Guido Biasco

E20527 Background: Liver and peritoneum are the most common site of recurrences from gastrointestinal stromal tumors (GISTs). Bone metastases represent a rare finding in clinical practice and their biological significance and clinical management remain unsettled. Methods: We reviewed all patients with advanced/metastatic GIST at our institution and focused on cases with bone metastases, describing the clinical and radiological features of these lesions. Results: Among 71 patients, four had bone metastases. Two were female (82 and 54 years of age) and two were male (62 and 44 years of age). All four of them had an high-risk primary GIST, localized in small intestine in three cases, and in the stomach in the other one. One patient harboured a KIT esone 11 mutation (T>A 69429(p.V559d), one patient had a WT GIST and in the other patients mutational analysis was not assessable. Bone tumour involvement was present at the time of initial diagnosis in two patients, and occurred during the follow-up period after 7 and 2 years in the other two. In all patients bone metastases were associated to other visceral site of relapse. Radiologically, the lesions were diagnosed by CT-scan and had a lytic pattern in all cases, with a widespread skeletal involvement. In no case a single lesion was found. Clinically, bone metastases were symptomatic in three patients and presenting with a pathological fracture in a single case. In only one patient bone lesions were asymptomatic and diagnosed as occasional finding. All patients received zoledronic acid in association with TK inhibitors, obtaining a long stable disease in two cases whereas there was a quick disease progression in the other two cases. Only one patient was also treated with radiotherapy for palliative purpose. Conclusions: Although bone metastases are an infrequent site of recurrence from GISTs, they may become more prevalent due to increased patient life expectancy as well as the improvement in imaging techniques. By now their biological background and prognostic value and their clinical management are unknown. Moreover standard imaging and metabolic criteria for diagnosis and tumor response assessment are still lacking. These data should be collected in large series.

E14590 Background: Biliary tract cancers are rare malignancies with a very poor prognosis. Several studies comparing chemotherapy and supportive care showed significant survival prolongation and improvement in life quality with chemotherapy. The association of gemcitabine and a platinum compound is considered the standard first-line treatment but there are no adequate data supporting any second-line therapy. Our retrospective study aims at evaluating retrospectively the impact of second-line chemotherapy in this setting. Methods: We reviewed medical records of patients (pts) affected by histologically confirmed biliary tract cancer, treated in our institution from 2005 to 2010 with a second line chemotherapy after first-line treatment progression. Results: We identified 121 pts progressing after first-line chemotherapy. 56 (46%) of these were considered fit for a second-line treatment and 49 (40%) were effectively treated. Of these 49, 34 had relapsed after radical surgery and 15 were unresectable at diagnosis. The primary site was intrahepatic in 25 pts, extrahepatic in 18 and gallbladder in 6. The first-line schedules were mainly gemcitabine plus a platinum compound (n=35) or gemcitabine plus capecitabine (n=8). As second line, gemcitabine plus a platinum compound and gemcitabine plus capecitabine were delivered respectively in 15 and 14 pts. The remaining 20 were treated with other regimens (6 gemcitabine plus irinotecan, 14 monochemotherapy). Median time to progression was 3.5 months (IC_95% 2.0-5.0) and median overall survival, calculated since second-line treatment start, was 8.1 months (IC_95% 4.3-11.9). Median overall survival calculated since first-line start was 18.7 months (IC_95% 13.9-23.5). Conclusions: Many patients affected by biliary tract cancer have still a good performance status after first-line chemotherapy and could be treated in second-line with potential survival benefit. The optimal regimen has not yet been identified.

10056 Background: Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic mutations of KIT and PDGFRA receptors. However about 10%-15% of adult GISTs do not harbor any KIT or PDGFRA gene mutation and are defined as wild-type (WT). Studies reported that gene expression profile and genome are strongly different between WT and mutated cases. On the contrary, up to now, really few data are available on microRNAs. We studied the miRNA profiles in GISTs correlated with kinase genotype. Methods: We analyzed the miRNA profile of 13 GIST tumor samples, of which 9 carry either KIT or PDGFRA mutations, and 4 do not carry any KIT and PDGFRA mutations. Total RNA was labeled, hybridized to Agilent microRNA microarrays; differential microRNAs were selected by a two-fold change cutoff and Benjamini and Hochberg-corrected unpaired t test. Gene targets of differentially expressed miRNAs were selected if supported by at least five Prediction Algorithms out of six (miRDb, PicTar, Elmmo, MiRanda, PITA, TargetScan). Results: The miRNA profile is different in WT and mutated GIST samples, with 56 microRNAs differentially expressed (0.05 cutoff p-value). By correlation with gene expression profiling data, we identified the miRNA target genes differentially express between WT and mutated tumors. hsa-let-7b and hsa-let-7c microRNAs are the most significantly differentially expressed miRNAs, resulting less expressed in WT samples and able to target IGF1R receptor. We reported that WT-GISTs overexpress IGF-1R, in spite of Copy-Number-Analysis by high-resolution mapping arrays demonstrated no IGF-1R amplification. These data suggest that overexpression of IGF1R in WT-GISTs could be associated to the downregulation of microRNAs targeting these gene. Validation studies on the significance of the identified miRNAs are ongoing. Conclusions: WT-GISTs have a different miRNA profile compared with mutated patients. hsa-let-7b and hsa-let-7c, targeting IGF1R, are the most significantly differentially expressed. microRNA expression difference between WT and mutated GISTs needs to be more investigated in order to understand the biological role on the pathogenesis, clinical behavior and treatment responsiveness.

10046 Background: Recently SDH variations were identified in KIT and PDGFRA wild type GISTs. However the causative genomic alterations of these GISTs still remain unknown. Massively parallel sequencing allows identifying novel putative variants. Methods: Whole transcriptome paired-end RNA sequencing was performed by Illumina GAIIx system using a 75 bases paired-end strategy on tumor samples of two young adults patients (P1 and P2) affected by gastric WT-GIST (age 28 and 30 years). Sequences were aligned with BWA against exon+junction references. SNVmix2 was used for SNP calling, identifying by this 2045 and 1780 coding non-synonymous novel single nucleotide variants (SNVs) in P1 and P2, respectively. After checking misalignments by SAMTools, the variants were filtered to increasing SNPcall confidence: SNVs with quality read score > 30 (error probability of 0.1%), total coverage > 40, and ratio between coverage of alterate base and total coverage >0.3 were labeled high confidence. Single point mutations were translated at the protein level and their likelihood of being disease-associated was computed with SNP&GO and confirmed with Sanger sequencing. Results: In both patients common mutations in SDHA and different private SNV were highlighted. Among the private disease-related SNVs we identified mutations in MYH9 (myosin heavy chain 9) and TPI1 (triosephosphate isomerase 1) in patient P1, and a mutation in OGDHL (oxoglutarate dehydrogenase-like) in P2. MYH9 is involved in cell motility and cytokinesis, while OGDHL is an enzyme of the Krebs cycle, as the SDH complex, and TPI1 is involved in glycolysis. Ongoing validation by Sanger sequencing on DNA from tumor and peripheral blood (PB) already confirmed the presence of heterozygous MYH9 K1775E in P1 and OGDHL V815M in P2, both in tumor and PB, showing that these mutations are germinal. Conclusions: Massively parallel RNA sequencing, followed by data analysis, allows to discover novel single nucleotide variants and to identify new potential target genes in WT-GIST.

E14608 Background: The antiangiogenic drug sorafenib is standard treatment for advanced hepatocellular carcinoma (HCC) not suitable for surgical or loco-regional therapies in patients with preserved liver function. Many second-line drugs are currently under investigation, but none has proven effective so far. Metronomic chemotherapy is characterized by antiangiogenic activity as well and good tolerability. Therefore we started a phase II clinical study to assess efficacy of metronomic capecitabine in advanced HCC patients. Methods: This phase II trial allowed inclusion of 90 HCC patients (pts) in two sub-studies: first-line systemic treatment (60 pts) and second-line after sorafenib (30 pts.) Main inclusion criteria were Child-Pugh liver function class A and progressive disease not candidate for surgery or loco-regional treatments. Treatment schedule was capecitabine 500 mg bid after meals continuously. In case of severe toxicity up to two weeks off-therapy were permitted for recovery; no dose adjustment was allowed. Primary endpoint is three-months progression-free survival rate (PFS_3mo), assessed by RECIST 1.1. This preliminary analysis aims at evaluating efficacy and toxicity of metronomic capecitabine after progression on sorafenib treatment. Results: Patients’ accrual ended in December 2010. A total of 30 pts pre-treated with sorafenib have been included; of these, 12 had relapsed after surgery and 24 had been previously managed with percutaneous ablations or chemoembolizations. 23 pts are currently assessable for efficacy. We observed no objective responses, 9 stable disease and 14 progressive disease, for a PFS_3mo of 39%. Overall and progression-free survival will be evaluated in a further analysis with longer follow-up. All patients are assessable for toxicity. The most common adverse events were asthenia, nausea, diarrhoea and pruritus, generally mild and tolerable. Conclusions: Metronomic capecitabine is well tolerated and can be active in HCC patients after progression under sorafenib treatment. The extent of clinical benefit will be determined in the final analysis.

10045 Background: About 10%-15% of GISTs do not harbour KIT/PDGFRA mutations (wild-type, WT). We analyzed the tumor transcriptome of two sporadic young adults WT GISTs patients using massively parallel sequencing approach in order to identify novel genetic mutations. Methods: Patients were a 28 years-old female (GIST_07) and a 30 years-old male (GIST_10). Primary tumors were in the stomach, multi-centric in GIST_07 and uni-centric in GIST_10, and presented metastases at diagnosis. RNA-Seq paired-end libraries were sequenced on the Illumina GAIIx system using a paired-end strategy at a read length of 75bp. Bioinformatic analysis highlighted 9 candidate genes common to both patients. Mutations in these genes were then filtered with SNP&GO, a tool suited to compute the likelihood of a mutation to be disease-related or not, depending on the protein sequence and its functional annotation. The identified candidate gene was analyzed by Sanger sequencing on tumor (T) and peripheral blood (PB) DNA from GIST_07 and GIST_10 patients, and on additional 8 tumor and 3 PB DNA from 8 sporadic WT GISTs patients. Immunohistochemistry (IHC) was also performed. Results: Only mutations in the sequence of SDHA were identified as disease-related with a high reliability index. GIST_07 tumor DNA was homozygous for an exon 9 p.S384X nonsense mutation; GIST_10 patient showed compound heterozygosity for a germinal exon 2 p.R31X nonsense mutation and a somatic exon 13 p.R589W missense mutation.Heterozygous nonsense mutations were already present in germline DNA in both patients. All mutations lead to functional inactivation of the protein. IHC revealed that both patients showed the down regulation of SDHA. The expanded study showed that 2/10 carried SDHA mutations in the tumor: compound heterozygosity for an exon 9 p.G419R and exon 13 p.E564K mutations in one patient, and a somatic heterozygous exon 5 p.R171C mutation in the other. Conclusions: Recently, SDHB and SDHC mutations were also reported in these patients. This the first study that identifies mutations on SDHA, in 4/10 WT GIST patients. This gene should be evaluated in larger seriesand be added in the sequencing screening of WT GISTs.

299 Background: Biliary tract cancer is a rare malignancy with poor prognosis. Surgery is the only potential curative approach, but even if surgical intervention is performed correctly the relapse risk remains very high. In adjuvant setting only few randomized trials using no standard treatments and producing controversial data are reported in literature. Methods: This is a retrospective analysis on 144 consecutive biliary tract cancer patients (pts), undergone potentially curative resection in our institution (109 pts with R0 surgery and 31 pts with R1 surgery). The series included 57 intrahepatic cholangiocarcinomas (ICC), 68 extrahepatic cholangiocarcinomas (ECC), 19 gallbladder cancers (GBC) (Table). Median age was 63, 80 pts were males and 64 pts were females. 80 (60 R0 and 20 R1) out of 144 pts received adjuvant chemotherapy and the remaining 64 pts (49 R0 and 15 R1) started a follow-up program. Adjuvant chemotherapy schedule was gemcitabine 1000 mg/m² at day 1, 8, 15 every 28, for six months. Results: Median time to relapse (TTR) in the treatment group and in the follow-up group were 18 months and 11 months respectively (p=0.038). No grade 3-4 chemotherapy-related adverse events were observed and only grade 1-2 thrombocytopenia occurred. This hematologic toxicity did not affect treatment dose intensity. Conclusions: Our preliminary retrospective analysis suggests a significant advantage of adjuvant treatment on TTR in radically resected biliary tract cancer patients, although further placebo-controlled double blind trials are required. [Table: see text] No significant financial relationships to disclose.