Abstract 4248: A long tail of sub-clonal TP53 mutations emerged by ultra-deep sequencing of newly diagnosed multiple myeloma (MM)
Authors: Martello Marina , Martinelli Giovanni , Dico Angela Flores , Santacroce Barbara , Borsi Enrica , Procacci Mauro , Haferlach Torsten , Mignone Flavio , Saggese Igor , Zamagni Elena , Tacchetti Paola , Pantani Lucia , Brioli Anna Maria , Rocchi Serena , Pezzi Annalisa , Cavo Michele , Terragna Carolina 
Published: August 2015

Abstract
TP53 is rarely reported as being affected either by deletions or mutations in MM, even if chr17p13 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 TP53 gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact.
A cohort of 92 MM, receiving up-front velcade (vel)-based regimens, followed by autologous stem cell transplantation, was included in the study. TP53 gene mutational status was analysed by amplicon-targeted deep NGS approach. An upcoming NGS data analysis software was employed to detect vars (var) and to compare them to IARC database. p53 activity was evaluated by immunoblot assays.
Ultra-deep TP53 coding sequence analysis (median depth: 1060) highlighted the presence of a median of 1.8 vars per samples in 73/92 (79%) MM: a total of 131 nucleotide substitutions emerged, with vars allele frequencies (VAF) ranging from 1 to 99% (median 1.3%). TP53 vars, were defined either neutral or deleterious mutations, according to their predicted effect at amino acid level. 36 cases carried 42 deleterious vars (VAF = 1.2%), 67% of which were predicted as non-functional at protein level. 9 samples carried 21 neutral vars (VAF = 1.1%), mainly affecting the DNA binding domain. 28 samples carried 68 SIFT unclassified vars, among which several polymorphisms were counted (VAF = 94.6%). SNPs arrays were performed on 83/92 samples: 14/83 (17%) carried a TP53 CN loss 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 var) or deleted TP53 (p = 0.006). By immunoblot assay we showed the absence of phosphorilation both in 3 TP53 deleted cases and in 3 MM carrying deleterious vars; on the contrary p-p53 was observed in 4 non-mutated cases.
Clinical correlations were performed on 81 MM showing that the presence of either TP53 hemizygous deletions or at least one TP53 deleterious var was more likely associated to the response to vel-based induction therapy (frequencies of ≤partial response: 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%).
In conclusion, the TP53 coding sequence analysis by ultra-deep NGS of a cohort of newly diagnosed MM highlighted an unexpected, still un-explored, high rate of TP53 vars. The impact of TP53 damage on MM disease course has to be confirmed in randomized clinical trial.
Supported by Roche Diagnostics and FP7 NGS-PTL project
Citation Format: Marina Martello, Giovanni Martinelli, Angela Flores Dico, Barbara Santacroce, Enrica Borsi, Mauro Procacci, Torsten Haferlach, Flavio Mignone, Igor Saggese, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Anna Maria Brioli, Serena Rocchi, Annalisa Pezzi, Michele Cavo, Carolina Terragna. A long tail of sub-clonal TP53 mutations emerged by ultra-deep sequencing of newly diagnosed multiple myeloma (MM). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2015-4248