A Branching Evolution Model at Relapse Characterizes Multiple Myeloma Patients Who Responded to up-Front Combination Therapy Including New Drugs
Authors: Terragna Carolina , Martello Marina , Santacroce Barbara , Solli Vincenza , Pantani Lucia , Zamagni Elena , Tacchetti Paola , Zannetti Beatrice Anna , Mancuso Katia , Marzocchi Giulia , Testoni Nicoletta , Ameli Gaia , Termini Rosalinda , Dico Flores A , Borsi Enrica , Martinelli Giovanni , Cavo Michele 
Published: October 2019

Abstract
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.