenia in rheumatoid cachexia: Definition, mechanisms, clinical consequences and potential therapies. Rev Bras Reum. 2009;49, 294–30.[3]de Santana FS, da Cunha Nascimento D, de Freitas JPM, Miranda RF, Muniz LF, Neto LS, et al. Assessment of functional capacity in patients with rheumatoid arthritis: Implications for recommending exercise. Rev Bras Reumatol. 2014;54(5):378–85.Table 1.Associations between the quadriceps muscle morphology by ultrasound (pennation angle) with clinical features, muscle strength, functional capacity and physical function in rheumatoid arthritis patients.VariablesnComponents of the quadriceps muscleRp-valueAge (y)81RF-0.2900.009VINSNSVL-0.2720.014Disease duration (y)81RFNSNSVINSNSVLNSNSDas-28 (CRP)81RF-0.415VI-0.3040.006VL-0.2370.033Handgrip strength test (kg)81RF0.2240.044VINSNSVLNSNSChair stand test (s)58RF-0.3720.004VI-0.2810.033VL-0.2900.027HAQ (score)65RF-0.4040.001VI-0.3020.015VLNSNSTUG test (s)68RFNSNSVINSNSVLNSNSSPPB (score)58RF0.2620.047VINSNSVLNSNSRF: rectus femoris; VI: vastus intermedius; VL: vastus lateralis; n: number; y: years; s: seconds; kg: kilogram; DAS28: Disease Activity Score 28; CRP: C-reactive protein; TUG: Timed-up-and-go; SPPB: Short Physical Performance Battery; NS: Not significant.AcknowledgementsWe thank the Coordination for the Improvement of Higher Level Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES) institution, the Foundation for Research Support of the Rio Grande do Sul State (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul-FAPERGS), the Research and Events Incentive Fund (Fundo de Incentivo à Pesquisa e Eventos-FIPE) of HCPA and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq).Disclosure of InterestsNone declared

Background Pre-clinical and clinical evidence suggests a rationale for the use of anti-angiogenic agents, including sorafenib, in recurrent and/or metastatic salivary gland carcinomas (RMSGCs). This study evaluates the activity of sorafenib in patients with RMSGCs and also investigates whether the activity of sorafenib could be related to its main tailored targets (i.e. BRAF, vascular endothelial growth factor receptor 2 [VEGFR2], platelet-derived growth factor receptor α [PDGFRα] and β, RET, KIT). Patients and methods Patients received sorafenib at 400 mg BID. The primary end-point was response rate (RR) including complete response or partial response (PR); secondary end-points included RR according to Choi criteria, disease control rate (DCR), overall survival (OS), and progression-free survival (PFS). Results Thirty-seven patients (19 adenoid cystic cancers, ACC) were enrolled. Six PRs were recorded. RR was 16% (95% confidence interval [CI]: 6–32; 11% in ACC and 22% in non-ACC). Choi criteria could be applied in 30 out of 37 cases with a RR of 50% (95% CI: 31–69%); DCR was 76% (95% CI: 59–88%). Incidence of ≥G3 adverse events was 29.7%. Median PFS and OS for the entire population were 5.9 months and 23.4 months, respectively. Median PFS and OS were 8.9 and 26.4 months for ACC versus 4.2 and 12.3 months for non-ACC patients. All the cases showed expression of PDGFRβ in the stroma and VEGFR2 in endothelial cells; PDGFRα positivity was found in the stroma of four (27%) cases. All except for two cases showed no PDGFRβ, VEGFR2 and PDGFRα expression in the tumour cells. KIT expression was restricted to ACC and a weak RET expression was limited to one adenocarcinoma, not otherwise specified (NOS). No BRAF mutation was found. No correlation was observed between the sorafenib activity and the expression of its markers although all six responders (two ACC, one adenocarcinoma, NOS, one salivary duct cancer [SDC], one high-grade mucoepidermoid [HG-MEC] and one poorly-differentiated cancer) are enriched in the stromal component showing a PDGFRβ immunodecoration. In ACCs, immunohistochemistry revealed MYB protein expression in 15/16 cases (94%) and the MYB-NFIB fusion oncogene was observed in 9/14 (64%). Conclusions Sorafenib is the first anti-angiogenic agent to demonstrate activity in RMSGC patients, particularly in some histotypes such as HG-MEC, SDC and adenocarcinoma, NOS. The PDGFRβ-positive rich stromal component characterising these histotypes and the lack of correlation between the activity of sorafenib and its targets suggests anti-angiogenic effect as the prevalent mechanism of action of sorafenib in SGCs.

Chordomas are rare, malignant bone tumors of the skull-base and axial skeleton. Until recently, there was no consensus among experts regarding appropriate clinical management of chordoma, resulting in inconsistent care and suboptimal outcomes for many patients. To address this shortcoming, the European Society of Medical Oncology (ESMO) and the Chordoma Foundation, the global chordoma patient advocacy group, convened a multi-disciplinary group of chordoma specialists to define by consensus evidence-based best practices for the optimal approach to chordoma. In January 2015, the first recommendations of this group were published, covering the management of primary and metastatic chordomas. Additional evidence and further discussion were needed to develop recommendations about the management of local-regional failures. Thus, ESMO and CF convened a second consensus group meeting in November 2015 to address the treatment of locally relapsed chordoma. This meeting involved over 60 specialists from Europe, the United States and Japan with expertise in treatment of patients with chordoma. The consensus achieved during that meeting is the subject of the present publication and complements the recommendations of the first position paper.

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 37.5 mg/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.

Background Myoepithelial tumors of soft tissues (MT) and Extraskeletal Myxoid Chondrosarcoma (EMC) are closely related pathological entities whose overlapping features, in terms of morphology and immunoprofile, make differential diagnosis challenging. Different fusion genes have been described in MT. Instead, the rearrangement of NR4A3 is conventionally considered an exquisite feature of EMC. Nevertheless, whether there is a biological overlap between MT and EMC is still controversial. In order to shed light on this issue we compared the transcriptional profiles of the two entities. Methods A series of EMC (12 cases) and MT (7 cases), retrieved from the pathology files, was selected for the study. The diagnosis was made according to the WHO classification. FISH analyses confirmed that all EMC harbored NR4A3 rearrangement (7 EWSR1-NR4A3 and 5 TAF15-NR4A3); 4 EWSR1 and 1 FUS rearrangement were detected in MT. No rearrangement was detected in 2 cases. RNA was extracted from FFPE specimens with tumor cellularity >70%. RNA-sequencing was carried out on an Illumina Hiseq1000 platform (average 70 million reads/sample). Diverse algorithms and bioinformatic suites were employed to identify fusion transcripts and functional annotation analysis. Results RNA-seq analysis confirmed the rearrangements detected by FISH and identified one PTCH1-GLI1 fusion in a MT. Principal component analysis and unsupervised hierarchical clustering indicated that MT and EMC feature a distinct transcriptional profile. Functional annotation of the genes differentially expressed highlighted Hedgehog (HH) and WNT signaling as significantly enriched pathways in MT compared to EMC, with canonical GLI1 and WNT target genes upregulated in MT. Ectopic expression in cell models of the PTCH1-GLI1 chimeric transcript identified in the MT sample correlated with the induction of GLI1 target genes. Conclusions This study corroborates the notion that MT and EMC represent two distinct biological entities, with MT featuring a distinctive activation of HH and WNT pathways. The PTCH1-GLI1 fusion represents one possible mechanism of HH pathway activation in MT. Legal entity responsible for the study The authors. Funding Fondazione AIRC per la Ricerca sul Cancro, CRO Intramural Grant, Italian Ministry of Health. Disclosure P.G. Casali: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Deciphera; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eisai; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eli Lilly; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Nektar Therapeutics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Advenchen Laboratories; Research grant / Funding (institution): Amgen Dompe; Research grant / Funding (institution): AROG Pharmaceuticals; Research grant / Funding (institution): Blueprint Medicines; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Epizyme Inc.; Research grant / Funding (institution): Glaxo; Research grant / Funding (institution): Karyopharm Pharmaceuticals; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): PharmaMar. A.P. Dei Tos: Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer. All other authors have declared no conflicts of interest.

Background To date, no treatment modality has been identified as more effective for oropharyngeal cancer (OPC), and no predictive factors are known to guide treatment decision for this disease. This retrospective study evaluates the differential effects of diverse treatment options for OPC according to patient risk profiles. Patients and methods We considered two series of locally advanced squamous cell OPC patients treated with either surgery followed by radiotherapy (surgical series) or chemoradiation (CRT) with/without induction docetaxel, cisplatin and 5-fluorouracil (TPF) chemotherapy (CRT series). Smoking habits, tumor p16 expression/human papillomavirus (HPV) status and T and N stage were analyzed to stratify the patients according to Ang's risk profile (low, intermediate and high risk). Overall survival (OS) and disease-free survival were calculated with the Kaplan–Meier method. Results Globally, 171 patients were considered, 56 in surgical and 115 in CRT series. Patients were stratified in low- (20% of surgical and CRT groups), intermediate- (23% and 41%) and high-risk (57% and 39%) groups. In the surgical series, 5-year OS was 54.5%, 46.9% and 40.0% in low, intermediate and high Ang's risk profiles, respectively, whereas in the CRT series those were 100%, 78.9% and 46.7%, respectively. In the multivariable analyses, adjusting for inhomogeneity between the treatment group, the CRT effect was significantly higher in the low- and intermediate-risk groups (P-value for the interaction treatment risk group = 0.034 in the OS analysis). Conclusions In this retrospective analysis, low- and intermediate-risk OPC patients had a better survival when treated with CRT compared with open surgery followed by radiation therapy. These data suggest that different treatment approaches might be essential in determining outcome results.

Background: Plasma circulating tumour-specific microRNAs (miRNAs) are promising biomarkers of tumour presence and recurrence, especially for diseases whose best chance of successful treatment requires early diagnosis and timely surgery of an already malignant but not yet invasive tumour, such as colorectal cancer (CRC). Methods: Expression levels of miRNAs previously found to be differently expressed in tumour vs normal colon tissues were investigated by quantitative real-time PCR in plasma from CRC patients and from healthy donors and confirmed in independent case control series. The validated miRNAs were also measured after surgery. Analyses were repeated on the subsets of haemolysis-free samples. Results: We identified four miRNAs differently expressed between the compared groups, two (miR-21 and miR-378) of which were validated. miR-378 expression decreased in non-relapsed patients 4–6 months after surgery and miR-378 ability to discriminate CRC patients from healthy individuals was not influenced by haemolysis levels of plasma samples. Conclusion: The miRNA analysis on plasma samples represents a useful non-invasive tool to assess CRC presence as well as tumour-free status at follow-up. Plasma levels of miR-378 could be used to discriminate CRC patients from healthy individuals, irrespective of the level of haemoglobin of plasma samples.

Sanger sequencing primers. Schematic representation of the primers used for PCR and Sanger sequencing. (PPTX 302 kb)

Trabectedin clustering analysis. Unsupervised clustering analysis of trabectedin treated samples and control samples. The colors of the tree branches indicate distinct groups calculated by the clustering algorithm. Green boxes around the sample names indicate control samples, while red boxes show trabectedin-treated samples. (PPTX 68 kb)

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.

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators. Cancer Res; 70(6); 2235–44

Background Trabectedin has been approved in Europe as second-line therapy for advanced soft tissue sarcomas. A previous analysis showed that myxoid liposarcomas (MLS) are particularly sensitive to the drug. We report on the long-term efficacy of trabectedin in a subgroup of that series. Methods Since September 2002, 32 advanced pretreated MLS patients received trabectedin at our center. Data were reviewed focusing on their long-term outcome. Results Trabectedin was given as a 24-h continuous infusion every 21 days. A total of 376 and a median of 12 courses per patient (range 2–26; interquartiles range (IQR) 8–15) were delivered. Response rate per RECIST was 50% [95% confidence interval (CI) 32% to 68%], median progression-free survival (PFS) was 17 months (95% CI 13.5–30.1) and median overall survival is still not reached. In 10 patients, therapy was stopped in the absence of any evident disease, mostly after complete surgery of residual lesions. In these 10 patients, at a median follow-up of 25 months, PFS was 28.1 months (95% CI 25.6–36.4) from treatment start. Discussion These data indicate that the high response rate of MLS to trabectedin translates into prolonged PFS. Surgery of residual metastatic disease is already used quite extensively in metastatic MLS. Trabectedin may give further significance to this kind of surgery.

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.

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.

Expression profiles of breast carcinomas are difficult to interpret when they are obtained from tissue in toto , which may contain a large proportion of non-cancer cells. To avoid this problem, we microscopically isolated cells from a primary invasive ductal carcinoma of the breast and from an axillary node harboring a metastatic breast carcinoma, to obtain pure populations of carcinoma cells (≈500) and used them for serial analysis of gene expression. The expression profiles generated from both populations of cells were compared with the profile of a disease-free mammary epithelium. We showed that the expression profiles obtained are exclusive of carcinoma cells with no contribution of non-epithelial cells. From a total of 16,939 unique tags analyzed, we detected 559 statistically significant changes in gene expression; some of these genes have not been previously associated with breast cancer. We observed that many of the down-regulated genes are the same in both cancers, whereas the up-regulated genes are completely different, suggesting that the down-regulation of a set of genes may be the basic mechanism of cancer formation, while the up-regulation may characterize and possibly control the state of evolution of individual cancers. The results obtained may help in characterizing the neoplastic process of breast cancer.

Purpose To assess the role of TP53 status in predicting pathologic complete remission after primary chemotherapy in patients with ethmoidal intestinal-type adenocarcinoma (ITAC). Patients and Methods Thirty patients with ethmoidal ITAC enrolled onto a phase II study received chemotherapy with cisplatin, fluorouracil, and leucovorin (PFL) followed by surgery and radiation. On surgical specimens, absence of viable tumor cells was defined as pathologic complete remission (pCR). TP53 status/p53 function, analyzed on pretreatment biopsies, were retrospectively correlated with pathologic results and patient outcome. Results Twelve patients achieved a pCR; 18 patients did not (overall response rate, 40%). In patients with wild-type (wt) TP53 or functional p53 protein, the pCRs were 83% and 80%, respectively; in patients with mutated TP53 or impaired p53 protein, pCRs were 11% and 0%, respectively (P ≤ .0001). At a median 55-month follow-up, all pCR patients were disease-free; 44% of nonresponding patients experienced relapse (P = .0061). Conclusion The results indicate the existence of two genetic ITAC subgroups, defined by differences in TP53 mutational status or protein functionality, that strongly influence pathologic response to primary chemotherapy and, ultimately, prognosis. PFL seems to be highly effective in terms of pCR and disease-free survival in the presence of a wt or a still-efficient p53 protein, even when encoded by a mutated TP53 gene (eg, early-stop codon mutation), but ineffective in ITACs carrying a disabled p53 protein. Whether this model is extensible to other head and neck cancers needs appropriate investigation.

1. Introduction Gastrointestinal stromal tumors (GISTs) are the most common primary mesenchymal tumors of the human gastrointestinal tract, and most GISTs express constitutively activated c-Kit oncoproteins.1 Several clinical studies demonstrate that tumors showing mutation in exon 11 (encoding the juxtamembrane domain) respond better than all the other GIST tumoral genotypes. The KIT wild-type sequence folds into a series of β- hairpin structures. The structure, stability and folding of β- hairpin structures has been the object of many studies. Recently, several works reported successful simulations of reversible hairpin folding of different peptides in explicit water at native folding conditions through self-guided molecular dynamics (SGMD) simulations.2 2. Results and Discussion Using SGMD simulations we study the reversible folding events of wild-type and mutated c-Kit JMX domains. The mutations considered were the two-residue deletion Δ559-560 (Fig.1) and the missense point mutation V560G, both known to constitutively activate c-Kit in GISTs. This work aids to support the clinical evidence of a better response to Imatinib, an ATP-competitive TKIs, of KIT exon 11 mutant in comparison with WT receptor and provides the first atomistic description of the output of several clinical studies of GIST treated with Imatinib.

SCF is a potent pro-proliferative cytokine crucial for haematopoiesis, which binds to c-kit and activates its tyrosine kinase activity. Inactivating mutations of either SCF or c-kit have been described in mice and lead to increased sensitivity to treatment with ionising radiation. Imatinib is a tyrosine kinase inhibitor with high affinity for c-Abl, PDGFR and c-kit. In this study we investigated the effect of concomitant administration of imatinib and idarubicin, an anthracycline with haematosuppressive activity, in nu/nu mice and murine bone marrow cells. Double-treated animals showed significantly increased mortality compared to mice that received imatinib or idarubicin alone only when idarubicin and imatinib were given simultaneously. The combined treatment induced a more severe neutropenia with a slower recovery when compared to mice treated with idarubicin alone. The myeloid metaplasia usually observed in the spleen after idarubicin treatment was absent in mice co-treated with imatinib. Bone marrow from double-treated animals also showed decreased numbers of megakaryocytes and myeloid precursor cells. In vitro culture of murine bone marrow cells in the presence of imatinib inhibited SCF-induced proliferation and recovery from treatment with idarubicin. Our results indicate that the simultaneous administration of imatinib enhances idarubicin-induced haematopoietic toxicity in vivo and in vitro.

Detection of bladder cancer by multitarget multicolour FISH: comparative analysis on archival cytology and paraffin-embedded tissue We have evaluated the possibility of using the same specimen for both cytological diagnosis and multitarget multicolour FISH (MtMcFISH) analysis in order to determine whether the routinely processed specimens used for diagnosis were also suitable for this ancillary procedure. For this purpose 18 positive samples (11 voided urine and seven bladder washings) were selected, together with a representative section of the corresponding immediately previous or subsequent histological specimens. Two negative cytology slides were added as negative controls. FISH analysis revealed a normal pattern for each probe in the two negative controls and an abnormal pattern in the 18 positive cases. In the latter the same FISH alterations were found in the cytology samples and in the corresponding histological sections, and superimposable cytological/histological features were observed in two cases where two different histology samples were analyzed. The results clearly show that MtMcFISH may be successfully applied to destained routinely processed cytology slides.

A case series of 64 synovial sarcomas was characterized for the SYT-SSX fusion transcripts and statistically analysed in order to correlate molecular data with prognosis and morphology. SYT-SSX1 fusion transcript appeared to be an independent, though not reaching statistical significance (P = 0.183), prognostic factor clearly associated with a reduced metastasis-free survival. Regarding the association between transcript type and histologic subtype we found, a borderline P value (P = 0.067) between the SYT-SSX1 transcript and the biphasic subtype which, subsequently expanding the analysis to 70 cases, turned out to be significant. However, we could not confirm the prediction value of the biphasic subtype for the presence of the SYT-SSX1 transcript since in our hands 6 out 33 (18%) biphasic tumours carried the SYT-SSX2 transcript.© 2001 Cancer Research Campaign http://www.bjcancer.com

In a previous immunophenotypic molecular-based analysis it was shown that bcl2 over-expression characterizes the SS gene profile in addition to the non-random translocations. Here we show that the over-expression of an additional potentially antiapoptotic gene, the c-KIT gene, is associated with this tumour. Interestingly, whereas bcl2 over-expression appears to be restricted to the spindle cell tumoral component, c-kit mainly involves the epithelial component of biphasic SS. Twenty-three primary and metastatic samples from 21 patients were analysed by immunophenotyping (23/23), immunoprecipitations and Western blotting (3/23), and RT-PCR (23/23). Ten cases were biphasic and 13 monophasic in sub-type. Twelve, 10 and 1 case carried the SYT-SSX1, SYT-SSX2 and SYT-SSX4 fusion transcript, respectively. Co-presence of both c-Kit and SCF mRNA was observed in almost all cases (20/23), suggesting the occurrence of an autocrine loop. Immunophenotyping, confirmed by biochemical analyses, showed a modulation of c-Kit expression which was faint in the spindle and strong in the epithelial component, respectively. The study was complemented by c-Met/HGF receptor/ligand expression and c-Met protein analysis with results superimposable to those already reported. Since in each tumour, epithelial and spindle cell components harbour the same type of translocation t(X;18) the present findings suggest a shifting of the anti-apoptotic role from BCL2 to c-KIT gene during the transition from the uncommitted spindle to the differentiated epithelial cells. © 2001 Cancer Research Campaign http://www.bjcancer.com

Synovial sarcoma (SS) is cytogenetically characterized by the translocation t(X;18)(p11.2-q11.2) generating a fusion between the SYT gene on chromosome 18 and one member of the SSX family gene (SSX1; SSX2; SSX4) on chromosome X. Here, we report for the first time that 2 forms of SYT mRNA are present in both normal tissues and SSs. By amplifying the full-length SYT cDNA of two SSs, we detected 2 bands, here designated N-SYT and I-SYT. The latter, previously undescribed, contains an in-frame insertion of 93 bp. Its sequencing revealed a 100% homology with the mouse SYT gene. These two SYT forms were present, although in different amounts, in all human normal tissues examined, including kidney, stomach, lung, colon, liver and synovia. Coexistence of N-SYT and I-SYT (both fused with SSX) was detected in a series of 59 SSs (35 monophasic and 24 biphasic) and in a SS cell line. A preliminary analysis of the differential expression levels of N-SYT and I-SYT in SSs revealed that the latter was consistently overexpressed, suggesting a role in SS pathogenesis. © 2001 Cancer Research Campaign http://www.bjcancer.com

Synovial sarcomas (SS) are characterized by a chromosomal translocation t(X;18)(p11.2;q11.2) which usually fuses the SYT gene from chromosome 18 to SSX1 or SSX2 genes on chromosome X. Also, a Variant SYT-SSX4 fusion gene has recently been shown in a single SS case. In addition to these cytogenetic changes, bcl-2 expression, as assessed by immunohistochemistry, has been reported to be an almost general constitutive alteration of SS. In the present work, we analyze a series of 36 SS surgical samples (from 34 patients) by RT-PCR for the presence of the SYT-SSX1 or the SYT-SSX2 fusion transcript. The analysis was extended to SYT-SSX4 on SYT-SSX1-negative and SYT-SSX2-negative cases only. Our results showed a significant correlation between the SYT-SSX2 fusion and the monophasic SS histologic subtype. SYT-SSX1 fusion transcripts were present in both monophasic and biphasic tumors. The SYT-SSX4 fusion type was detected in a single monophasic SS. In the same series of SS cases, we also confirmed and extended the previously reported constitutive expression of bcl-2 protein, by using both immunohistochemical and western blot analysis. Moreover, we demonstrated that the BCL-2 gene is not rearranged or amplified at genomic level, indicating that the high levels of bcl-2 expression observed in SS might result from transcriptional activation of the gene and/or protein stabilization. Finally, we show that bcl-2 is not phosphorylated in tumors from patients who had been preoperatively treated with radio/chemotherapy, in tumors from untreated patients, or in an SS cell line (CME-1) after in vitro treatment with cytotoxic concentrations of DNA-damaging agents or taxanes. These data indicate that SS cells are unable to activate an apoptosis pathway involving bcl-2 phosphorylation/inactivation and may provide a possible explanation for the limited effectiveness of conventional pharmacological treatments of this tumor type.

The hormonal milieu at time of tumor surgery seems to have a significant impact on survival in premenopausal breast cancer patients. Indeed, surgery performed during the follicular phase of the menstrual cycle was suggested to correlate with a poor prognosis. To investigate the relationship between prognosis and menstrual cycle at time of surgery, we analyzed the expression of some markers associated with tumor aggressiveness, such as the hormone receptors, HER2, p53, Bcl2, and cathepsin D in breast carcinomas obtained from 198 premenopausal women who underwent surgery during different phases of the menstrual cycle. HER2 overexpression was found to fluctuate in hormone receptor-positive tumors. In actual fact, 20% of the tumors removed during the follicular phase scored HER2-positive, versus 8% of those removed during the luteal phase. Similarly, a number of hormone receptor-positive tumor specimens, obtained from the same patients during follicular and luteal phases, were scored HER2-positive when the sample was removed during the follicular phase and HER2-negative when removed in the luteal phase. Southern blot analysis of the HER2 gene indicated that, in hormone receptor-positive cases, the overexpression of HER2 is often not associated with gene amplification. The finding that overexpression of the HER2 gene, associated with tumor aggressiveness, can fluctuate according to the hormonal milieu may explain the increased survival of patients operated during the luteal phase. It is also relevant to the selection and treatment of patients most likely to benefit from anti-HER2 antibody therapy.

Aims Circumstantial evidence suggests that genetic changes may lead to tumor progression within the myxoid liposarcoma tumors (MLTs) carrying non-random chromosomal translocation t(12;16). Methods To address this subject an immunophenotypic analysis, applying antibodies against proteins encoded by TP53, MDM2 and CDK4 genes, complemented by molecular analysis of eight suitable cases, was performed on 104 consecutive cases. Chromosomal translocations were assessed either by cytogenetic analysis or by RT-PCR in 9 suitable cases and chimeric transcripts were found in all cases but two pleomorphic liposarcomas. Results Based on immunophenotyping and tumor site, the case material consisted of three groups. The first one was made up of 92 non-retroperitoneal cases carrying a null p53, mdm2, cdk4 immunophenotype, which remained unchanged over the time of recurrences and along the gamut of histologic subtypes. The second group was represented by five p53+, mdm2-, cdk4- non-retroperitoneal cases, 4 of which were further analysed by PCR-SSCP for p53 mutation. The im-munophenotipic profile of these cases, complemented by the molecular findings, supported a role of TP53 in tumor progression in three high-grade MLTs. The third group, consisting of 7 retroperitoneal cases, showed a heterogeneous immunophenotype, sharing immunophenotypic and molecular features with the well-differentiated/evoluted (dedifferentiated) liposarcoma group. Conclusions TP53 mutations seem to play a role in tumor progression in a few cases of MLTs (2.8%) showing more aggressive histologic characteristics. The unexpected finding that a number of retroperitoneal LMTs display the immunophenotypic profile of the well differentiated/evoluted (dedifferentiated) liposarcomas, deserves further investigation.

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.

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 mm3. 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.

To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.

The mapping near Kras2 of pulmonary adenoma susceptibility 1 (Pas1), a major locus affecting inherited predisposition to lung cancer in mice prompted us to test the homologous human region (12p12) for association with lung adenocarcinoma, by a population-based study. We genotyped 213 lung adenocarcinoma patients and 219 healthy blood donor subjects for five polymorphic markers mapping in the putative region of interest. Three marker polymorphisms, located in a region spanning approximately 700 kb, were significantly associated with lung adenocarcinoma risk. Furthermore, polymorphisms in KRAS2 and PTHLH loci were also associated with tumor prognosis. These results suggest the existence of a human Pas1 homologous locus on chromosome 12p12.

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 phosphoarrays, 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. © The Author 2013. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.

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.

Twenty-nine samples from 28 cases of vulvar squamous cell carcinoma, of which 13 fulfilled the criteria of the bowenoid subtype (mean age 45 years, range 31-68) and 16 of the usual subtype of invasive squamous cell carcinoma (ISCC) (mean age 67.5 years, range 34-83) were investigated for human papillomavirus (HPV) DNA, TP53 alterations, and mdm2 and bcl-2 gene product deregulation. Microscopically all the bowenoid subtype cases (group I) showed a high-grade intraepithelial (VIN 3, carcinoma in situ) lesion associated with early invasive carcinoma in six cases and overt invasive carcinoma in one. By contrast, no evidence of early carcinoma was present in the ISCCs (group II). By in situ hybridization and/or Southern blot hybridization or polymerase chain reaction (PCR), HPV DNA was detected in all cases of group I and in four of 16 cases (25%) of group II, two only by Southern blot after PCR. By single-strand conformation polymorphism and immunocytochemistry only wild-type TP53 and absence of detectable p53 product, respectively, were found in all cases of group I, i.e., in high-risk HPV-positive carcinomas, whereas mutations and/or p53 overexpression accounted for 75% in group II, i.e., in mainly HPV-negative carcinomas. The TP53 gene mutations observed in invasive carcinomas were significantly related to node-positive cases (p = 0.04). Taken together and in agreement with in vitro data, these results support the view that an alteration of TP53, gained either by interaction with viral oncoproteins or by somatic mutations, is a crucial event in the pathogenesis of vulvar carcinomas, but that TP53 mutations are mainly associated with disease progression. Finally, a preliminary immunocytochemical analysis seems to speak against the possible involvement of both MDM2 and BCL-2 gene products in the development of vulvar carcinoma.

An analysis of loss of heterozygosity for markers on both the short and the long arm of chromosome 11 was performed in 24 sporadic Wilms tumors. Six cases (25%) showed allelic losses involving the entire chromosome. In one case (4%) the loss was restricted solely to the WT1 gene on band p13. Two cases (8%) displayed allelic losses for WT1 and for markers on band p15.5, where the putative tumor suppressor gene WT2 has been mapped, but retained heterozygosity for markers on the long arm. In three tumors (13%) the loss of heterozygosity involved markers mapped to chromosomal regions p15.5 and q23.3-qter, but did not affect WT1 and markers on q12-q13. Altogether, the proportion of cases showing allelic losses at the distal region of 11q (37%) was comparable to that of cases with LOH affecting the WT1 (37%) or the WT2 (46%) loci, thus suggesting the existence of a third chromosome 11 tumor suppressor gene involved in the pathogenesis of Wilms tumors.

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.

Background Epidemiology and outcome of oropharyngeal cancer (OPC) are changing in the last decades, due to the role of HPV infection. No different treatment modality has been identified as more effective in treating OPC according to HPV or smoking status. Material and methods Two series of locally advanced (stage III-IV) squamous cell OPC patients (pts) treated at our Institution were considered: 1) treated with surgery followed by radiotherapy (dose 50-66 Gy), from 1/1991 to 7/2000 (“surgical series”) 2) receiving concurrent chemoradiation (CTRT) (RT dose = 66-70 Gy), with/without induction docetaxel, cisplatin, 5-fluorouracil (TPF) chemotherapy (CT), from 7/2004 to 3/2011 (“CTRT series”) Smoking habits and tumoral p16 expression were analyzed in order to stratify each series according to Ang risk profile (low, intermediate, high risk). Overall survival (OS) and disease free survival (DFS) were calculated with Kaplan-Meier method. Results Globally, 171 pts were considered, 56 in surgical and 115 in CTRT series. In CTRT series, 40% of the pts received induction TPF chemotherapy; in surgical series 57% of the pts had extracapsular extension and/or microscopically involved surgical margins. Surgical series CTRT series p16 expression 39% 59% Stage III 13% 7% Stage IV 87% 93% Low risk 20% 20% Intermediate risk 23% 41% High risk 57% 39% Five-year (yr) OS for p16 positive pts was 50% in surgical and 88% in CTRT series, while for p16 negative was 38% and 49% respectively (p When stratifying for risk profile, 5-yr OS of low risk CTRT pts was 100% vs 54% of surgical pts (p = 0.0042) and 5-yr DFS was 93% vs 53% (p = 0.0079); 5-yr OS of intermediate risk CTRT pts was 76% vs 46% of surgical pts (p = 0.0141) and 5-yr DFS was 79% vs 38% (p = 0.0359). High risk CTRT pts had a 5-yr OS of 51% vs 36% of surgical pts (p = 0.1902) and a 5-yr DFS of 24% vs 36% (p = 0.6411). Discussion In this retrospective analysis, low and intermediate risk OPC pts had a greater survival benefit when treated with CTRT compared with surgery followed by RT. Although with the limits of different RT techniques and lack of CT in adjunct to postoperative RT, these data should be considered as hypothesis generating for new trials design. Disclosure All authors have declared no conflicts of interest.

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

Background To evaluate neoadjuvant trabectedin (1.5 mg/m2 24-h i.v. infusion every 3 weeks; three to six cycles) in patients with locally advanced myoxid liposarcoma (ML) previously untreated with chemotherapy or radiation. Patients and methods Primary efficacy end point was pathological complete response (pCR) or tumoral regression rate. Objective response according to RECIST (v.1.0) was a secondary end point. Results Three of 23 assessable patients had pCR [13%; 95% confidence interval (CI), 3% to 34%]. Furthermore, very good and moderate histological responses were observed in another 2 and 10 patients, respectively. Histological decrement in the cellular and vascular tumor component and maturation of tumor cells to lipoblasts were observed in both myoxid and myoxid/round cell variants. Seven patients had partial response according to RECIST (objective response rate of 24%; 95% CI, 10% to 44%). No disease progression was reported. Neoadjuvant trabectedin was usually well tolerated, with a safety profile similar to that described in patients with soft tissue sarcoma or other tumor types. Conclusion Trabectedin 1.5 mg/m2 given as a 24-h i.v. infusion every 3 weeks is a therapeutic option in the neoadjuvant setting of ML.

Angiosarcoma is a highly aggressive soft tissue sarcoma. Responses to anthracyclines plus/minus ifosfamide, and taxanes alone or in combination with gemcitabine are well documented. Very few data are available on gemcitabine as a single agent.We retrospectively reviewed all cases of advanced progressive angiosarcoma treated with gemcitabine as a single agent (1000 mg/m(2) i.v. every week for 3 weeks every 4 weeks), at Istituto Nazionale Tumori and within the Italian Rare Cancers Network from January 2008 to November 2010.Twenty-five patients [mean age: 52 years; radiation therapy (RT)-related: 8] received gemcitabine. Best tumor response by RECIST was as follows: complete response = 2, partial response = 14, stable disease = 2, progressive disease = 7 cases, for an overall response rate (PR + CR) of 68%. Six of eight post-RT angiosarcomas responded to treatment. Median overall survival (OS) was 17 months. Median progression-free survival (PFS) was 7 months (range 1-40 months). One patient with a locally advanced thyroid angiosarcoma became resectable after 5 months of gemcitabine, with10% residual viable tumor cells seen on surgical specimen. Overall, gemcitabine was well tolerated.Gemcitabine is active in both RT- and non-RT-related angiosarcoma, with dimensional and possibly long-lasting responses. A formal phase II study on gemcitabine as a single agent is warranted.