420 Flash RAS study: RAS testing assessment in patients with metastatic colorectal cancer in 2014

Abstracts C-term AR staining to determine the presence of truncated AR variants (including, but not specific to, AR-V7). Of 52 samples, 17 (33%) were eligible for C-term AR testing (11 responders, 6 non-responders), and displayed a range of 14 to 100% AR truncations as determined by the presence/absence of C-terminal AR staining on CTCs. 11/17 (65%) pts showing response to PSMA ADC had CTCs expressing AR variants (range: 35–100%), with 6/11 (55%) samples having predominantly (>50%) truncated AR variant expression. In contrast, 29/35 (83%) poor responders were either CTC-negative (n = 4), AR-negative (n = 14), or had low counts of AR+ CTCs (1−2 AR+ CTC/mL; n = 11). 6/35 (17%) poor responders had CTCs expressing AR variants (range: 14–100%), with 4/6 (67%) samples showing predominantly AR truncated expression on CTCs. Conclusions: These preliminary results suggest that while additional treatment with ADT is unlikely to be effective, pts with AR+ CTCs (wild type or variant AR) may respond to PSMA ADC. While response to PSMA ADC was unaffected by AR variant status after treatment with abi/enz, pts with AR− CTCs appeared to have significantly poorer responses which could indicate a neuroendocrine phenotype. Determinations of AR and AR variant status may be useful parameters to guide treatment selection for future PSMA ADC clinical trials. Analysis of AR-V7 specific variants is ongoing. Conflict of interest: Other Substantive Relationships: Vincent A. DiPippo and Robert J. Israel are employees of Progenics Pharmaceuticals. Shannon L. Werner, Sarah Baird, Amanda K. L. Anderson, and Dena Marrinucci are employees of Epic Sciences. 420 POSTER Flash RAS study: RAS testing assessment in patients with metastatic colorectal cancer in 2014 A. Lievre1 , J.L. Merlin2 , P. Laurent-Puig3 , P. Artru4 , A. Seronde5 , ˆ C. Gicquel6 , J.C. Sabourin7 , M. Ducreux8 . 1 Institut Curie − Hopital Rene´ ´ Huguenin, Oncology, Saint Cloud, France; 2 Institut de Cancerologie ˆ de Lorraine, Biopathology, Vandoeuvre-les-Nancy, France; 3 Hopital ´ ˆ Europeen Georges Pompidou, Biochemistry, Paris, France; 4 Hopital 5 Prive´ Jean Mermoz, Digestive oncology, Lyon, France; Merck Serono, 6 Medical Affairs Oncology, Lyon, France; Merck Serono, Regional Clinial Operations, Lyon, France; 7 CHU Charles-Nicolle, Biopathology, Rouen, France; 8 Institut Gustave Roussy, Gastro-enterology, Villejuif, France In addition to KRAS exon 2 mutation, it was shown in 2013 that mutations in KRAS exons 3 and 4, or NRAS exons 2 to 4 had a similar impact on antiEGFR (Epidermal Growth Factor Receptor) therapy efficacy. The resulting cetuximab prescribing restriction to mCRC (metastatic ColoRectal Cancer) patients without so called “RAS mutations” thus enabled to better define the category of population likely to benefit from this treatment. FLASH-RAS (following the FLASH-KRAS study performed in 2011) is an observational retrospective French multicenter study. The primary objective was to describe RAS testing prescribing and performing practices in patients with a recent diagnosis of mCRC in 2014. The secondary objectives were to describe the changes in biomarker testing prescribing practices from 2011 to 2014, the circuit and time required to obtain the test results and their impact on the therapeutic strategy. 104 centers participated in the FLASH-RAS study: 375 patients diagnosed with mCRC after 1 March 2014 and initiating a 1st line treatment between 1 March 2014 and 30 June 2014 were analyzed. 90.1% (IC95%= [87.1%; 93.2%]) of them benefited from a genotyping request regarding one or all RAS mutations when they were managed in 1st line. The percentage of patients for which a RAS genotyping request was made has increased since 2011 (81.1% in FLASH-KRAS, p < 0.001). For 75% of patients, this request was made before or at least one month after the first diagnosis of the metastatic disease. For the remaining 24.5%, the request was made more than one month after the first diagnosis of the metastatic disease which is not compatible with an informed choice of 1st line treatment according to the patient’s biomarker status. The genotyping report was available in 96.4% of cases. The median and the mean time to obtain the genotyping report in 2014 (20 and 24.6±17.2 days) were not longer than in 2011 (19 and 23.6±28.2 days) despite the increased number of exons to test (1 exon in 2011 versus 6 in 2014). It was noticed that the standard deviation of the mean time was decreased compared to 2011, meaning that the time to obtain RAS status could tend to be standardized. In 2014, RAS genotyping is routinely performed as part of mCRC patient management. The number of RAS genotyping requests has increased since 2011. Nevertheless, for 24.5% of patients, the request is made more than one month after the first diagnosis of the metastatic disease which is not compatible with an informed choice of 1st line treatment according to the patient’s biomarker status. The median and the mean time to obtain the RAS genotyping report were similar in 2011 and 2014, the decreased standard deviation of the mean time could indicate a trend toward standardization. This study showed how much every stakeholder

S85 involved in mCRC patient management was responsive in implementing these new predictive tests. Conflict of interest: Advisory Board: Amgen, AstraZeneca, Bayer, Boehringer-Ingelheim, Integragen, Lilly, Merck Serono, Novartis, Pfizer, Roche, Sanofi. 421 POSTER Heterogeneous HER2 antigen expression among individual circulating tumor cells within breast cancer patients 1 ¨ , M. Vetter1 , S. Nejlund2 , J. Smith1 , H. Stender2 , T. Gluckstadt 5 A. Fabisiewicz3 , A. Jagiello-Gruszfeld4 , T. Hillig5 , G. Sol . ¨ etormos ´ 1 Metropolitan University College, Department of Technology, Copenhagen, Denmark; 2 CytoTrack, CTC Center of Excellence, Lyngby, Denmark; 3 The Maria Sklodowska, Curie Memorial Cancer Centre and Institute of Oncology, Department of Translational and Molecular Oncology, Warsaw, Poland; 4 The Maria Sklodowska, Curie Memorial Cancer Centre and Institute of Oncology, Department of Breast Cancer and Reconstruction Surgery, Warsaw, Poland; 5 North Zealand Hospital, Department of Clinical Biochemistry, Hillerød, Denmark

Background: Today’s treatment of metastatic breast cancer is guided by characterization of the primary tumor despite molecular profiles of metastases can differ. Characterization of the human epidermal growth factor receptor 2 (HER2) expression profile on circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients can potentially personalize adjuvant therapy. This study describes a novel method beyond detection and enumeration of CTCs, which determines HER2 surface antigen expression on individual CTCs in breast cancer patients. Materials and Methods: Four cancer cell lines with known HER2 expression were used to develop a HER2 immunofluorescence staining method. Using the CytoTrack system, tumor cells were detected and enumerated in blood samples and subsequently immunostained for expression of the HER2 antigen. The HER2 expression profiles were hence evaluated for the individual tumor cells by retracing of the detected and enumerated tumor cells. An exploratory clinical study using blood samples from 11 metastatic breast cancer patients was performed to measure HER2 expression profiles of individual CTCs within each patient. Results: CTC enumeration followed by HER2 characterization using cell lines and donor blood spiked with cell lines yielded results in accordance with HER2 expression profiles of the cell lines. In seven of 11 patient specimens 1–189 CTCs were detected. Individual patients showed significant HER2 expression heterogeneity ranging from 0% to 100%. Conclusion: A novel method for CTC enumeration and HER2 characterization is described. CTC heterogeneity was observed in breast cancer patients. Future clinical studies are on-going to examine the clinical significance of these findings. No conflict of interest. 422 POSTER Our experience with HER2 screening of the bioptic breast cancer samples using the method fluorescence in situ hybridization in the year 2014 − application of the updated ASCO/CAP guideline recommendations J. Zmolikova1 , S. Pitronova2 , M. Uvirova1 , B. Kubova3 , J. Simova1 , N. Strossova2 , D. Ziak4 , J. Dvorackova5 . 1 University of Ostrava, Faculty of Medicine, Ostrava, Czech Republic; 2 CGB Laboratory Inc., Laboratory of Cytogenetics, Ostrava, Czech Republic; 3 CGB Laboratory Inc., Laboratory of Molecular Genetics, Ostrava, Czech Republic; 4 CGB Laboratory Inc., Laboratory of Pathology, Ostrava, Czech Republic; 5 University Hospital Ostrava, Institute of Pathology, Ostrava, Czech Republic Background: The criteria of human epidermal growth factor receptor (HER2) testing and analysing in breast cancer using in situ hybridization were updated by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) in 2013. This new testing criteria define HER2 positive status when there is HER2/CEP17 ratio >2.0 or average HER2 copy number 6.0 signals/cell − in these cases the patients unequivocally meet the criteria of HER2 positivity and are suitable for the anti-HER2 targeted therapy. But there is also a group of patients with HER2/CEP17 ratio <2.0 and average HER2 copy number ranging between 4.0−6.0 signals/cell at the same time. This group represents equivocal category, because in these cases it is hard to determine true polysomy 17 from the coamplification of the two adjacent regions − centromeric region of chromosome 17 and gene HER2 (17q12). According to the new guideline recommendations it is necessary to perform reflex testing using a different control DNA probe for chromosome 17.