F-C Bidard

Abstract S3-01: IMENEO: International MEta-analysis of circulating tumor cell detection in early breast cancer patients treated by NEOadjuvant chemotherapy

Background We performed an international meta-analysis of individual patient data to assess the clinical validity of circulating tumor cell (CTC) count in non-metastatic breast cancer (BC) patients (pts) treated by neoadjuvant chemotherapy (NCT). Methods A protocol pre-specified the study objectives. We performed a literature & abstracts search up to Dec 2014, then contacted all centers deemed to have eligible data (published or not): early BC pts treated with NCT with CTC count by CellSearch®. The primary endpoint was overall survival (OS); secondary endpoints included distant disease-free survival (DDFS), locoregional relapse-free interval (LRFI) and pathological complete response (pCR). Non-overlapping CTC time points were: baseline (5-0 weeks before NCT), 1-8 weeks after NCT start, 5-0 weeks before surgery and 1-52 weeks after surgery. We used Cox regression models, stratified by study, and the landmark method to establish the prognostic value of CTC count/changes during treatment and survival. Results We collected 2,156 individual pt data from 21 studies and 16 centers worldwide. With ≥1/≥2/≥5 CTC/7.5ml as thresholds, CTC positivity rate was 25/13/6% at baseline, 17/6/3% after NCT start, 15/5/1% before surgery and 11/4/1% after surgery (decrease, p 301, 418 and 157 events were reported for OS, DDFS and LRFI, respectively. In univariate analyses, ≥1 CTC at baseline was a prognostic factor for OS (HR=2.6 [1.9-3.4], p Finally, in multivariate analyses, baseline CTC detection (whatever the CTC threshold used : ≥1/≥2/≥5 CTC) was an independent prognostic factor for OS, DDFS and LRFI, together with pCR, cT, cN and tumor subtype, (e.g. for OS: CTC≥2 HR=4.2 [3.0-5.9] p Conclusions Our study demonstrates with the highest level of evidence that CTCs are a prognostic biomarker in early BC treated by NCT. This impact was independent to that of pCR and was observed on OS, DDFS and also -for the first time- on LRFI. CTC count can usefully complement standard prognostic factors and pCR to improve the prognostication of early BC pts. Citation Format: Bidard F-C, Michiels S, Mueller V, Riethdorf S, Esserman LJ, Lucci A, Naume B, Horiguchi J, Gisbert-Criado R, Sleijfer S, Toi M, Garcia-Saenz JA, Hartkopf A, Generali D, Rothe F, Smerage J, Muinelo L, Stebbing J, Viens P, Magbanua M, Hall CS, Engebratenm O, Takata D, Vidal-Martinez J, Onstenk W, Fujisawa N, Diaz-Rubio E, Taran F-A, Cappelletti MR, Ignatiadis M, Name N, Proudhon C, Wolf D, Bowman Bauldry J, Borgen E, Nagaoka R, Caranana V, Kraan J, Maestro M, Brucker SY, Weber K, Reyal F, Amara D, Gopalkrishna Karhade M, Ruud Mathiesen R, Tokiniwa H, Llombart-Cussac A, D9Hollander K, Cottu P, Park JW, Loibl S, Pierga J-Y, Pantel K. IMENEO: International MEta-analysis of circulating tumor cell detection in early breast cancer patients treated by NEOadjuvant chemotherapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S3-01.

Abstract OT3-4-06: Circulating tumor cells to guide the choice between chemotherapy and hormone therapy as first line treatment for hormone receptors positive metastatic breast cancer patients: the STIC CTC METABREAST trial

Background: Baseline CTC count already demonstrated its accuracy as an independent prognostic marker in metastatic breast cancer (MBC), before the start of any treatment (Cristofanilli, NEJM 2004; Pierga Ann Oncol 2011) for progression free survival and overall survival. Multivariate analyses performed in both a pooled analysis (Liu, ASCO 2011) and in the IC 2006–04 study showed that the other independent prognostic factors were the performance status and hormone-receptor (HR) status. Oppositely, the other criteria that are frequently used to choose between hormone therapy and chemotherapy for the treatment of first line hormone receptor-positive MBC patients (e.g. number of metastatic sites, visceral disease, metastasis-free interval…) were not independent prognostic factors. As CTC count is highly correlated to PFS, we designed a large pivotal phase III trial to evaluate the use of CTC to determine the disease aggressiveness and the choice of first line treatment in potentially hormonosensitive MBC. Trial design: First line metastatic breast cancer patients will be randomized between the clinician choice and CTC count-driven choice. In the CTC arm, patients with ≥5 CTC/7.5ml will receive chemotherapy whereas patients with Eligibility criteria: Main inclusion criteria include hormone receptor-positive metastatic breast cancer patients with no previous treatment for the metastatic disease, and who can receive either hormone therapy or chemotherapy as first line treatment. Statistical methods: As every patient will receive a treatment, this pivotal trial has been designed to show a non-inferiority of the CTC arm for PFS (primary clinical endpoint) and a superiority of the CTC arm for the medico-economics study (co-primary endpoint). Present accrual and target accrual: This trial began in March 2012 and more than 15 French cancer centers participate to this study. The accrual of 996 metastatic breast cancer patients should be completed in 2 to 3 years. 20 patients have been included in 2 months in the first opened center. Contact information for people with a specific interest in the trial The STIC CTC METABREAST trial has been funded by the French Ministry of Health (STIC program) and Veridex. The promoter is the Institut Curie (Paris). Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-4-06.

Meningeal carcinomatosis in patients with breast cancer: clinical features and prognostic factors in 91 patients from a single institution.

Abstract #1090 Background Breast cancer is the leading solid tumor responsible for meningeal carcinomatosis (MC) and the incidence is rising. To date, there is no standard of care and few prospective trials. Intrathecal treatment is provided but the modalities remain controversial.
 Methods From our data base we recorded 91 patients with breast MC admitted to Institut Curie between 2000-2007. Lumbar punction was performed in all patients for cerebrospinal fluid (CSF) cytology, protein level and dosage of CYFRA 21. Intra-thecal treatment: methotrexate 15 mg day1 to 5 and hydrocortisone acetate day 1 with oral folinic acid rescue day 1 to 5 every two weeks.
 Results Median age was 53 years (range 30-78). Tumor type was ductal carcinoma (63%), lobular (28%), ER positive 74%, HER2 overexpressed 10% and triple negative 31%. All patients presented other metastatic site: bone (67%), liver (42%) lung (29%), brain (38%). The median interval between primary tumor and diagnosis of MC was 60 months.
 Amongst the 80 patients treated (87%) with intra-thecal MTX, clinical response was observed in 88% of cases. There was a strong correlation between clinical response and survival (p 1 year) or patients for whom treatment will have no impact on survival ( Conclusion : This score has to be confirmed in prospective study and new modalities of treatments need to be evaluated according to the biology of disease. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1090.

Abstract PD6-6: A pooled analysis of the prognostic relevance of circulating tumor cells in early breast cancer

Background: While there is unequivocal evidence regarding the prognostic relevance of circulating tumor cells (CTCs) in peripheral blood of patients (pts.) with metastatic breast cancer (BC), less data is available for the prognostic relevance at time of primary diagnosis. Methods: We conducted a pooled analysis with individual data of 3172 pts with non-metastatic (Stage I-III) BC from five academic institutions. Prevalence and number of CTCs in the peripheral blood were assessed at time of primary diagnosis using the FDA-approved CellSearch System (Veridex, USA). Patient outcomes were analyzed using univariate log-rank tests and multivariate Cox regressions. The median follow-up time was 61 months. Results: At least one CTC was detected in 20.2% of the pts. The presence of CTCs was associated with larger tumors, more axillary lymph node metastases, and higher histological tumor grade (all p < 0.005). The presence of CTCs was significantly related to poor progression-free (PFS, log-rank test, p < 0.001, hazard ratio [HR] 2.0) and overall survival (OS, p < 0.001, HR 2.6). Multivariate Cox regressions, including tumor size, nodal status, histological tumor grade, hormone receptor status and HER2 status, and CTC prevalence confirmed that the presence of CTCs was an independent prognostic factor for both poor PFS (HR 1.8) and OS (HR 2.1, both p < 0.001). In the univariate subgroup analysis concerning OS, the HR for pts. with positive HRS was 3.1 (p< 0.0001), and 2.0 (p = 0.001) for pts. with a negative HRS. The prognostic relevance of CTC status was similar for patients with HER2 negative and HER2 positive tumors (HR 2.6 and 2.5, respectively). The HR in CTC-positive pts. with HRS positive/HER2 positive tumors was 3.4 (p = 0.003). For patients with more than 3 CTCs, the hazard ratio for death was increased to 5.2 (1.5 - 2.8) in univariate and to 4.0 (2.6 - 6.2) in multivariate analysis. View this table: Cox Regression Analysis for Overall Survival Conclusions: In patients with early breast cancer, the presence of CTCs in peripheral blood is an independent predictor of poor progression-free and overall survival. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-6.

Abstract S6-06: High-depth massively parallel sequencing reveals heterogeneity between primary tumor and metastatic deposits in de novo metastatic breast cancer patients prior to exposure to systemic therapy

Background: Breast cancers are often composed of mosaics of tumor cells that in addition to the founder genetic events harbor private genetic aberrations. Previous studies comparing the repertoire of mutations in primary breast cancers and their metachronous metastatic deposits that developed after systemic therapy revealed differences in their clonal composition and mutational repertoire. It is unclear, however, whether the differences documented could be attributed to the metastatic process itself or because of selective pressure from systemic therapies. Hence we sought to investigate whether the metastatic process would constitute a biological ‘bottleneck’ resulting in the selection of clones fittest to metastasize. We subjected primary breast cancers and their synchronous metastatic deposits from patients who presented with de novo metastatic disease and who had not received any systemic therapy to gene copy number analysis and high-depth massively parallel sequencing. Materials and Methods: Frozen primary tumor and distant metastases biopsies were obtained from 7 patients with de novo metastatic disease (i.e. stage IV breast cancer at presentation) enrolled in the ESOPE study (Institut Curie, Paris). DNA samples extracted from microdissected tumors and from peripheral blood were subjected to high-depth (250x) whole exome sequencing and SNP6 copy number profiling. The impact of spatial heterogeneity was further assessed by targeted sequencing of paraffin-embedded samples from additional, independent pre-treatment biopsies of the primary tumor and matched metastasis from the same patients. Driver mutations were defined by bioinformatic methods; for single nucleotide variants (SNVs), CHASM and FATHMM were employed and for insertions/ deletions (indels), only frameshift or truncating mutations in genes normally expressed in breast tissue were included. Results: In de novo metastatic breast cancers, without any pretreatment, significant genomic differences were observed between primary and metastatic deposits in all cases. A median number of 105 (32-224) and 54 (10-57) SNVs and indels were found, respectively, of which 36 (9-139) and 11 (1-19) were shared between the primary tumors and the de novo metastases, respectively. Although a substantial proportion of driver SNVs and indels were found in common between primary tumors and their respective metastatic deposits (median: 29% (17%-38%)), 50% (25%-78%) of driver SNVs and 79% (60%-90%) of the potentially pathogenic indels were restricted either to the primary or the metastatic deposit, including driver mutations affecting epithelial-to-mesenchymal transition (EMT)-related genes in 3 patients, namely TGFB1, SMAD4 and TCF7L2. Conclusions: This is, to the best of our knowledge, the first study reporting on the differences in the mutational repertoire between primary tumors and metastatic deposits in de novo metastatic breast cancer patients who have not received systemic therapy. Our findings suggest that the breast cancer metastatic process likely constitutes a biological bottleneck with selection of subclones harboring specific driver genetic aberrations, often affecting EMT-related genes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-06.

Abstract OT3-4-05: Use of early CIRculating tumor CElls count changes to guide the use of chemotherapy in advanced metastatic breast cancer patients: the CirCe01 randomized trial

Background: After the SWOG0500 trial, the CirCe01 trial (NCT01349842) is the second randomized attempt to demonstrate that patients who received a first cycle of chemotherapy and whose CTC count did not drop should be switched off this chemotherapy. The clinical setting used here is a population with high chemoresistance prevalence, i.e. patients starting a third line chemotherapy; the CTC test will be also repeated at the beginning of every new chemotherapy line in patients randomized in the CTC arm ( i.e. to evaluate the 3 rd , 4 th , 5 th … lines). In the CTC arm, it has been hypothesized that many patients with chemoresistant tumor will experience repeated early chemotherapy changes (e.g. 3 chemotherapy regimens tested in 9 weeks), giving support to the discontinuation of inefficient, toxic and costly chemotherapies and the start of palliative care. It is believed, for this subgroup of patients, that such de-escalating management will benefit both patients and healthcare systems. For some other patients, it is also expected that they will benefit from the early chemotherapy turn-around and find earlier an efficient therapy among all those tested. Methods: The main inclusion criteria are metastatic breast cancer patients starting a third line of chemotherapy and an elevated CTC count at baseline. 304 metastatic breast cancer patients will be randomized between the standard arm, in which the treatment management is made following the current standard of care, and the CTC-driven arm, in which the “response” after every first cycle of any new chemotherapy line is assessed by CTC count made before the second cycle. Chemotherapies which did not lead to a significant reduction of CTC count are discontinued, and the patient might be offered another chemotherapy regimen (which will be also evaluated by CTC). The medical primary endpoint of the trial is the overall survival, whereas a medico-economic study as a co-primary endpoint. Several secondary endpoints are pre-planned, including progression-free survival, quality of life, anxiety/depression and comparison with serum markers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-4-05.

Transcriptomic Profiles and Circulating Tumor Cell Detection in Primary Breast Cancer.

Background: We have previously demonstrated that circulating tumor cells (CTC) detection by the CellSearch system is strongly associated with metastatic outcome in non-metastatic breast cancer (BC) treated by neoadjuvant chemotherapy (NACT) [Pierga, Clin Cancer Res 2008]. Transcriptomic analysis of primary tumors may uncover molecular phenotypes associated with CTC detection. Methods: Both CTC detection in blood at diagnosis and transcriptomic analysis of the primary tumor have been performed prospectively in 58 non-metastatic BC patients (pts) treated by NACT in a phase II trial (REMAGUS02). We searched for an association between CTC detection and (i) intrinsic molecular subtypes, (ii) stemness signature, (iii) other published signatures, and (iv) expression of molecular markers involved in CTC detection. CTC-associated genes were also studied (v). Results: (i) CTC detection was not statistically associated with an intrinsic molecular subtype: 29% in basal-like (n=5/17pts), 33% in HER2+ (3/9pts), 13% in luminal A (2/13pts), 20% in luminal B (2/10pts) and 29% in normal-like (2/7pts) BC. (ii) No association was found between CTC detection and the stemness signature. (iv) CTC detection was also not related to the “stemness profile”; it was independent of EpCAM, CK8 and CK18 transcriptomic expression. Other results (iii, v) will be disclosed at the meeting. Conclusion: Our study is the first to compare tumor gene expression profiles and blood dissemination of cancer cells in early BC pts. In these pts, CTC detection by the CellSearch system does not depend on intrinsic molecular subtypes, contrary to a published report based on in vitro-grown cells lines [Sieuwerts, J Natl Cancer Inst 2009]. Being independent from molecular prognostic factors, CTC detection is likely to play a critical role in early BC management. Supported by PHRC AOM/2OO2/02117, Pfizer inc., Roche, sanofi-aventis.ISRCTN10059974 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3005.

Single Circulating Tumor Cell Detection and Overall Survival in Non Metastatic Breast Cancer.

Background: Circulation of cancer cells in the blood is a necessary step of hematogeneous metastasis while circulating tumor cells (CTC) have been reported to have a low metastatic efficiency in preclinical animal models. After a median follow-up of 18 months, we previously reported that CTC detection influences the distant metastasis-free survival (DMFS) in non-metastatic breast cancer (BC) patients (pts) treated by neoadjuvant chemotherapy (NACT) in a multicenter prospective trial. Updated results are presented here, focusing on overall survival (OS) and predictors of metastatic relapse. Methods: In 115 localized BC pts, CTC were prospectively screened (CellSearch) before and after NACT (REMAGUS02). We analyzed their outcome after a median follow-up of 36 months. Results: At baseline, 23% of pts were CTC-positive, but only 10% had more than 1 CTC per 7.5ml of blood. At an individual level, CTC detection before chemotherapy, used as a test to predict metastatic relapse, exhibited a global accuracy of 77%, higher than that of tumor grade (54%), tumor size (57%), lymph node invasion (40%), triple negative phenotype (76%) and pathological complete response (27%). Multivariate analyses for OS and DMFS showed that CTC detection before chemotherapy was a strong independent prognostic factor for both DMFS (p=0.01, RR=5.0, 95%CI[1.4-17]) and OS (p=0.007, RR=9, 95%CI[1.8-45]), along with tumor size and triple-negative phenotype, while post-chemotherapy CTC detection had a lower significance for both endpoints (p=0.07 and p=0.09 respectively). Conclusion: Biologically, the metastatic efficiency of CTC could be higher than previously thought. Clinically, besides confirming our previously reported results, this study shows that CTC detection may become the main prognostic factor in BC pts treated with NACT. Implementing this technique in everyday management might help to identify high-risk pts in whom innovative strategies should be investigated.Supported by PHRC AOM/2OO2/02117, Pfizer inc., Roche, sanofi-aventis.ISRCTN10059974 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3017.

Abstract P4-09-02: Disseminated tumor cells as predictive factor of benefit of lymph node irradiation to prevent loco-regional relapse

Background: Early stage breast cancer patients with micrometastatic spread (cM0(i+) per the 2010 TNM staging), detected either in the bone marrow (disseminated tumor cells, DTC) or in the blood (circulating tumor cells, CTC) are at higher risk of distant relapse and death. Loco-regional relapses were also more frequently observed in patients with DTC and, recently, with CTC (IMENEO study, Bidard et al , SABCS 2016). In that context, we analyzed whether DTC detection would be a predictive factor for the benefit of comprehensive loco-regional irradiation. Methods: Patients with localized breast cancer were eligible for this IRB-approved prospective cohort after informed written consent. DTC status was prospectively assessed by trained pathologists after immunocytostaining following ISHAGE criteria, at time of surgery or prior to any primary systemic therapy. Irradiation volumes (breast or chest wall +/- regional lymph nodes) were defined per standard of care. Locoregional relapse was defined as documented ipsilateral invasive relapse occurring in the breast, chest wall and/or in regional lymph nodes, prior to any distant metastatic relapse. Locoregional relapse-free interval (LRFI) was defined as the time elapsed between breast surgery and locoregional relapse. Cumulative incidence rates and hazard ratio were obtained using both Cox and Fine-Gray models, taking into account metastatic relapse and death as competitive events. Interaction tests were performed to confirm the predictive value of DTC status in a multivariate analysis. Results: From 11.1998 to 09.2005, a total of 620 patients with non-metastatic breast cancer were included in this prospective cohort. Median FU was 11.7 years. Overall, 94 patients (15.1%) were DTC-positive and 50 patients (8.1%) experienced a locoregional relapse during follow-up. DTC detection was significantly associated with shorter LRFI in univariate and multivariate analyses (Cox, HR=2.6 [1.4;4.8], p=0.004 ; Fine-Gray, HR=1.76 [1.04;3.0], p=0.04). In the multivariate subgroup analysis, locoregional lymph node irradiation was associated with a longer LRFI for DTC-positive patients, but not for DTC-negative patients (interaction test in multivariate analysis: p=0.03). Similar results were obtained when taking locoregional relapses synchronous with distant metastatic disease into account (interaction test: p=0.02). Importantly, the predictive value of DTC status for the benefit of locoregional irradiation was independent of other clinical and pathological characteristics, including locoregional nodal (pN) status. Conclusion: This long term analysis confirms the independent long-term prognostic value of DTC on locoregional relapses. Moreover, the finding that cM0(i+) status is a predictive marker for the efficacy of locoregional lymph node irradiation promises a new opportunity to better tailor adjuvant radiation therapy in early stage breast cancer patients. Citation Format: Bidard F-C, Mignot F, Poortmans P, Dureau S, Berger F, Loirat D, Proudhon C, Vincent-Salomon A, Pierga J-Y, Kirova Y. Disseminated tumor cells as predictive factor of benefit of lymph node irradiation to prevent loco-regional relapse [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-09-02.

Abstract P2-01-02: Capturing intra-tumor genetic heterogeneity in cell-free plasma DNA from patients with oligometastatic breast cancer

Background: The analysis of cell-free tumor DNA (ctDNA) from plasma has been heralded as a non-invasive technique for disease monitoring and as a means to overcome the challenges posed by intra-tumor genetic heterogeneity. ctDNA levels have been shown to correlate with tumor burden in breast cancer patients. Hence, we sought to define whether massively parallel sequencing of cell-free plasma DNA would capture the entire repertoire of somatic mutations present in the primary tumors and/ or metastases from patients with oligometastatic breast cancer. Methods: Frozen diagnostic biopsies from primary tumors and their distant metastases were obtained from five prospectively accrued treatment-naive patients with stage IV breast cancer at presentation (1 estrogen receptor (ER)+/HER2+, 2 ER+/HER2-, 2 ER-/HER2+). A second, independent formalin-fixed paraffin-embedded (FFPE) diagnostic biopsy was obtained from the primary tumor and metastasis from 4 patients. Plasma samples were obtained from all patients. DNA samples from microdissected frozen tumors and peripheral blood, as well as plasma from one patient, were subjected to high-depth whole exome sequencing. DNA samples from all biopsies (frozen/FFPE), plasma and peripheral blood were subjected to targeted capture massively parallel sequencing, with baits for all somatic mutations detected by whole exome sequencing and all exons of the 100 genes most frequently mutated in breast cancer. Driver mutations were defined by state-of-the-art bioinformatic methods and literature search. Results: We identified and confirmed a median of 54 (range 25-75) and 53 (range 26-85) non-synonymous mutations in the primary tumors and metastases from the 5 cases analyzed, respectively. By sequencing the plasma DNA to a median depth of 248x (range 92-431x), state-of-the-art mutation callers revealed 0-4 mutations (0%-8% of mutations) per patient, and direct interrogation of the sequencing data, based on prior knowledge of the mutations present in the lesions, resulted in the identification of 2-18 mutations (3%-38% of mutations) per patient. Of the bona fide driver mutations, 2/3 TP53 mutations, 0/1 PIK3CA hotspot mutation, 0/1 BRCA2 frameshift mutation, 0/1 GATA3 frameshift mutation and 0/1 ERBB3 activating mutation were captured in the plasma DNA. A SMAD4 pathogenic mutation and a TCF7L2 truncating mutation were found in two diagnostic biopsies of metastatic lesions but not in two biopsies of the primary tumors in one patient each. Whilst the SMAD4 mutation was detected in the plasma DNA from the respective patient, the TCF7L2 mutation was not. Of the 62 mutations restricted to the primary tumors (0-42 per patient) and 74 restricted to the metastatic tumors (1-41 per patient), 4 and 7, respectively, were captured in the plasma DNA. Conclusions: Massively parallel sequencing assessment of plasma DNA allows for the identification of mutations found in primary tumors and/ or their metastases, however, only a subset of these could be detected at up to 431x depth. These observations suggest that current approaches for whole exome or targeted massively parallel sequencing may not be sufficient to capture the genetic heterogeneity of breast cancers in patients with oligometastatic disease. Citation Format: Ng CKY, Bidard F-C, Piscuoglio S, Lim RS, Pierga J-Y, Cottu P, Vincent-Salomon A, Viale A, Norton L, Sigal B, Weigelt B, Reis-Filho JS. Capturing intra-tumor genetic heterogeneity in cell-free plasma DNA from patients with oligometastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-02.