STRATEGIES TO PREDICT TREATMENT RESPONSE AND SELECT THERAPIES IN METASTATIC BREAST CANCER PATIENTS USING A NEXT GENERATION SEQUENCING (NGS) MULTI-GENE PANEL

The standard of care for many patients with advanced breast cancer (BC )is gradually evolving from empirical treatment based on clinicalpathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. In the last decade, an increasing number of molecularly targeted drugs have been developed for the treatment of metastatic BC. These drugs target specific molecular abnormalities that confer to cancer cells a survival advantage [1]. Interestingly, the ability to perform multigene testing for a range of molecular alterations may provide an opportunity to clarify the mechanisms of treatment response, to find the strategies to overcome treatment resistance and thus, to identify patients who are more likely to develop relapse and who may be candidates for matched targeted therapies [2-3]. The main aim of this study is to find prognostic and predictive molecular biomarkers for the management of metastatic BC patients in clinical practice. MATERIALS AND METHODS The amplicon-sequencing analyses took advantage of the Ion AmpliSeq™ technology (Thermo Fisher, Waltham, MA, USA). A custom panel was designed with the help of the Designer online tool (www.ampliseq.com), which was employed to generate optimized primers encompassing the coding DNA sequences (with 100bp of exon padding and the UTRs regions) of 25 genes in the Human Reference Genome (hg19); these genes were selected searching and screening scientific literature for treatments resistance in BC and are reported in Table 1. Primer pairs were divided into two pools to optimize multiplex PCR conditions and the coverage, that assessed to 89.02%. The customized Ion AmpliSeq panel was employed on samples from 7 primary BC samples and matched metastatic sites (3 skin, 3 lymph node and 1 lung metastases). They were all processed using the Ion AmpliSeq Library Kit 2.0, starting from 15 nanograms of FFPE extracted DNA/pool. Samples were barcoded with the Ion Express Kit to optimize matched patients pooling on the same 318 Chip v2 sequencing chip. The template-positive Ion Sphere Particles were sequenced on a Personal Genome Machine (Thermo Fisher, Waltham, MA, USA). RESULTS The mutation profiles of paired primary and secondary tumors of the seven patients enrolled in this study are presented in Table 2. Ten different genes (PTEN, PIK3CA, mTOR, ERBB2, ERBB3, MET, INPP4B, MAP2K1, CDK6, KRAS) in 6 different patients showed possible damaging variants as shown in Table 2. • Four patients (number 1, 3, 5 and 6) showed no additional or different mutations in secondary tumors if compared to primary samples. • In patient number 2, the metastatic site presented new mutations if compared to the primary tumor. • Finally in patient number 4 and 7 we did not detect in metastases some of the mutations found in the primary tumor. DISCUSSION In 5 patients (71,4%) the mutational status of primary tumor could explain treatment resistance and thus predict relapse, in one patient the mutational status of the new subclones could be relevant for guiding differently the subsequent treatment choices. In 2 patients (28,5%) we were not able to detect in metastases some of the mutations found in the primary tumor. This could be explained by considering the clonal evolution of metastases. These preliminary data suggest that the multi-gene panel analysis of primary and secondary tumors may help clinicians: • in discriminating BC patients HR+ and/or HER2+ with mutations predicting an increased risk of adjuvant treatment resistance and thus relapse • in guiding treatment selection strategies in the metastatic setting. The study is still open and we are currently recruiting other patients.

The main aim of this study is to find prognostic and predictive molecular biomarkers for the management of metastatic BC patients in clinical practice. The preliminary data suggest that the multi-gene panel analysis of primary and secondary tumors may help clinicians: • in discriminating BC patients HR+ and/or HER2+ with mutations predicting an increased risk of adjuvant treatment resistance and thus relapse • in guiding treatment selection strategies in the metastatic setting. The study is still open and we are currently recruiting other patients.