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1592 Background: The germline variant APC I1307K is one of the most commonly identified pathogenic variants on germline genetic testing panels. The purpose of the Molecular Epidemiology of Colorectal Cancer study was to quantify the risk of colorectal cancer among carriers, characterize the clinical, pathologic, and molecular features of colorectal cancers arising in patients with APC I1307K, and to describe the overall and disease-specific survival of carriers with colorectal cancer. Here, the final results of the Molecular Epidemiology of Colorectal Cancer Study are reported with respect to APC I1307K. Methods: We consented 6,006 incident, pathologically confirmed cases of colorectal adenocarcinoma and 5,023 age, sex, and ethnicity matched controls without colorectal cancer between March 31, 1998 and July 1, 2017 within a geographically defined area of Northern Israel. Comprehensive, in-person epidemiologic interviews were conducted for cases and controls, with uniform histopathologic review, detailed molecular analysis, medical record review and clinical follow-up for up to 21 years. Results: The demographic and clinical features of incident colorectal cancer cases matched the population distribution of colorectal cancer in Israel. APC I1307K was identified in 429 (7.1%) of cases and 201 (4.0%) of controls. The estimated relative risk of colorectal cancer among carriers was 1.89 (95% confidence interval, 1.59 - 2.24), p < 0.0001. The prevalence and odds ratios differed by ethnic group. Homozygous carriers were at especially high risk, with an odds ratio of 3.90 (95% confidence interval 1.11–13.71). APC I1307K carriers were significantly less likely to have microsatellite instable tumors (p = 0.04). Overall survival of APC I1307K carriers was not significantly different than survival of non-carriers, after adjustment for age, stage, sex, ethnicity, and microsatellite instability. Conclusions: APC I1307K is an actionable germline mutation that confers meaningful lifetime risk of colorectal cancer in heterozygous and homozygous carriers. APC I1307K is not an independent prognostic factor for overall survival or disease specific survival and is not associated with the MSI phenotype. Cumulative lifetime risk estimates inform genetic counseling and provide data for policies regarding the timing and frequency of screening and other preventive strategies.

TPS9122 Background: Lung cancer (LC) is a rare disease among young adults. Polymorphisms in genes involved in carcinogenic metabolism of tobacco and/or xenobiotic metabolism have been identified in those patients. The growing use of next generation sequencing (NGS) unravelled germline mutations in genes associated with hereditary cancer in patients with LC. However, germline DNA mutations have not been systematically studied in young adults with LC. Early onset LC patients are likely to harbor actionable somatic mutations. Broad hybrid NGS can identify actionable genomic alterations in LC patients deemed driver negative by routine molecular analysis. Trial Design: This is a multicentre, prospective, single-cohort study to determine whether young patients with LC harbor germline mutations and to evaluate the impact of NGS using Foundation One platform on the therapeutic options and overall survival for this specific patient population. Major inclusion criteria: 1) patients already or newly diagnosed of non-small cell lung cancer at any stage at age < 51; 2) to have sufficient tumor sample to perform standard molecular diagnosis ( EGFR/ALK/ROS1/BRAF); 3) to provide written informed consent for the study. Methods: After signing the informed consent form, family history of cancer and smoking history will be collected and a peripheral blood sample will be obtained. Germline targeted sequencing will be carried out in our centre using a customized hereditary cancer panel (I2HCP) of 136 genes designed to cover the coding exons and intron-exon boundaries of genes associated with moderate or high risk of hereditary cancer. Patients with clinical criteria for hereditary cancer or harboring pathogenic or probably pathogenic germline alteration will be referred to the genetic counselling unit. Patients will follow the usual clinical pathway for biomarker analysis and in case of remaining tumour material it will be used for conducting comprehensive genomic profiling through Foundation One platform. When tumour material available will not be sufficient to perform NGS, a tumour re-biopsy will be considered. Enrolment begun on June 2018 and, to date, a total of 41 patients have been included in the study.

3559Background: Mutational analysis of RAS in mCRC has showed an improvement in patient (pts) selection for anti-EGFR drugs. The standard techniques (Techs) have an analytical sensitivity (Sns) of ...

e17071Background: Since the approval of the PARP inhibitor olaparib, the TFGT indications for OC have dramatically increased. Furthermore, several guidelines include genetic testing for all non-muc...

e14613 Background: Mutational analysis of RAS in mCRC has demonstrated an improvement in patient selection for anti-EGFR drugs. The techniques for the detection of point mutations have an analytica...

e12579 Background: Individuals at high risk of developing breast cancer benefit from an annual surveillance with contrast enhanced breast magnetic resonance imaging (CEMRI). However, this technique...

e22057 Background: Metronomic chemotherapy has been suggested as a maintenance administration strategy after the Maximum Tolerated Dose (MTD) treatment in a multi-targeted chemo-switch schedule (C-S). We report the effectiveness of this chemo-switch schedule in mice models of human pancreatic and ovarian cancer. Methods: In pancreas cancer models, Gemcitabine (G) was administered on four different schedules: metronomic (METG), Maximum Tolerated Dose (MTDG), chemo-switch schedule (C-SG, MTDG dosing followed by the METG) and anti-angiogenic (MTDG followed by the administration of monoclonal anti-VEGF receptor antibody DC101). In ovarian cancer model, animals were treated following a Cyclophosphamide chemo-switch schedule (C-S CTX). Results: In all these models C-S schedule had the most favorable effect, reaching at least 80% of tumor growth inhibition in absence of increased toxicity. Moreover, in the pancreas cancer model while peritoneal metastases were observed in control and MTD groups, no dissemination was observed in the MET and C-S groups. C-S treatment caused a decrease in angiogenesis and its effect on tumor growth was similar to obtained by the MTD followed by anti-angiogenic DC101 treatment. At the molecular level, C-S treatment combined an increase in thrombospondin-1 expression with a decrease in number of CD133+ cancer cells and triple positive CD133+/CD44+/CD24+ cancer stem cells. Conclusions: These findings provide confirmation that the chemo-switch schedule is a challenging clinical strategy with demonstrated inhibitory effects on tumor dissemination, angiogenesis and cancer stem cells.

3629 Background: Different clinico-pathological factors are used to define a group of patients with high-risk stage II colon cancer (CC) that may benefit of adjuvant treatment. Moreover, several molecular markers, such as microsatellite instability (MSI) and BRAF, have been widely investigated as prognostic factors. Recently a high stroma component (EMT+ subtypes) has also been associated with poor outcome. The aim of the study is to analyze the prognostic value of MSI, BRAF and tumor-stroma ratio in a prospective series of stage II CC patients. Methods: FFPE tissue from 432 stage II CC patients operated at Hospital Universitari de Bellvitge (1996 - 2006) were included in the study. MSI status was assessed by the analysis of 5 mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). BRAF V600E mutation was analyzed by single strand conformation polymorphism technique. Tumor-stroma ratio was analyzed by immunohistochemistry. Associations between molecular factors and clinical features were assessed by Chi-Squared (X2) tests. A Cox regression model was used to evaluate the Relapse Free (RFS) and the Colon Cancer specific Survival. Results: MSI status could be determined in 350 patients. 48 (14%) had MSI high (MSI-H) tumor. BRAF status could be assessed in 380 cases. 58 (15%) cases were BRAF mutated. Tumor-stroma ratio was analyzed in 407 tumor samples. 176 (43%) tumors had more than 50% intra-tumor stroma and were classified as stroma-high. MSI-H tumors were significantly located in right colon (X2 p-value = 1.03e-5), were poorly differentiated (X2 p-value = 0.003) and had more than 12 lymph nodes resected (X2 p-value = 0.037). MSI-H tumors were associated with BRAF mutation (X2 p-value = 0.02) and stroma-low (X2 p-value = 0.0005). When a molecular prognostic risk classification was performed, patients with MSI-H /stroma-low suggested a low risk of relapse comparing to MSI-H/stroma-high, microsatellite stable (MSS)/stroma-low and MSS/stroma-high patients (HR = 3.15; 95 CI, 0.76 – 13.1, p-value = 0.0602). Conclusions: MSI-H tumors have BRAF mutation as well as low intra-tumor stroma. Our results suggest that tumor-stroma ratio can explain differential prognosis in MSI-H stage II tumors.

11056 Background: The microRNA-34b/c (miR-34b/c) is a transcriptional target of TP53 that is frequently mutated in primary lung adenocarcinoma (AC). We investigated the clinical implications of miR-34b/c methylation in early stage lung AC patients and the functional role of miR-34b/c re-expression in lung AC cell lines. Methods: DNA methylation of miR-34b/c promoter was assessed by real-time PCR temperature dissociation in 15 lung AC cell lines and 140 early stage lung AC resected at the Bellvitge Hospital (Barcelona) and at the University of Michigan (Ann Arbor). Patient characteristics: 65% males, 88% smokers, 68.5% stage I and 31.5% stage II. Expression of miR-34b/c was determined by TaqMan RT-PCR. Two lung AC cell lines (NCI-H1838 and SK-LU-1) were transfected with an expression vector containing both miRs and the effects upon cell proliferation, migration and invasion were determined. Results: MiR-34b/c was methylated in 40% of cell lines and in 46% of primary tumors with significant association with higher tumor stage (P=0.033), recurrence (P=0.017) and death (P=0.005). In the training set (n=58), patients with higher levels of miR-34b/c methylation had significantly shorter median DFS (28.5 months) compared to low to medium levels (not reached, log-rank P=0.016). In the test set (n=82), higher levels of miR-34b/c methylation were also associated to shorter median DFS (19 months) compared to patients with low to medium levels (not reached, log-rank P=0.005). MiR-34b/c methylation remained an independent prognostic marker for DFS after adjusting by age, gender and stage. Tumors harboring TP53 mutations and miR-34b/c methylation expressed significantly lower levels of miR-34b/c (P≤0.001). Stable cells expressing miR-34b/c had lower proliferation rate relative to cells transfected with empty vector (P≤0.001). Expressing miR-34b/c in SK-LU-1 cells reduced migration and invasion ability. Conclusions: Epigenetic inactivation of miR-34b/c by DNA methylation is an independent prognostic marker in early stage lung AC. Ectopic expression of miR-34b/c generated a less aggressive phenotype in lung AC cell lines suggesting a potential for therapeutic targeting.

57 Background: It has been suggested that the sequential analysis of the mutational spectra at loci involved in the pathogenesis of tumors (mutational load distribution analysis [MLDA]) may reflect the biology of the tumor and/or the risk of cancer before the tumor emerges. Metrics derived from MLDA could assist quantifying risk, in early diagnosis, and in monitoring response to therapy. The aim of this study was to assess the efficacy of the NGS platform to obtain clinically relevant metrics derived from MLDA. Methods: Two sets of DNA samples were assessed: (i) 6 paired tumor/normal colorectal carcinomas and 8 colorectal adenomas; and (ii) 21 pancreatic carcinoma and 6 non-matched normal pancreas. Thirty amplicons from 6 genes (KRAS, BRAF, EGFR, CDKN2A, TP53, and PI3KCA) were sequenced using the Roche 454 GS FLX-TI platform. Data analysis was carried out by Variant Identification Pipeline (VIP) software and R (computing environment) commands. Total mutational load (TML) was calculated as the sum of all variants frequency for each sample. Results: In all, 18,626 variants were identified. Optimal filters applied to discard false positives were: (1) variants with an absolute frequency < 10x, (2) variants in homopolymer (HP) ≥ 6 nucleotides, (3) insertions and deletions (INDELs) in HP ≥ 5 nucleotides, and (4) INDELs in only one strand. Filtering yielded 870 variants from all samples remained as valid, 92 being unique. After discarding polymorphisms, variants classified as neutral by the Condel software and variants preferentially detected in normal tissues, 46 variants were used to compute TML. TML in colonic carcinomas and adenomas was higher than normal mucosa (0.83 vs. 0.607 vs. 0.128, p = 0.01). In pancreatic disease, TML for pancreatic carcinoma was higher than normal pancreas (0.716 vs. 0.135, p = 0.01). KRAS and TP53 analysis provide most of the information in both tumors, while PI3KCA adds information for colorectal carcinomas exclusively and CDKNA2 is informative in pancreatic samples. Conclusions: NGS provides a robust and quantitative measure of genetic alterations in tumor samples. MLDA is an effective tool for the classification of GI tumors.

466 Background: Microsatellite Instability (MSI-H) occurs in 10-20% of colon tumors and has been attributed predominantly to gene silencing of DNA mismatch repairs (MMR) genes by mutation or methylation, including MSH2, PMS2 and in particular MLH1. Methods: MSI status for 276 primary stage II and III colorectal tumors have been determined (n=29 MSI-H, n=247 MSS and MSI-L) by immunohistochemically staining of MLH1 and PMS2 (for 90 patients) or by PRC amplification of six microsatellite DNA regions from paired normal and tumor tissues (for 186 patients). Full genome gene expression data for the same 276 tumors was available and used to identify genes that correlate with MSI-H status. All samples had full clinical information including 5-year follow-up and ColoPrint results (as described in Salazar et al. JCO 2011). Results: Most MSI-H patients were classified as ColoPrint low risk (26 of 29 patients; 90%) however this is only as subgroup of the 195 patients identified by ColoPrint as low risk. While ColoPrint had great prognostic power in this dataset (HR 2.37, p=0.0006), it does not identify MSI-H patients with high specificity. We therefore developed a MSI-gene signature of 64 genes using a 10-fold cross validation procedure to complement ColoPrint. The MSI-signature identifies MSI-H patients with high sensitivity (93.1% (27/29)) and high specificity (87.9% (217/247)) and an overall accuracy of 88.4% (244/276). The MSI-signature has prognostic power in the subset of stage II patients (n=215) with HR = 2.38 (95% CI 1.15-4.93, p=0.06. However, in multivariate analysis of MSI status and all clinical factors, ColoPrint was the only significant prognostic factor (HR 3.2; p=0.0014). The MSI-signature is currently validated in an independent patients set of 132 stage II patients (31 MSI-H, 101 MSS). Conclusions: The diagnostic MSI signature identifies patients with MSI-H status with high accuracy. In combination with the prognostic power of ColoPrint, the identification of MSI-H patients might add additional information for treatment of stage II patients.

e22037 Background: About 13% of Familial Adenomatous Polyposis (FAP) families and 70% of Attenuated FAP families remain with unknown molecular pathogenic cause after APC and MYH mutational analyses. Also, mutations can affect specific allele expression (ASE) at the germline level. The aim of the study was to determine the presence of germline ASE in the APC gene in FAP and AFP with and without detectable APC or MYH mutations. Methods: Germline RNA from fresh frozen and/or cultured lymphocytes of 17 APC/MYH-negative Polyposis (7 FAP, 10 AFAP) families (21 individuals) and 35 APC-mutated Polyposis (30 FAP, 5 AFAP) families (60 individuals) was analyzed. Fourteen controls were also studied. ASE was investigated by single nucleotide primer extension (SNuPE) of rs2229992 APC coding SNP. Results: In controls ASE was 1.04± 0.3. We found that 17% (3 of 17) APC/MYH(-) FAP and AFAP families showed ASE (range=1.17–1.39) and ASE co-segregated with disease. ASE was more intense in short-cultured lymphocytes except for two cases and completely reversed by puromycin treatment. Eleven of 35 (31%) APC-FAP/AFAP harbored ASE (range=1.20–7.76), and the mutant allele was underexpressed in each case. ASE was restricted to splicing (4 families), nonsense (3 families) and frameshift (3 families) mutations outside of exon 15. Puromycin reversed ASE in all cases analyzed. Conclusions: APC ASE is present in a significant proportion (17%) of APC/MYH(-) FAP or AFAP. ASE, due to nonsense-mediated decay (NMD), is present in APC-FAP and is associated with specific mutation location, similar to reports for other hereditary syndromes. No significant financial relationships to disclose.

e22181 Background: I1307K is a missense APC variant with incomplete penetrance that has been found in 6% of Jewish Ashkenazi population and confers a two-fold increased risk to develop multiple adenomas and colorectal tumours. It is believed that it creates a hypermutable region within the gene that leads to an accumulation of mutations. It remains unknown whether the presence of this mutation modifies APC expression. Our goal was to study whether allele-specific expression (ASE) of I1307K is present at the germline and tumoral level. Methods: Paired germline/carcinoma RNA and DNA was studied from eleven I1307K carriers. To analyze changes in the mutant/wt allelic ratio we used single nucleotide primer extension (SNuPE) for the I1307K and the rs2229992 APCcoding SNP. Fourteen controls were also analyzed. Results: No germline allele specific expression was found in I1307K carriers (range=0.954–1.173). Significant ASE was observed in 8 of the 10 carcinomas analyzed. In 4 cases the I1307K allele was overexpressed (range=2.51–9.51) and in 4 cases was underexpressed (range=0.09–0.28). Tumor ASE correlated with the DNA mutant/wt allelic dose. Conclusions: I1307K variant is not associated with allelic specific expression at the germline level. I1307K overexpression is not selected for during tumor progression. No significant financial relationships to disclose.

e15036 Background: Previous studies have shown that assessment of promoter hypermethylation of a limited number of genes in tumor biopsies may identify all colorectal tumors analyzed. The aim of the present study was to assess the clinical usefulness of a panel of methylation biomarkers in stool DNA in the diagnosis of colorectal tumors using Methylation Curve (MC) analyses, a technique that simultaneously analyze all CpG residues within a promoter. Methods: Promoter methylation status of 5 tumor-related genes (RARB2, p16INK4a, MGMT, p14ARF and APC) was analyzed in DNA stool samples and corresponding tissues in an initial set of 12 newly diagnosed patients with primary colorectal carcinomas and 20 with colorectal adenomas using Methylation-specific PCR (MSP). Results were validated in a set of 88 patients (20 healthy subjects, 17 inflammatory bowel disease, 23 adenomas, 28 carcinomas) using MC analyses. Median age for every group was 63, 51, 66 and 67 y respectively. Results: In the initial set, the majority [10 of 12 (83%) carcinomas and 18 of 20 (90%) adenomas] of biopsies were positive for at least one marker. In stool DNA prevalence was 75% for carcinomas (9 of 12) and 60% for adenomas (12 of 20) with no false positive in stools. In the validation set MC was used. Analytical sensitivity of MC was 5% of methylated alleles for p16INK4a, p14ARF, RARB2 and APC and 10% for MGMT. In the validation set MC analyses of biopsies showed that at least one marker was positive in 22 of 28 (79%) carcinomas and 16 of 23 (70%) adenomas. In stool DNA, these percentages were 64% (18 of 28) for carcinomas and 42% (9 of 23) for adenomas. No aberrant methylation was observed in healthy subjects and in 2 of 15 (13%) of IBD patients aberrant RARB2 methylation was detected. Conclusions: Melting Curve analysis of a panel of methylation markers in stool DNA is a good alternative for the early non-invasive diagnosis of colorectal tumors. [Table: see text]

4036 Background: Between 25 and 35% of stage II CRC patients will experience a recurrence of their disease and may benefit from adjuvant chemotherapy. Official guidelines give suggestions but no clear recommendation for best risk stratification. Here we describe the development a robust signature that predicts disease relapse and can assist in treatment decisions. Methods: Fresh frozen tumor tissues from 180 patients with stage I, II and III colorectal cancer undergoing surgery were analyzed using high density Agilent 44K oligonucleotide arrays. Median FU was 70.2 months; 85% of patients did not receive adjuvant chemotherapy. Unsupervised hierarchical clustering based on full-genome gene expression measurement indicated the existence of 3 main colon molecular subclasses. Survival analysis of the 3 classes showed that subtype C (n= 27) had a poor outcome and subtype A (n= 48) good outcome. Only the intermediate group B (n=104) was used to develop a signature by using a cross validation procedure to score all genes for their association with 5-yr distant metastasis free survival (DMFS) and subsequently applied to all samples (n=180). The obtained gene signature was further validated on an independent cohort of 178 stage II + III colon samples. Results: A set of 38 prognosis related gene probes showed robust DMFS association in over 50% of all iterations in the Training Set of 180 samples. The gene signature was validated on an independent cohort of 178 samples from stage II + III colon cancer patients. The profile classified 61% of the validation samples as low-risk and 39% as high-risk. The low- and high-risk samples showed a significant difference in DMFS with a HR of 3.19 (P= 8.5e-4). Five-year DMFS rates were 89% (95%CI 83–95) for low-risk and 62% (95%CI 50–77) for high-risk samples. Moreover, the profile showed a significant performance within stage II (P=0.0058) and III (P=0.036) only samples. The performance of the profile was significant for both untreated (P=0.0082) and treated patients (P=0.016) suggesting that its power is independent of treatment benefits. Conclusions: ColoPrint is able to predict the prognosis of stage II and III colon cancer patients and facilitates the identification of patients who would benefit from adjuvant chemotherapy. [Table: see text]

e22046 Background: APC mutations are believed to constitutively activate the wnt pathway in colorectal tumors. Our goal was to comprehensively characterize Wnt signaling components in a set of APC-associated FAP tumors both at the DNA and RNA levels. Methods: Sixty adenomas from FAP cases harboring pathogenic APC mutations were included (10 adenomas and 1 normal mucosa per case). Somatic APC and KRAS alterations, β-catenin immunostaining and qRT-PCR of APC, MYC, AXIN2 and SFRP1 were analyzed. aCGH was also assessed in 26 FAP adenomas and 24 additional paired normal-adenoma-carcinoma samples. Results: A second APC alteration was present in 30 (50%) of adenomas (26 LOH and 4 point mutations). LOH was only occasionally associated with loss of genetic material. In 3 of 6 cases APC mRNA was overexpressed in macroscopically normal mucosa. In these cases diminished APC levels mRNA were observed in 11 of 30 adenomas analyzed.. In the remaining 3 cases APC mRNA was already underexpressed in normal mucosa with only 3 of 30 adenomas showing further underexpression. In FAP normal mucosae MYC was overexpressed (logratio range: 3.37–5.66). All adenomas showed further MYC (logratio range: 1.78–2.92) and AXIN2 overexpression (logratio range: 1.62–4.65), corregulating with APC mRNA levels (r=0.31 for MYC; r=0.59 for AXIN2). β-catenin nuclear immunostaining was detected in 80% of adenomas correlating with MYC mRNA levels. SFRP1 was underexpressed in all tumors (logratio range= -3.06–27). While copy number changes were rare in adenomas (median number :2.5; range 0–5). DNA changes were more often detected in areas containing wnt genes when FAP and sporadic tumors were jointly analyzed (p=0.02). Conclusions: Wnt pathway is altered, at the DNA and/or RNA level, in all APC mutant FAP tumors. MYC overexpression is a universal event that correlates with APC and AXIN2 expression levels as well as bcatenin nuclear accumulation. No significant financial relationships to disclose.

16070 Background: Germ cell tumors (GCTs) of the testis are highly curable, but patients refractory to cisplatin (CDDP) based chemotherapy have a poor prognosis. Several studies support an important role of angiogenesis in GCT, suggesting anti-angiogenic treatment as a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor with antiangiogenic and antitumoral activities. We evaluate the effect of sunitinib, CDDP or the combination of both compounds on tumoral growth using an orthotopic model of human testicular GCTs (Piulats et al., AACR 2006; abstract 2760). Methods: Mice were implanted with 4 different tumors: a yolk sac without prior exposure to CDDP; a choriocarcinoma from a CDDP-resistant patient; and finally both a choriocarcinoma without prior exposure to CDDP and its CDDP refractory variant induced in mice by a continous CDDP exposure. Mice were treated with sunitinib (40 mg/kg, daily for 15 days), CDDP (2 mg/kg three times at 5-day interval) or combination of both. Animals wer...

12031 Background: Human tumor xenografts in mice can be remarkably predictive of response in humans to cytotoxic chemotherapeutic drugs. Tumor endothelial cells are sensitive to the action of conventional cytotoxic drugs when they are regularly administrated at low doses. This concept, known as metronomic chemotherapy, has been demonstrated in preclinical studies using transplanted tumor models. We aim to investigate the potential anti-tumoral activity of Gemcitabine (G) when administered in a low-dose schedule in an ortothopic implantation model of human pancreatic carcinomas. Methods: Standard gemcitabine schedule: NP18 tumor orthotopically implanted nude mices were randomly distributed to experimental (n = 13, G100 mg/kg intraperitoneally on days 0, 3, 6 and 9 post-implantation) and control group (n = 13, saline). Animal were sacrificed after 4 weeks and we compared weigths (grams) and volume (cm3) of tumors betwen the two groups by the Mann-Whitney U test. Metronomic schedule: After a toxicological study an optimal metronomic dose of 1 mg G /kg per day was chosen. Thirty xenografted mices were randomly distributed to experimental group (n = 15, intraperitoneal G1 mg/kg) and control group (n = 15, saline) and treated for 30 days. Animal were analysed as described before. Results: Standard schedule: Tumor weight mean of treatment group was 0.01 grams ± 0.01 versus 0.54 grams ± 0.48 of the control group. Tumor volume mean in G group was 0.01 cm 3 ± 0.01 versus 0.51 cm 3 ± 0.67) in the control group.Treatment significantly inhibited NP18 tumour growth (p < 0.001). No differences in mice weight were observed between both groups. Metronomic schedule: Tumor weight mean in the treatment group was 0.04 grams ± 0.08 versus 0.53 grams ± 0.46 in control group. Tumor volume mean in G group was 011 cm 3 ± 0.19 versus 0.37 cm 3. Treatment with low-dose of G significantly inhibited NP18 tumour growth (p < 0.003). There were no differences in mice weight between the two groups. Conclusions: Our data show that G administered in a metronomic schedule is effective in inhibiting the growth of NP18 tumor orthotopically implanted in the nude mice. We now aim to study the angiogenic profile of tumors receiving the standard and metronomic schedule and to set up a new experiment to compare survival benefit in the animal model. No significant financial relationships to disclose.

3618 Background: Specific molecular profiles of exfoliated tumour cells may lead to non invasive diagnosis of cancer. We have developed a DNA chip-based methodology that allows for the simultaneous and quantitative detection of multiple allelic substitutions in bodily fluids. Total Mutational Load Distribution Analysis (MLDA) of DNA found in bodily fluids yields biometrics that may enable early tumor diagnosis. We wanted to ascertain whether the MLDA of selected K-ras and p53 mutations in fecal DNA distinguishes between absence of tumor and the presence of neoplastic disease and whether patients with adenomas can be distinguished from those bearing carcinomas. Method: DNA was obtained from solid stools or colonic lavage in 67 patients undergoing colonoscopy: non-neoplastic condition (n=24); adenomas (n=16); carcinomas in situ (n=6) and carcinomas (n=21). Quantitative and simultaneous detection of 8 substitutions at codons 12 and 13 of K-ras gene and 15 hotspots substitutions at exons 5 and 7 of p53 gene was performed using an oligonucleotide zip-code microarray with rolling circle amplification signal enhancement. Total Mutational Load (TML) and relative prevalence values of independent alleles were measured. Results: MLDA separated the three groups: Total Mutational Load (TML) of non-neoplastic conditions ranged from 5.3 to 7.15. TML of adenomas ranged from 16.50 to 22.24 and of carcinomas in situ from 22.30 to 36.29. TML of invasive carcinomas ranged from 25.06 to 67.9. In a training set of the first 40 samples analyzed a halfway cut-off value of 11.87 was established to discriminate between non-neoplastic and neoplastic disease. Sensitivity was 100% (95% CI 91.7–100) and specificity was 100% (95% CI 86.2- 100) in both sets. We have reproduced similar results with classic techniques like sequences and cloning. Conclusion: Stool MLDA appears to be a useful non-invasive marker of distal and proximal colonic neoplasms that deserves widespread validation. No significant financial relationships to disclose.

3588 Background: Adjuvant chemotherapy treatment, mainly based in the inhibition of thymidylate synthase (TS) enzyme with 5-FU (5-fluorouracil) is offered to all Dukes C and indications for Dukes B...

PURPOSE: p53 gene and K-ras mutations are among the most common genetic alterations present in colorectal cancer. The prognostic utility of such mutations remains controversial. The purpose of this study was to prospectively evaluate the prognostic significance of p53 and K-ras gene mutations in colorectal cancer. PATIENTS AND METHODS: One hundred forty patients were analyzed. Tumors belonging to the microsatellite mutator phenotype were excluded (n = 8). Mutations at the K-ras and p53 genes were detected and characterized by restriction fragment length polymorphism, single-strand conformation polymorphism, and sequencing, as appropriate. RESULTS: p53 mutations were detected in 66 (50%) and K-ras mutations were detected in 54 (41%) of the 132 patients. In 26 cases (20%), ras and p53 mutations coexisted; in 38 cases (29%), neither mutation was found. Multivariate analysis of the whole population analyzed (n = 132) showed that survival was strongly correlated with the presence of p53 mutations alone or in combination with K-ras mutations (P = .002; log-rank test). When only patients undergoing a radical resection were considered (R0; n = 101), p53 mutations were no longer of prognostic significance. CONCLUSION: p53 mutations alone or in combination with K-ras mutations are correlated with a worse outcome. However, the routine use of these mutations as prognostic markers in the clinical setting is not recommended.