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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.

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 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.

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.

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.

Aim: Clinical and radiological diagnostics are not able to predict nodal or extranodal extension known to negatively impact on disease outcome in oral cavity squamocellular cancer (OCSCC). We assessed whether a gene-expression signature from specimens obtained from primary tumour could predict for nodal status. Methods: A series of archival specimens from patients with stage III-IV OCSCC treated with surgery at T and N level as first treatment from 1989 to 2008 was collected. The histological samples were randomly chosen in order to have an equal number of cases with pathological negative nodes (pN0), positive nodes without extracapsular extension (pN + ECS-) and positive nodes with extracapsular extension (pN + ECS+). The histological specimens of primary disease were microdissected in order to obtain one sample from the central area of the tumour (pTcent) and one from the peripheral area (pTperiph). The samples of primary tumour were profiled for gene expression on DASLwg Illumina BeadChips. Results: We present hereafter the results of the first 29 patients in the trial (pN0 = 10; pN + ECS- = 9; pN + ECS + = 10). Gene expression profile resulted in a data matrix containing about 18600 detected genes. We focused our attention on the genes differentially expressed between pN0 and pN+ and between pN + ECS- and pN + ECS+. Imposing a significance threshold of false discovery rate (FDR) Conclusions: The research revealed different gene expression signatures on primary tumour useful to predict nodal status in OCSCC. Mature results of the analysis on a higher number of cases will be presented during the meeting. *Acknowledgment: Italian Ministry of Health call “Ricerca Finalizzata” (Project code: GR -2009 -1492184). Disclosure: All authors have declared no conflicts of interest.

Background In GIST, KIT/PDGFRA mutations correlate with response to TKI. The exon 18 PDGFRA-D842V mutation is known to be resistant to imatinib and sunitinib. Activation of PDGFRA involves two main signal transduction pathways, RAS/MEK/ERK, which involves BRAF, and PI3K-AKT. We used sorafenib in three pts with a PDGFRA-D842V mutated metastatic GIST. Methods Since January 2010, 3 PDGFRA-D842V mutated GIST pts (age: 57, 71, 72 yrs) have been treated with sorafenib 400 mg twice daily, on an individual use basis. Two pts were progressing on imatinib in association to mTOR inhibitors (sirolimus and everolimus), while the third had four previous lines of therapy. Molecular/biochemical analyses were performed on cryopreserved material taken before sorafenib treatment in two of the three patients (one biopsy and one surgical specimen). A primary cell culture was obtained from one patient. Results One pt had a Choi's response 3 mos after starting the treatment; the others a RECIST response at 2 mos. The first pt had a tumor progression after 12 mos from starting therapy and 9 mos from tumor response. The second pt had a partial response lasting 15 mos. Treatment was relatively well tolerated, with G3 skin toxicity in one pt, causing a dose reduction. Biochemical analyses on pretreatment tumors revealed activated S6K and S6, both downstream mTOR, as well as ERK1/2 strong activation. The primary cell culture was treated with sorafenib 0.5 and 1 µM and phosphorylation switch-off of both S6K and S6 (Ser 240/244), associated with a retained ERK1/2 expression/activation, was observed. Further analyses are ongoing to verify which pathways converging on mTOR are inhibited by sorafenib. Conclusions In the unresponsive PDGFRA-D842V mutated GIST we saw clinical signs of antitumor activity of sorafenib in 3 pts. This was consistent with biochemical analyses, which showed an inhibitory effect downstream mTOR. This clinical setting constitutes a high-priority unmet medical need, since very few clinical data are known and no functional analyses on sorafenib activity have been reported so far. Disclosure P.G. Casali: Advisory role and honoraria for lectures: Novartis Pharma, Pfizer. Advisory role: Bayer. All other authors have declared no conflicts of interest.

Background To report on anthracycline-based chemotherapy in a retrospective case series analysis of 45 solitary fibrous tumor (SFT) patients treated within the Italian Rare Cancer Network (RTR). Methods We retrospectively reviewed all SFT treated with chemotherapy at our Institution or within RTR since 2002, focusing on anthracyclines (i.e. doxorubicin or epirubicin), administered alone or in combination with ifosfamide. Responses to ifosfamide as a single agent were also evaluated. Pathologic diagnosis was centrally reviewed in all cases, by distinguishing typical, malignant (MSFT) and dedifferentiated (DSFT) SFT subtypes. Results Among 45 pts with SFT treated with chemotherapy, we could select 31 patients (M/F: 14/17 – mean age: 62 years - locally advanced/metastatic/adjuvant: 13/17/1 - front-line/ further line = 25/5; typical/MSFT/DSFT = 0/17/13) who received anthracycline-based chemotherapy (single agent in 6, combined with ifosfamide in 24). 28 pts are evaluable for response (adjuvant = 1; too early = 1; other = 1). Overall, best response according to RECIST was: partial response (PR) = 6 (21%), stable disease (SD) = 7, progressive disease (PD) = 15 cases. Responses were confirmed after 3 months. In 2 pts with SD, a minor dimensional response was detected. Median PFS was 4 (range 2-15) months, with 20% of patients being progression-free at 6 months. PR were found in 1/16 (6%) MSFT e 5/12 (41%) DSFT. 17 pts received high-dose prolonged-infusion ifosfamide (front-line/further line = 15/2; typical/MSFT/DSFT/not assessable = 0/11/3/3 with 2 PR (both MSFT), 4 SD, 11 PD. Conclusions Anthracycline-base chemotherapy is active in a proportion of SFT pts. However, a high response rate was observed in DFST. Comparatively, ifosfamide as a single agent has low activity. Disclosure S. Stacchiotti: Pfizer: research grant for studies in which my institution is involved; honoraria. M. Libertini: Pfizer: research grant for studies in which my institution is involved;. E. Palassini: Pfizer: research grant for studies in which my institution is involved;. B. Vincenzi: Pfizer: honoraria. A.P. Dei Tos: Pfizer: honoraria. P.G. Casali: Pfizer: research grant for studies in which my institution is involved; honoraria; advisory (compensated). All other authors have declared no conflicts of interest.

Laser-induced fluorescence from normal and neoplastic tissue differs in shape and intensity and can be used for diagnostic purposes. In this view a new device, LIFE imaging system (Xillix Co., Vancouver), is available for clinical application in the tracheo-bronchial tree ad it has been used in 140 patients at high risk of lung cancer at INT of Milan (Italy). After a white light bronchoscopy, the patients were submitted to fluorescence bronchoscopy and the findings of both examinations have been classified in 3 cathegories: class I or normal, class II for alterations suggesting inf1ammation or metaplasia and class III for neoplastic mucosa. All II and III sites have been biopsied for histologic examination: a comparison between histologic results and white light or fluorescence bronchoscopy has been performed for assessing sensitivity and specificity of the two exams. Squamous intraepithelial neoplasia has been detected in 19 patients with a sensitivity of 74% for white light bronchoscopy and 100% for fluorescence bronchoscopy. Specificity of white light and fluorescence bronchoscopy was 92% and 79%, respectively. These data suggest that fluorescence bronchoscopy improves accuracy of endoscopic examination in detecting early lung cancer.

In an attempt to reduce the recurrence rate and to improve survival in resected stage-II nonseminomatous testicular cancer, two consecutive series of patients were treated with adjuvant vinblastine-bleomycin (VB) continuous infusion, or cisplatin, vinblastine, bleomycin (PVB). 11 former patients who received no adjuvant chemotherapy and 5 who refused the adjuvant treatment were considered historical controls. After a median follow-up of 5 years, relapses were 4 in 15 patients treated with VB (27%), 1 in 29 treated with PVB (3%) and 8 in the untreated (50%). Adjuvant PVB significantly reduced the recurrence rate (p less than 0.001), while VB did not (p = 0.4). However, the advantage of adjuvant PVB was evident only in patients with retroperitoneal metastases larger than 5 cm, macroscopic extranodal spread, and tumor invasion into retroperitoneal veins, who were classified as pathologic stage II-C: no relapse and 100% survival following adjuvant PVB in 11 cases, versus 86% relapses and 28.5% survival in 7 untreated patients (p less than 0.01); 2 relapses and 2 survivors in 3 treated with VB. On the other hand, only 1 of the 39 patients with less extensive retroperitoneal disease died of testicular cancer, and he was in the VB adjuvant treatment group. We conclude that only aggressive adjuvant chemotherapy significantly reduces the recurrence rate and improves survival in resected stage-II nonseminomatous testis cancer. However, it is mandatory only in the very high risk subset II-C of patients, while the others could be carefully followed at monthly intervals and safely treated with aggressive chemotherapy only in the event of relapse.

A retrospective analysis of patients with pathologic stage I nonseminomatous germinal testis tumors treated with orchidectomy and retroperitoneal lymphadenectomy was done in an attempt to define the relapse pattern and to eventually identify risk factors predisposing to tumor recurrence. Of 102 patients, 91 (89.2%) remain disease free with a median follow-up of greater than 5 years (range 3-10 years), and 11 (10.8%) suffered relapse 3-35 months after lymphadenectomy (median free interval 6 months). 9 of 11 patients developed primarily intrathoracic recurrences. The tumor was so rapidly progressing at relapse, that it was diagnosed when clinically advanced in 7 of 11 cases. Nevertheless, 8 of 11 patients were salvaged with effective available chemotherapy and resection of residual disease. Only scrotal violation showed a significant increased risk of tumor recurrence (5 of 24 cases, versus 6 of 78 who had inguinal orchidectomy, p less than 0.05). We recommend a very close follow-up for all patients with pathologically assessed stage I nonseminomatous germinal testis tumors during the first 3 years following orchidectomy and retroperitoneal lymphadenectomy. With early recognition of relapse, an approximately 100% cure rate will be achieved with effective available chemotherapy.