Search

Breast cancer is a frequent disease for which the discovery of markers that enable early detection or prognostic assessment remains challenging. Circular RNAs (circRNAs) are single-stranded structures in closed loops that are produced by backsplicing. CircRNA and messenger RNA (mRNA) are generated co-transcriptionally, and backsplicing and linear splicing compete against each other. As mRNAs are key players in tumorigenesis, we hypothesize that a disruption of the balance between circRNAs and mRNAs could promote breast cancer. Hence, we developed an assay for a simultaneous study of circRNAs and mRNAs, which we have called splice and expression analyses by exon ligation and high-throughput sequencing (SEALigHTS). Following SEALigHTS validation for BRCA1 and BRCA2, our hypothesis was tested using an independent research set of 95 pairs from tumor and adjacent normal breast tissues. In this research set, ratios of BRCA1 and BRCA2 circRNAs/mRNAs were significantly lower in the tumor breast tissue compared to normal tissue (p = 1.6 × 10−9 and p = 4.4 × 10−5 for BRCA1 and BRCA2, respectively). Overall, we developed an innovative method to study linear splicing and backsplicing, described the repertoire of BRCA1 and BRCA2 circRNAs, including 15 novel ones, and showed for the first time that a disequilibrium between BRCA1 and BRCA2 circRNAs and mRNAs plays a role in breast cancer.

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

Supplementary Figure S7: Fibroblast transformation with each of the 8 somatic-mutation-harboring RTKs

Supplementary Table S1: Detailed annotation of the somatic mutations in the 58 RTKs, KRAS, PI3KCA, and TP53

Morphological, immunohistochemical, and molecular methods often need to be combined for accurate diagnosis and optimal clinical management of sarcomas. Here, we have developed, a new molecular diagnostic assay, for the detection of gene fusions in sarcomas. This targeted multiplexed next-generation sequencing (NGS)-based method utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR-NGS) to detect oncogenic fusion transcripts involving 137 genes, leading to 139 gene fusions known to be recurrently rearranged in soft-tissue and bone tumors. 158 bone and soft-tissue tumors with previously identified fusion genes by fluorescent in situ hybridization (FISH) or RT-PCR were selected to test the specificity and the sensitivity of this assay. RNA were extracted from formalin-fixed paraffin-embedded (n = 143) or frozen (n = 15) material (specimen; n = 42 or core needle biopsies; n = 116). Tested tumors encompassed 23 major translocation-related sarcomas types, including Ewing and Ewing-like sarcomas, rhabdomyosarcomas, desmoplastic small round-cell tumors, clear-cell sarcomas, infantile fibrosarcomas, endometrial stromal sarcomas, epithelioid hemangioendotheliomas, alveolar soft-part sarcomas, biphenotypic sinonasal sarcomas, extraskeletal myxoid chondrosarcomas, myxoid/round-cell liposarcomas, dermatofibrosarcomas protuberans and solitary fibrous tumors. In-frame fusion transcripts were detected in 98.1% of cases (155/158). Gene fusion assay results correlated with conventional techniques (FISH and RT-PCR) in 155/158 tumors (98.1%). These data demonstrate that this assay is a rapid, robust, highly sensitive, and multiplexed targeted RNA sequencing assay for the detection of recurrent gene fusions on RNA extracted from routine clinical specimens of sarcomas (formalin-fixed paraffin-embedded or frozen). It facilitates the precise diagnosis and identification of tumors with potential targetable fusions. In addition, this assay can be easily customized to cover new fusions.

Transarterial chemoembolization (TACE) is used to treat patients with unresectable hepatocellular carcinoma (HCC). We evaluated the clinical impact of a-fetoprotein (AFP) and circulating cell-free and tumor DNA (cfDNA and ctDNA) changes around the TACE procedure. Our prospective monocentric study enrolled consecutive patients treated with TACE, with samples collected at baseline (D - 1), Day 2 (D + 2) and 1 month (M + 1) after TACE. cfDNA was quantified by the fluorometric method, and ctDNA was quantified by digital polymerase chain reaction designed for two hotspot TERT mutations. Computerized tomography scans or magnetic resonance imaging were performed at M + 1 every 3 months following TACE and independently reviewed. The objective was to identify thresholds of cfDNA, ctDNA and AFP changes associated with progressive disease (PD) using receiver operating characteristic curves. Thirty-eight patients were included from March 2018 to March 2019. All markers significantly increased from D - 1 to D + 2 (P .005), and cfDNA and ctDNA significantly decreased from D + 2 to M + 1 (P .0001). The analysis of changes from D - 1 to M + 1 identified thresholds at +31.4% for cfDNA and 0% for ctDNA that were significantly associated with PD at M + 1 (44.4% [+31.4%] vs 3.8% [≤+31.4%] and 50.0% [0%] vs 5.0% [≤0%], respectively). No significant threshold was identified for AFP. Using a score combining cfDNA and ctDNA, the patients were classified into high- or low-risk PD groups at M + 1, with PD rates of 80.0% vs 4.3% (P = .001) and median progression-free survival times of 1.3 vs 10.3 months (P = .002). Our study suggests that cfDNA and ctDNA increases around the TACE procedure and are associated with therapeutic failure.

BACKGROUND: We previously reported that CEA kinetics are a marker of progressive disease (PD) in metastatic colorectal cancer (mCRC). This study was specifically designed to confirm CEA kinetics for predicting PD and to evaluate CA19-9, cell-free DNA (cfDNA), circulating tumour DNA (ctDNA) and circulating tumour cell (CTC) kinetics. METHODS: Patients starting a chemotherapy (CT) with pre-treatment CEA > 5 ng/mL and/or CA19.9 > 30 UI/mL were prospectively included. Samples were collected from baseline to cycle 4 for CEA and CA19-9 and at baseline and the sixth week for other markers. CEA kinetics were calculated from the first to the third or fourth CT cycle. RESULTS: A total of 192 mCRC patients were included. CEA kinetics based on the previously identified >0.05 threshold was significantly associated with PD (p

AIMS Secretory carcinoma (SC) is characterized by ETV6 rearrangements, most often ETV6-NTRK3 fusion. Given its histologic overlap with other salivary gland tumors (SGTs), SCs can be difficult to diagnose without genetic confirmation. A recently developed pan-TRK (tropomyosin receptor kinase) antibody shows promise for identifying tumors with NTRK (neurotrophic tyrosine kinase receptor 3) fusions. The aim of this study was to evaluate the utility of pan-TRK immunohistochemistry in distinguishing SCs from mimics and selecting patients eligible for TRK inhibitor clinical trials. We examined whole-tissue sections from 111 SGTs with molecular characterization, including 26 SCs (23 with ETV6-NTRK3 fusion and 3 with ETV6-RET fusion detected by ligation-dependent reverse transcription-polymerase chain reaction, next-generation sequencing and 85 non-SC SGTs (no ETV6-NTRK3 fusion). Immunohistochemistry was performed with a pan-TRK rabbit monoclonal antibody. When any pan-TRK staining (nuclear or cytoplasmic with any staining intensity) was considered to indicate positivity, 22 of 23 SCs with ETV6-NTRK3 fusion (95.7%) and 33 of 85 non-SC (38.8%) salivary neoplasms were positive, mainly basal cell adenoma, pleomorphic adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas. All SCs with ETV6-RET fusion were entirely negative. When only nuclear pan-TRK staining with any staining intensity was considered positive, 18 of 23 SCs with ETV6-NTRK3 fusion (78.3%) were positive, 11 among them with diffuse staining (>30% of cells). All non-SCs and SCs with ETV6-RET fusion were entirely negative. In comparison to molecular analysis (ligation-dependent reverse transcription-polymerase chain reaction, next-generation sequencing), nuclear pan-TRK IHC has a sensitivity of 78.3% and a specificity of 100% for diagnosing SCs with ETV6-NTRK3 fusion, 69% and 100% for SCs (all fusions). Pan-TRK is a reasonable screening test for diagnosing SCs among SGTs when taking only nuclear staining into account. Although pan-TRK expression is not entirely sensitive for SCs, nuclear staining is highly specific for SCs with ETV6-NTRK3 fusion. The lack of pan-TRK immunoreactivity in a subset of SCs is suggestive of atypical exons 4 to 14 or exons 5 to 14 ETV6-NTRK3 fusion or non-NTRK alternative fusion partners such as ETV6-RET. Pan-TRK staining can serve as a strong diagnostic marker to distinguish SC from it mimics and to select patients eligible for TRK inhibitor clinical trials.

Background Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. Methods We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. Results For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. Conclusion Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

The clinical implications of plasmatic cell-free and tumor DNA (cfDNA and ctDNA) are challenging in glioblastoma. This prospective study included 52 consecutive newly diagnosed glioblastoma (n = 49) or gliosarcoma (n = 3) patients treated with concomitant temozolomide and radiotherapy (RT-TMZ), followed by a TMZ maintenance phase. Plasma samples were collected at baseline, before RT-TMZ (pre-RT-TMZ) and at the end of adjuvant TMZ, or at the time of progression in cases of progressive disease (PD). The cfDNA concentration was measured with a fluorometric method, and ctDNA was detected using targeted droplet digital PCR. The main objectives were to analyze the associations between cfDNA and ctDNA measurements during the course of treatment with PD and survival. There was a significant decrease in median cfDNA concentration from baseline to pre-RT-TMZ—19.4 versus 9.7 ng/mL (p p = 0.037), respectively, while no difference was observed for nonprogressive patients. Neither the cfDNA concentration at baseline nor its kinetics correlated with survival. ctDNA was detected in 2 patients (3.8%) and only in gliosarcoma subtypes.Trial registration ClinicalTrial, NCT02617745. Registered 1 December 2015, https://clinicaltrials.gov/ct2/show/NCT02617745?term=glioplak&draw=2&rank=1.

2046 Background: The prediction of the relapse pattern is an important issue in glioblastoma for personalized approach. Molecular factors, such as MGMT promoter methylation, influence relapse in- or out-field of the initial radiotherapy volume. Recently, a recurrent mutation located at position 289 of the extracellular domain of the epidermal growth factor receptor ( EGFRA289mut) has been associated with a more infiltrative phenotype in glioblastoma. The primary objective of the present study was to explore the impact of EGFRA289mut on the pattern of relapse after a chemo-radiotherapy based treatment of patients suffering from glioblastoma. Methods: An ancillary study from a monocentric prospective cohort of patients suffering from glioblastoma was conducted. All patients received radiotherapy and concomitant temozolomide. The population was divided into two groups according to EGFRA289 status (mutated versus wild-type). Primary endpoint was the overlap score (varying from 0 to 1) between initial irradiated tumor volume (Vinit) and relapse volume (Vr). Vinit was the initial 95% isodose of the radiotherapy and Vr was delineated using the enhanced MRI T1-weighted part of the relapse tumor. Secondary endpoints explored the impact of other EGFR extracellular mutations, EGFR amplification and EGFRvIII on the relapse pattern, as well as the impact of EGFRA289mut on survival. EGFR alterations were identified using next-generation sequencing or droplet-digital PCR based methods on formalin-fixed paraffin-embedded samples collected at initial diagnosis. Results: One hundred patients were included: 11% of the population had EGFRA289mut glioblastoma (n = 11/100). EGFRA289mut glioblastomas had a relapse pattern more marginal compared to EGFRA289wt glioblastomas: a mean overlap score Vinit/Vr of 0.78 was observed in the EGFRA289mut group versus 0.94 in the EGFRA289wt group (p = 0.021). The proportion of EGFR A289mut glioblastomas in the outfield relapse (overlap score < 0.8) has a trend to be higher than in the infield relapse (overlap score > 0.8): 25% (n = 2/8) versus 9.8% (n = 9/92) for the EGFRA289wt population, p = 0.21. Neither EGFR amplification nor EGFRvIII did influence overlap score Vinit/Vr. In our population, EGFRA289mut did not influence survival. Conclusions: EGFR A289mut influences the relapse pattern in a population of patients suffering from glioblastoma. The role of EGFRA289mut as a decision-making biomarker for personalized radiotherapy should now be investigated in dedicated clinical trial. Clinical trial information: NCT02617745.

RT-qPCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method. We developed a multiplex RT-ddPCR assay, targeting six SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate Sars-CoV2 infection. The 6-plex RT-ddPCR assay was shown to have 100% sensitivity on nasopharyngeal swabs and a higher sensibility than RT-qPCR on saliva (85% versus 62%). Saliva samples from 2 individuals with negative results on nasopharyngeal swabs were found to be positive. These results show that multiplex RT-ddPCR should represent an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results, and its application to saliva an appropriate strategy for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2–7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma.

Background Endocrine therapy is recommended as a first-line treatment for hormone receptor-positive metastatic breast cancer (HR+MBC) patients. No biomarker has been validated to predict tumor progression in that setting. We aimed to prospectively compare the risk of early progression according to circulating ESR1 mutations, CA-15.3, and circulating cell-free DNA in MBC patients treated with a first-line aromatase inhibitor (AI). Methods Patients with MBC treated with a first-line AI were prospectively included. Circulating biomarker assessment was performed every 3 months. The primary objective was to determine the risk of progression or death at the next follow-up visit (after 3 months) in case of circulating ESR1 mutation detection among patients treated with a first-line AI for HR+MBC. Results Overall, 103 patients were included, and 70 (68%) had progressive disease (PD). Circulating ESR1 mutations were detected in 22/70 patients with PD and in 0/33 patients without progression (p ESR1-mutated patients, 18/22 had a detectable mutation prior to progression, with a median delay of 110 days from first detection to PD. The detection of circulating ESR1 mutations was associated with a 4.9-fold (95% CI 3.0–8.0) increase in the risk of PD at 3 months. Using a threshold value of 25% or 100%, a CA-15.3 increase was also correlated with progression (p p = 0.003, respectively). In contrast to ESR1, the CA-15.3 increase occurred concomitantly with PD in most cases, in 27/47 (57%) with a 25% threshold and in 21/25 (84%) with a 100% threshold. Using a threshold value of either 25% or 100%, cfDNA increase was not correlated with progression. Conclusion The emergence of circulating ESR1 mutations is associated with a 4.9-fold increase in the risk of early PD during AI treatment in HR+MBC. Our results also highlighted that tracking circulating ESR1 mutations is more relevant than tracking CA-15.3 or cfDNA increase to predict progression in this setting. Trial registration ClinicalTrials.gov, NCT02473120. Registered 16 June 2015—retrospectively registered after one inclusion (first inclusion 1 June 2015)

We conducted a prospective study to assess the prognostic impact of selected copy number variations (CNVs) in stage II-III microsatellite stable (MSS) colon cancer. A total of 401 patients were included from 01/2004 to 01/2009. The CNVs in 8 selected target genes, DCC/18q, EGFR/7p, TP53/17p, BLK/8p, MYC/8q, APC/5q, ERBB2/17q, and STK6/20q, were detected using a quantitative multiplex polymerase chain reaction of short fluorescent fragment (QMPSF) method. The primary end-point was the impact of the CNVs on the 4-year disease-free survival (DFS). The recurrence rate at 4 years was 20.9%, corresponding to 14% stage II patients vs 31% stage III patients (p

Background Spinal myxopapillary ependymoma (SP-MPE) is a subgroup of ependymomas in which after initial gross tumor resection, recurrences occur in more than half of the patients. Anaplastic transformation may also occur and contributes to intraneural and extraneural metastatic dissemination. Extraneural metastases from SP-MPE are rare and worsen the prognosis. In this situation, the noninvasive detection of recurrent somatic mutations in the circulating tumor DNA (ctDNA) from plasma is challenging. Telomerase-reverse transcriptase gene promoter (TERTp) mutation has been identified in a subset of ependymomas with aggressive behavior. Case Description We report on a patient with TERTp mutated SP-MPE presenting with an extraneural anaplastic metastatic dissemination after iterative local recurrences. From the initial SP-MPE to pleural anaplastic lesion, TERTp C228T mutation was present with allele frequency varying from 33% to 39%. Interestingly, TERTp mutation was also detected by droplet digital polymerase chain reaction in the plasma with a frequency of 2.1% at the time of pleural metastases, highlighting that ctDNA is released in plasma of patients suffering from SP-MPE with extraneural metastatic dissemination. Conclusions Despite the rarity of this evolution, plasmatic liquid biopsy appears to be a useful diagnostic and follow-up tool in a subset of primary brain tumors.

BACKGROUND Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one ( CONCLUSIONS The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

Classical Hodgkin lymphoma (cHL) accounts for 30% of all lymphomas [1]. Currently, 20–25% of cHL cases relapse or are refractory after first-line standard treatments, leading to the need to better ...

The 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.The aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA.Digital 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.Digital 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 (p0.001). 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).Chip-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.

Background Heparin is often used as a blood anticoagulant for tumor marker analysis but results in the inhibition of PCR detection of circulating tumor DNA (ctDNA), which has been deemed a potential “liquid biopsy”. We aimed to evaluate the impact of heparinase addition on heparinized plasma samples to allow ctDNA analysis. Methods Plasma samples were collected in heparinized (n = 194) and EDTA (n = 8) tubes from hormone receptor-positive metastatic breast cancer (HR + MBC) (n = 144) and pancreatic adenocarcinoma (PA) patients (n = 50). Circulating ESR1 and KRAS mutations were detected with or without heparinase by digital PCR in HR + MBC and PA patients, respectively. Patients were classified into 2 subgroups i) inhibition, I + and ii) no inhibition, I − based on a threshold of 200 copies/μL for PCR inhibition by heparin. Results In the I + subgroup (91/144 HR + MBC and 26/50 PA), heparinase treatment significantly improved PCR efficacy, enabling ctDNA detection in 22/91 and 13/26 patients. Moreover, comparable results for ctDNA detection (4/8) were obtained with heparinized and EDTA PA samples. In the I − subgroup, heparinase addition did not quantitatively and qualitatively alter ctDNA detection. Conclusion Heparinase addition removes the heparin inhibition and allows accurate ctDNA detection in heparinized samples. These findings could make the samples from heparinized blood suitable for ctDNA analysis.

International audience; Background: The direct comparison of CA19.9, circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) using endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has never been performed for the diagnosis of solid pancreatic tumours (SPTs).Methods: We included 68 patients with a SPT referred for EUS-FNA. CTCs were analysed using size-based platform and ctDNA using digital PCR. The sensitivity, specificity, negative and positive predictive values were evaluated for each marker and their combination.Results: SPTs corresponded to 58 malignant tumours (52 pancreatic adenocarcinoma (PA) and 6 others) and 10 benign lesions. The sensitivity and specificity for PA diagnosis were 73% and 88% for EUS-FNA, 67% and 80% for CTC, 65% and 75% for ctDNA and 79% and 93% for CA19.9, respectively. The positivity of at least 2 markers was associated with a sensitivity and specificity of 78% and 91%, respectively. CtDNA was the only marker associated with overall survival (median 5.2 months for ctDNA þ vs 11.0 months for ctDNA , P 1⁄4 0.01).Conclusions: CA19.9 alone and in combination with ctDNA and/or CTC analysis may represent an efficient method for diagnosing PA in patients with SPTs. Further studies including a larger cohort of patients with both malignant and benign lesions will be necessary to confirm these promising results.

In standard pre-analytical conditions, an isolation step is required for circulating tumor DNA (ctDNA) analysis. The need for this step remains unclear with the development of ultrasensitive detection technologies such as digital PCR (dPCR). The aim of our study was to evaluate the ctDNA detection by dPCR platform either directly from plasma (plasma group, PG) or after an isolation step (isolation group, IG).We included 17 patients corresponding to a selection of 43 blood samples in metastatic colorectal cancer patients. For each sample, ctDNA was analyzed with or without isolation step (IG and PG, respectively) using KRAS, NRAS and BRAF mutations identified from the tumor tissue. ctDNA detection was performed after a preamplication step using dPCR platform (QuantStudio™ 3D Digital PCR System). ctDNA detection rate and mutant allelic frequencies (MAF) were compared between IG and PG.Our results showed a detection rate at 93% in IG vs. 88% in PG. The concordance rate between the two groups was 91% (39/43) for ctDNA detection with the four discordant cases occurring in patients with low MAF (0.5%). The mean value of MAF were 16.9±18.9 and 18.5±18.9 for IG and PG, respectively (p=0.24). The correlation coefficient rIn conclusion, our results show that direct detection of ctDNA from unpurified plasma is a feasible approach, particularly from sample with high MAF (0.5%).

International audience; Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA ispresent only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichmentmethod to detect and identify mutations present at low-abundance in clinical samples. However, recentstudies have shown the importance to accurately quantify low-abundance mutations as clinicallyimportant decisions will depend on certainmutation thresholds. The possibility for an enrichmentmethod to accuratelyquantify the mutation levels remains a point of concern and might limit its clinical applicability.In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectalcancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by Eice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordantfor mutation levels below the clinical relevant threshold.E-ice-COLD-PCR can accurately detect and quantify low-abundantmutations in ccfDNA and has a shorter time toresults making it compatible with the requirements of analyses in a clinical setting without the loss of quantitativeaccuracy.

International audience

e12534 Background: Circulating cell free DNA (cfDNA), CA 15.3 and circulating tumor DNA (ctDNA) have been associated with survival in MBC. These 3 biomarkers have never been compared. Methods: We retrospectively included all patients treated in our center between 2010 and 2012 for a HR+ MBC, who had clinical progression during the first line of aromatase inhibitor (AI) and available plasma samples. CfDNA level (ng/mL), CA 15.3 level (U/mL) and ESR1 circulating mutation level (Y537N, Y537S, Y537C and D538G, identified by ddPCR) were determined at time of clinical progression (Tp) and 3 months before (Tp-3). Biomarkers values at Tp and Tp-3 as well as their variations were analyzed according to clinical progression and overall survival (OS). Results: 91 patients were analyzed. CA 15.3 was elevated ( > 30 U/mL) in 71/91 (78%) at Tp and 62/91 (68%) at Tp-3. Higher CA 15.3 was associated with worse OS, especially at Tp (HR = 1.0006/unit at both times, p = 0.009 at Tp; p = 0.08 at Tp-3). Higher cfDNA was associated with worse OS, both at Tp and Tp-3 (HR = 1.006/unit, p < 0.0001 and HR = 1.007/unit, p = 0.02, respectively). An ESR1 mutation was detected in 14/91 at Tp-3 (15%) and 28/91 at Tp (31%). ESR1 mutation detection was strongly correlated with OS both at Tp and Tp-3 (HR = 2.4, p = 0.0005 and HR = 1.97, p = 0.03, respectively). Between Tp-3 and Tp, overall median CA 15.3 and ESR1 mutation level increased (p < 0.0001 and p = 0.007, respectively) and were related to poorer OS (HR = 1.007/unit, p = 0.0002 and HR = 3.1, p = 0.05). In contrast, CfDNA was not correlated at all with clinical progression (36/91 increase, 36/91 decrease, and 19/91 stable cfDNA). ROC curve analysis identified a 59% CA 15.3 increase as the best cut-off for predicting progression, but with poor performance (AUC = 0.64). Multivariate analysis showed that CA 15.3 at Tp (HR = 1.0006/unit, p = 0.006), cfDNA at Tp (HR = 1.005/unit, p = 0.01), ESR1 detection at Tp (HR = 2.7, p = 0.0002) and CA 15.3 increase between Tp-3 and Tp (HR = 1.006/unit, p = 0.003) were associated with poor OS. Conclusions: All 3 biomarkers are correlated with OS in MBC treated with AI. CfDNA variation is poorly related to clinical progression while CA 15.3 variation or ESR1 mutation detection are much more reliable. Overall the better performance is observed with ESR1 mutation detection.

// Florian Clatot 1,2,3 , Anne Perdrix 3,4 , Laetitia Augusto 1 , Ludivine Beaussire 3,5 , Julien Delacour 3,5 , Celine Calbrix 4 , David Sefrioui 3,5,6 , Pierre-Julien Viailly 2 , Michael Bubenheim 7 , Cristian Moldovan 1 , Cristina Alexandru 1 , Isabelle Tennevet 1 , Olivier Rigal 1 , Cecile Guillemet 1 , Marianne Leheurteur 1 , Sophie Gouerant 1 , Camille Petrau 1,3 , Jean-Christophe Thery 1,5 , Jean-Michel Picquenot 1,2,4 , Corinne Veyret 1 , Thierry Frebourg 5 , Fabrice Jardin 2 , Nasrin Sarafan-Vasseur 3,5,* and Frederic Di Fiore 1,3,5,6,* 1 Department of Medical Oncology, Henri Becquerel Centre, Rouen, France 2 INSERM U918, Henri Becquerel Centre, Rouen, France 3 EquIpe de Recherche en Oncologie, Rouen, France 4 Department of Biopathology, Henri Becquerel Centre, Rouen, France 5 INSERM U1079, Faculty of medecine, Rouen, France 6 Department of Gastroenterology, Rouen University Hospital, Rouen, France 7 Department of Biostatistics, Rouen University Hospital, Rouen, France * These authors have contributed equally to the work Correspondence to: Florian Clatot, email: // Keywords : ESR1 mutation, digital PCR, breast cancer, aromatase inhibitor, kinetics Received : October 12, 2016 Accepted: October 24, 2016 Published: October 27, 2016 Abstract Purpose: To assess the prognostic and predictive value of circulating ESR1 mutation and its kinetics before and after progression on aromatase inhibitor (AI) treatment. Patients and methods: ESR1 circulating D538G and Y537S/N/C mutations were retrospectively analyzed by digital droplet PCR after first-line AI failure in patients treated consecutively from 2010 to 2012 for hormone receptor-positive metastatic breast cancer. Progression-free survival (PFS) and overall survival (OS) were analyzed according to circulating mutational status and subsequent lines of treatment. The kinetics of ESR1 mutation before (3 and 6 months) and after (3 months) AI progression were determined in the available archive plasmas. Results: Circulating ESR1 mutations were found at AI progression in 44/144 patients included (30.6%). Median follow-up from AI initiation was 40 months (range 4-94). The median OS was decreased in patients with circulating ESR1 mutation than in patients without mutation (15.5 versus 23.8 months, P =0.0006). The median PFS was also significantly decreased in patients with ESR1 mutation than in patients without mutation (5.9 vs 7 months, P =0.002). After AI failure, there was no difference in outcome for patients receiving chemotherapy (n = 58) versus non-AI endocrine therapy (n=51) in patients with and without ESR1 mutation. ESR1 circulating mutations were detectable in 75% of all cases before AI progression, whereas the kinetics 3 months after progression did not correlate with outcome. Conclusion: ESR1 circulating mutations are independent risk factors for poor outcome after AI failure, and are frequently detectable before clinical progression. Interventional studies based on ESR1 circulating status are warranted.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments.

511Background: We recently reported that the detection of somatic and circulating activating ESR1 mutations is related to AI resistance in hormone receptor positive (HR+) mBC (Sefrioui et al., 2015). We aimed to evaluate the predictive and prognostic values of circulating ESR1 mutations detection at time of progression in mBC patients under AI. Methods: This study is a monocentric retrospective analysis of all consecutive patients treated with first-line AI for HR+mBC from 2008/01 to 2010/12. ESR1 circulating mutations rates (D538G, Y537S/N/C) were assessed by droplet digital PCR on plasma samples at progression. The impact of the mutational status on OS, PFS and clinical response to subsequent treatments was determined by univariate and multivariate analysis. Results: A total of 144 HR+mBC patients was analyzed. Median follow-up from initiation of AI was 40 months (range 4-94). At progression, 44 patients (30.6 %) presented at least one ESR1 circulating mutation (D538G 54%, Y537S 45%, Y537N 34%, Y537C 4%...

3560Background: CEA, cDNA and CTC have been reported as useful markers in mCRC pts treated with chemotherapy (CT)-based regimen. We previously reported that CEA kinetic (threshold 0.05) was associa...

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.