ALBERT

Description: INTRODUCTION In newly diagnosed Multiple Myeloma (MM) patients (pts), Copy Number (CN) losses of chromosome 17p13, carrying the TP53 tumor-suppressor gene, are strong predictors of poor outcomes. On the contrary, the prognostic relevance of TP53 mutations at the onset of the disease is less clear, due to the very limited frequency of clonal lesions, as revealed by Sanger sequencing. To address this poorly investigated issue, we used an ultra-deep sequencing (UDS) approach to characterize the TP53 structural architecture in both newly diagnosed and relapsed MM pts and to assess the prognostic role and evolution over time of small TP53 mutated sub-clones. SAMPLES AND METHODS A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and autologous stem cell transplantation, was included in this molecular study. In 29 cases, samples were collected both at diagnosis and at relapse(s). DNA was obtained from CD138+ highly purified plasma cells. TP53 gene mutational status was analysed by using an amplicon-targeted UDS approach (GSJ, 454 Roche Life Sciences). In order to discriminate between low frequency sub-clonal TP53 variants and sequencing errors, sequencing raw data were filtered according to cut-off values based on different ranges of sequences' coverage depth. Additional filters were also applied, based on both quality and biological cut-offs, to obtain a final confident list of variants. Analysis of CN alterations (CNAs) was performed by SNPs array and results analysed with ChAS software. RESULTS With a median coverage of 1386X, a list of 129 correctly called TP53 variants (either missense, or nonsense or splice ones), including 20 INDELs, was detected. Only deleterious and N/A variants (according to SIFT classification) were included in the list. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts), with 45/99 (45%) carrying non-mutated TP53. Pts carrying TP53 sub-clonal variants bared also TP53 CN hemizygous losses (20%), CKS1B gains (56%) and cdkn2c losses (14%). According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with

Description: Introduction. The role of TP53 tumor-suppressor gene in mediating cellular basic tumor suppressive mechanisms is crucial: its utmost importance is underscored by a 50% mutational rate among most human cancers. In MM, TP53 is rarely reported as being affected either by deletions or mutations, even if chromosome (chr) 17p13 copy number (CN) loss defines a samples subgroup with a particularly poor prognosis. Here we aim at retrospectively analyzing by Next Generation Sequencing (NGS) the TP53gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact. Samples and methods. A cohort of 92 MM, receiving up-front velcade (vel)-based regimens, followed by autologous stem cell transplantation, was included in this molecular study. Response to therapy was evaluated according to the IMWG criteria. DNA was obtained from CD138+ highly purified plasma cells and profiled by SNPs array (Affymetrix). TP53gene mutational status was analysed employing an amplicon-targeted deep NGS approach (GSJ, 454 Life Sciences). A novel upcoming NGS data analysis software was employed to detect variants from raw sequencing data and to compare them to IARC database. As a validation, 30 randomly chosen samples have been re-sequenced. p53 activity was evaluated by p-p53 and p53 immunoblot assays. Results. Ultra-deep TP53coding sequence analysis (median depth: 1060) highlighted the presence of a median of 1.8 variants per samples in 73/92 (79%) MM: a total of 131 nucleotide substitutions emerged, with variants allele frequencies (VAF) ranging from 1 to 99% (median 1.3%). The polymorphism c.215C〉G (validated as SNP1042522) was the most prevalent detected variant, recurring in ~40% of the analysed samples. TP53variants were assessed according to the SIFT classification, in order to identify either neutral or deleterious mutations, on the basis of their predicted effect at amino acid level. 36 cases carried 42 deleterious variants (VAF=1.2%): they were mostly missense substitutions affecting mainly the DNA binding domain; most deleterious variants (67%) were predicted as non-functional at protein level. 9 samples carried 21 neutral variants (VAF=1.1%), which were mostly missense mutations, again mainly affecting the DNA binding domain. Finally, 28 samples carried 68 SIFT unclassified variants – among which several polymorphisms were counted – (VAF=94.6%). Of these, 29 were silent substitutions affecting the DNA-binding domain, 32 were missense variants affecting the SH3-like/Proline- rich domain. To assess the genomic background of deep-sequenced MM, SNPs arrays were performed on 83/92 samples: 14/83 (17%) carried a TP53 CN loss (1N) on chr17p13.1. The incidence of TP53 hemizygous deletion was higher among cases carrying deleterious mutations, as compared to cases carrying either neutral or unclassified mutations (25%, 12,5% and 12%, respectively). 2/17 non-mutated samples carried TP53 hemizygous deletion, as well. Interestingly, an Rb1 tumour suppressor gene CN loss on chr13q14.2 significantly characterized samples carrying either mutated (deleterious variant) or deleted TP53 (p=0.006). The activation state of p53 was evaluated by p-p53 immunoblotting assay, showing the absence of phosphorilation either in 3 TP53 deleted cases, or in 3 cases carrying deleterious variants; on the contrary p-p53 was observed in 4 non-mutated cases. Finally, clinical correlations were performed on 81 MM, for whom clinical data were available, showing that the presence of either TP53 hemizygous deletions or at least one TP53 deleterious variant was more likely associated to the response to vel-based induction therapy (frequencies of ≤partial response were 35% and 64% among samples carrying or not impaired TP53, respectively; p=0.05). Conversely, the frequency of progression events was slightly higher among MM carrying impaired TP53 (69% vs. 50%). Conclusions. The analysis by ultra-deep next generation sequencing of TP53 coding sequence in a cohort of newly diagnosed MM highlighted an unexpected, still un-explored, high rate of TP53 variants, recurring with a wide range of frequencies among samples. The impact of TP53 damage on MM disease course has to be confirmed in randomized clinical trial. Acknowledgments: Roche Diagnostics for supporting us in the realization of this project; ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Cavo:Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy; Roche: Honoraria.