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: Background Multiple Myeloma (MM) is a biologically complex disease, whose genetic plasticity favors the coexistence of genetically heterogeneous subclones, selected in a Darwinian fashion throughout the disease course. Based on a disease evolution model, it is likely that therapy acts like an evolutionary bottleneck, applying a selective pressure on the clones' genomic background. However, a formal correlation between the modulation of intra-clonal heterogeneity and the treatment has yet to be demonstrated. Aim To explore the existence of different clonal evolution patterns in MM, eventually driven by therapeutic selective pressure, we longitudinally analyzed a cohort of patients (pts) by using a high-throughput technology, able to finely dissect the genomic intra-clonal changes occurring in each pts, across disease progression. Patients and methods Thirty-three pts with symptomatic MM were included in this study. Most pts (28) were up-front treated with regimens including a proteasome inhibitor (PI), either in combination with an immunomodulator (IMiD) (thalidomide or lenalidomide) or with cyclophosphamide. The remaining pts (5) were up-front treated with IMiDs-based regimens, not including PI. For each pts, paired bone marrow samples were collected both at diagnosis and at relapse. SNPs array analyses were performed on the CD138+ enriched cell fractions (Affymetrix 6.0 and CytoscanHD) and data were analyzed with ChAS v3.1 and Nexus software, to obtain Copy Number Alterations (CNAs) results. Results: The genomic landscape's modulations were evaluated in details in each pair of samples included in the study: by one side, by monitoring the variations of macro CNAs types; by the other, by focusing on changes of CNAs frequencies, as observed in 27 genes of interest (selected according to their recognized role in MM pathogenesis). Both approaches were consistent in highlighting three major evolution patterns: in 7/33 (21%) pts, the genomic background at relapse was almost identical to that of diagnosis, suggesting that no evolution affected the diagnosis' genomic architecture. In 13/33 (39%) pts, an overall increase in the frequencies of the same CNAs as observed at diagnosis was detected at relapse, suggesting a linear evolution of the diagnosis' predominant clone. Finally, in 13/33 (39%) pts, either increased or decreased frequencies of several CNAs, as well as several differences in the CNAs type's prevalence were observed in samples collected at relapse, as compared to those collected at diagnosis, suggesting a branching evolution of sub-clones not-detectable at diagnosis. Of interest, even if an overall increase in the median number of CNAs was observed in the CD138+ cell fractions collected from the whole population of newly diagnosed and relapsed pts (226 vs 507, respectively) - supported by acquisition of CNAs either commonly described as secondary genomic events (i.e. del17p13, amp1q21, del1p23), or associated to the resistance to bortezomib (i.e. del8p21) - nevertheless any peculiar CNAs resulted significantly prevalent in the three identified subgroups of pts. The subgroup of pts who underwent a branching evolution was characterized by a high rate (92%) of achievement of VGPR or better quality of response to upfront therapy, regardless of incorporation of PI into induction regimens. On the contrary, although pts who experienced either linear or no evolution were mostly treated with highly effective PI-based triplet combinations (19/20 pts), the rate of VGPR in this subgroup was only 20% and PR or SD were observed in 9 and 7 pts, respectively. Finally, the median time to first progression of this subgroup of pts was significantly shorter as compared to that of pts with branching evolution (24 vs 35 months, range 4-41 and 7-123 months, respectively, p=0,01). Conclusion The genomic architecture of a subgroup of relapsed MM pts, who were up-front responsive to new drugs-based combination therapies, resulted overall different from that of diagnosis, suggesting a branching evolution of the disease, sustained by the shrinking of the most prevalent clone (therapy-sensitive), as well as by the expansion of subclones (therapy-resistant) not already evident at diagnosis. This observation raises the question whether re-treatment of relapsed pts should be appropriate in the case of branching evolution. Acknowledgments: AIRC (MC), Fondazione Berlucchi (CT), FUV (EB). Disclosures Cavo: Takeda: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.