D. Sefrioui

Clinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancer.

BACKGROUNDnThe detection of circulating DNA is considered a promising strategy in cancer patients. Digital PCR has emerged as a sensitive method able to quantify both circulating free and tumour DNA.nnnAIMnThe aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA.nnnMETHODSnDigital PCR was used in 34 metastatic colorectal cancer patients to detect and quantify circulating free and tumour DNA based on K-ras mutational status. Clinical outcomes were analyzed according to circulating DNA measurements.nnnRESULTSnDigital PCR yielded a detection rate of 69% for circulating tumour DNA. The median concentrations of circulating free and tumour DNA were 20 and 6.8 ng/mL, respectively, with significant correlation between both biomarkers (p<0.001). Median overall survival was 4.8 months in patients with high circulating free DNA (>75% quartile) versus not reached in patients with a low level (<25% quartile) (p=0.029). Moreover, median overall survival was significantly decreased in patients with detectable circulating tumour DNA compared to those without (respectively 11.8 months versus not reached, p=0.04).nnnCONCLUSIONSnChip-based digital PCR is a simple and non-invasive method allowing the efficient detection of circulating DNA. Our results highlight that levels of these circulating markers may have a potential prognostic value.

Genetic variations of the A13/A14 repeat located within the EGFR 3′ untranslated region have no oncogenic effect in patients with colorectal cancer

BackgroundThe EGFR 3′ untranslated region (UTR) harbors a polyadenine repeat which is polymorphic (A13/A14) and undergoes somatic deletions in microsatellite instability (MSI) colorectal cancer (CRC). These mutations could be oncogenic in colorectal tissue since they were shown to result into increased EGFR mRNA stability in CRC cell lines.MethodsFirst, we determined in a case control study including 429 CRC patients corresponding to different groups selected or not on age of tumor onset and/or familial history and/or MSI, whether or not, the germline EGFR A13/A14 polymorphism constitutes a genetic risk factor for CRC; second, we investigated the frequency of somatic mutations of this repeat in 179 CRC and their impact on EGFR expression.ResultsNo statistically significant difference in allelic frequencies of the EGFR polyA repeat polymorphism was observed between CRC patients and controls. Somatic mutations affecting the EGFR 3′UTR polyA tract were detected in 47/80 (58.8%) MSI CRC versus 0/99 microsatellite stable (MSS) tumors. Comparative analysis in 21 CRC samples of EGFR expression, between tumor and non malignant tissues, using two independent methods showed that somatic mutations of the EGFR polyA repeat did not result into an EGFR mRNA increase.ConclusionGermline and somatic genetic variations occurring within the EGFR 3′ UTR polyA tract have no impact on CRC genetic risk and EGFR expression, respectively. Genotyping of the EGFR polyA tract has no clinical utility to identify patients with a high risk for CRC or patients who could benefit from anti-EGFR antibodies.

229PLIQUID BIOPSY BASED ON CIRCULATING TUMOUR CELLS (CTC) DETECTION IS A DIAGNOSTIC AND PROGNOSTIC MARKER IN PATIENTS WITH PANCREATIC SOLID TUMOURS

ABSTRACT Aim: The pancreatic cytology by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is considered as thestandard procedure for the diagnostic of pancreatic tumour. We recently showed that detection of circulating tumour cells (CTC) hada diagnostic accuracy of 70% for pancreatic adenocarcinoma (Am J Gastroenterol 2013;108:152-155). The aim of the study was toevaluate both diagnostic and prognostic impact of CTC detection in an extended series of patients referred for a EUS-FNA for apancreatic solid tumour. Methods: It was a single center study including all consecutive patients referred from 01/2011 to 07/2013 for a EUSFNAprocedure in a context of pancreatic solid mass. EUS-FNA was performed with a 22 gauge needle and analysed by twopathologists. A 10u2003ml peripheral blood sample was collected in each patient before the EUS-FNA procedure. Samples were filteredusing the ScreencellsCyto method®, stained with Giemsa and analyzed by a cytologist blinded to clinical data and FNA results. The CTC detection was positive according to the presence of the following parameters: nuclear diameter > 7m, anisocytosis, membraneirregularities, presence of a large nucleolus. Results: A total of 69 patients were included. Amon them, 57 (83%) have a confirmed pancreatic tumours corresponding to 47primitive adenocarcinoma, 4 others primitive tumours and 6 metastatic lesions. The sensitivity and the specificity of EUS-FNA was83% and 100%, respectively. CTC were positive in 36/69 (52%) patients. The sensitivity and specificity of CTC was respectively64.4% and 73.3% in patients with pancreatic cancer and 64.1% and 81.8% in patients with all types of cancer. The presence of CTCwas significantly associated with the diagnostic of cancer (p = 0.01) and with the presence of distant metastases (p = 0.004). In contrast,tumour size, arterial involvement and CA19-9 serum level were not associated with CTC. The 18 months survival rate wassignificantly lower in patients with positive CTC as compared to those without detectable CTC (33 vs 44%, p = 0.03). Conclusions: Ours results highlighted that liquid biopsy based on circulating tumour cells (CTC) detection may be a diagnosticand prognostic marker in patients with pancreatic solid tumours. Disclosure: All authors have declared no conflicts of interest.

1617PCLINICAL INTEREST OF DIGITAL PCR FOR ROUTINE DETECTION OF CIRCULATING DNA IN METASTATIC COLORECTAL CANCER

ABSTRACT Aim: Liquid biopsy based on circulating DNA is considered as a promising issue in cancer patients to assess key somatic alterations involved in disease progression or in treatment sensitivity. However, the clinical relevance of sensitive detection methods such as Digital PCR remains to be established. The aim was to evaluate the clinical interest of Digital PCR in circulating DNA detection including cell-free (cfDNA) and circulating tumour (ctDNA) DNA in patients treated for a metastatic colorectal cancer (MCRC). Methods: A prospective single-center study was conducted from April to July 2013 in 34 patients treated for a MCRC. DNA was extracted from 1 mL of plasma using the QIAamp ® kit Circulating Nucleic Acid. ctDNA was detected and quantified (fragments per mL of plasma) by Digital PCR (Digital 3D ™ QuantStudio ®, Life Technologies) from KRAS mutations identified in the primary tumor. cfDNA was also quantified by Digital PCR and expressed in ng/mL of plasma. Response to chemotherapy and survival were analyzed according to both cfDNA and ctDNA. cfDNA was integrated as a dichotomized covariate (above versus below 75% percentile) for response and overall survival (OS) analysis. Results: The mean cfDNA concentration was 106 ng/mL (range, 3-1443 ng/mL). KRAS mutations were identified in tumour from 16/34 patients (c.34G>T, n=1; c.35G>A, n=8; c.35G>C, n=3; c.35G>T, n=2; c.38G>A, n=2) with a sensitivity and specificity for ctDNA detection of 63% (10/16) and 100% (0/18), respectively. The mean level of ctDNA was 21972 fragments/mL (range, 136 and 308,000). There was a significant correlation between cfDNA and ctDNA (r=0.993, p Conclusions: Digital PCR is a simple and non-invasive method allowing the routine detection of circulating DNA. Our results also highlight that cfDNA and ctDNA are correlate markers with a significant prognostic impact in MCRC patients. Disclosure: All authors have declared no conflicts of interest.

565PA BAR CODE OF SELECTED GENE COPY NUMBER ALTERATIONS IS ASSOCIATED WITH DISEASE-FREE SURVIVAL IN STAGE II-III MICROSATELLITE STABLE (MSS) COLON CANCER

ABSTRACT Aim: Genomic quantitative alteration is a backbone event during the carcinogenesis process in microsatellite stable colon cancer. However, the prognostic value of loss or gain of the main genomic regions is still controversial. We conducted a prospective study to assess the prognostic impact of the main genomic quantitative alterations in stage II-III MSS colon cancer (NCT02110329). Methods: Patients treated for stage II-III colon cancer from 01/2002 to 01/2009 with available frozen tissue were included. The Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) method was used for molecular screening (1). The QMPSF was based on the simultaneous amplification, from normal and tumoral tissues, of 8 selected target genomic sequences according to literature data: DCC (18q21); EGFR (7p12); TP53 (17p13.1); BLK (8p23-p22); MYC (8q24.12); APC (5q22.2); ERBB2 (17q12); STK6 (20q13.31); and two control: PCBD2 (5q31.1); HMBS (11q23.3). The primary end-point was to determine the association between molecular alterations and disease-free survival (DSF). Combinations of alterations were defined according to univariate results (p Results: A total of 401 patients were included with a median follow-up of 48 months. There were 236 stage II and 165 stage III and the recurrence rate was 30.2%. Loss of DCC/18q, TP53/17p, BLK/8p, and APC/5q were detected in respectively 33.2%, 26.7%, 22.7%, 9.7% and gain of EGFR/7p, MYC/8q, ERBB2/17q and STK6/20q were observed in 31.7%, 35.7%, 18.7% and 48.9% of cases, respectively. There was no significant interaction between alterations and disease stage. DFS was significantly associated with loss of DCC/18q (57.5 vs 70.6%, p = 0.02) with a trend for BLK/8p loss (59.8 vs 68.6%, p = 0.08) and ERBB2/17q gain (59.2 vs 68.5%, p = 0.14). In multivariate analysis, the combination of loss DCC/18q and/or loss BLK/8p and/or gain ERBB2/17q was significantly associated with DFS. The DFS significantly decreased from 74.2% to 61.7% (HR = 1.78, 95CI: 1.14-2.77) and to 57.2% (HR =2.06, 95CI: 1.26-3.37) in patients with respectively none, 1 and at least 2 of these 3 alterations. Conclusions: Combination of DCC/18q, BLK/8p and ERBB2/17q gene copy number alterations is associated with disease-free survival in stage II-III colon cancer. Disclosure: All authors have declared no conflicts of interest.