Fausto Catena

Systemic response to cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) causes the activation of endocrine, metabolic, hemodynamic and inflammatory processes. The aim of this work is to describe and analyze the time course of the inflammatory markers concentration during CRS+HIPEC in plasma and peritoneal fluids and the association with hemodynamic and metabolic parameters. Pre-, Intra- and Post-operative data were collected. Tumor necrosis factor (TNF), interleukine 6, procalcitonine (PCT), cancer antigen 125 (CA-125) in blood and in peritoneal fluids were evaluated. Thirty-eight patients included, 29 (76.3%) female. Mean/median PCI: 9.2/5. Primary malignancy: 5 colo-rectal (13.2%), 5 gastric (13.2%), 23 ovarian (60.5%) and 5 others (13.2%). CCR 0-1 reached in all patients. Cardiac Index, Heart rate and Central Venous Pressure, increased during the procedure while Stroke Volume Variation showed a decrease. Mean Arterial Pressure and Superior Vena Cava Oxygenation were stable through the whole procedure. TNF and CA-125 were steady during the whole procedure; IL-6 had a relevant increase from baseline to start of perfusion (p<0.01); PCT had a steady increase at every time point. Peritoneal sampling showed a statistically significant increase (p<0.01) between start and end of the perfusion phase for all markers but TNF. Serum and peritoneal marker concentration were similar for TNF, PCT and CA-125. IL-6 showed a sharp difference. The most significant variations are those of IL-6 and PCT. The cytokines level parallel the hemodynamic derangements. Treatment during HIPEC should mimic the established treatment during sepsis and septic shock.

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.

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.