Mutational Profile and Prognostic Relevance of Circulating Tumor Cells in Multiple Myeloma
Authors: Mishima Yuji , Paiva Bruno , Shi Jiantao , Massoud Mira , Manier Salomon , Flores Ludmila , Perilla-Glen Adriana , Aljawai Yosra , Huynh Daisy , Takagi Satoshi , Roccaro Aldo M , Sacco Antonio , Alignani Diego , Mateos Maria Victoria , Bladé Joan , Lahuerta Juan Jose , Richardson Paul G. , Laubach Jacob P. , Schlossman Robert L. , Anderson Kenneth C , Munshi Nikhil C. , Prosper Felipe , Michor Franziska , San Miguel J.F. , Ghobrial Irene M. 
Published: October 2019

Abstract
Introduction: Massive parallel sequencing of tumor cells obtained from the bone marrow (BM) of patients with multiple myeloma (MM) has demonstrated significant clonal heterogeneity with a median of five clones present in each sample. However, it could be envisioned that such clonal diversity may be even higher since single BM samples only represent a small fraction of the whole BM compartment, and the pattern of BM infiltration in MM is typically patchy. Accordingly, it remains unknown whether using liquid biopsies (i.e.: patients' genetic characterization performed in peripheral blood -PB- samples) can provide a more complete profile of MM clonal diversity. Moreover BM biopsies and cannot be repeated multiple times during the course of therapy, indicating a need for less invasive methods to genomically characterize MM patients. We aimed to determine the overall applicability of performing genomic characterization of MM patients non-invasively, and define if the mutation profile of circulating tumor cells (CTCs) reflected that of patient-paired BM clonal PCs.
Methods: We performed CTC enumeration using multiparameter flow cytometry (MFC) in 50 newly-diagnosed patients with symptomatic MM who were prospectively enrolled on the Spanish clinical trial PETHEMA/GEM2010MAS65 as well as 64 patients with MM with relapsed disease or in remission/on maintenance therapy seen at the Dana-Farber Cancer Institute. For whole exome sequencing studies, we obtained 8 samples of newly-diagnosed untreated patients whose bone marrow, CTC and germline T lymphocytes were available and selected for exome sequencing. We sequenced the whole exome of BM clonal PCs and CTCs up to 200x, and germline cells up to 50x. Whole genome amplification (WGA) was performed for CTCs, and two independent libraries were constructed from the sample, followed by sequencing up to 100x for each duplicate. For samples with WGA, only single nucleotide variants (SNVs) shared in both parallel libraries were used.
Results: Before investigating if CTCs could represent a reliable non-invasive alternative to perform genomic characterization of MM patients, we first aimed to define its true applicability at different disease stages. Using sensitive MFC, we showed that CTCs were detectable in 40/50 (80%) newly-diagnosed MM patients, and in 71/130 (55%) of multiple sequential samples from patients with relapsed disease or in remission/on maintenance. As for the prognostic value of CTC enumeration, 19 of the 40 newly-diagnosed cases displaying PB CTCs had relapsed (median time-to-progression of 31 months); by contrast, only 1 of the 10 patients with undetectable CTCs has relapsed (median time-to progression not reached; P=.08). Afterward, we investigated whether dynamic changes in the kinetics of CTCs in sequential PB samples from patients with relapsed disease or in remission/on maintenance therapy was also predictive of outcome. Accordingly, increasing CTC counts were associated with poor overall survival (P= .01), indicating that both the absolute numbers of CTCs and trend of CTC are predictive of outcome in MM.
After demonstrating that CTCs can be readily detected in the majority of MM patients, we then determined the mutational profile of CTCs and compared it to that of patient-paired BM clonal PCs. We identified a median of 223 and 118 SNVs in patient-paired BM clonal PCs and CTCs, respectively. The concordance of somatic variants found in matched BM clonal PCs and CTCs was of 79%. Noteworthy, upon investigating specific mutations implicated in MM (eg. KRAS, NRAS, BRAF) a total of 18 nonsynonymous SNVs (NS-SNVs) in 13 genes were identified in our cohort, and most of these NS-SNVs were simultaneously detected in matched BM clonal PCs and CTCs from the same patients. That notwithstanding, we also identified several unique mutations present in CTC or BM clonal PCs; of those, up to 39 NS-SNV were identified as CTC specific, and 6 NS-SNVs in 4 genes (CR1, DPY19L2, TMPRSS13, HBG1) were detected in CTC from multiple patient samples. A significant concordance for the pattern of copy number variations (CNVs) between matched BM and PB tumor cells was also observed.
Conclusion: This study defines a new role for CTCs in the prognostic and molecular profiling of MM patients, and provides the rational for an integrated flow-molecular algorithm to detect CTCs in PB and identify candidate patients for noninvasive genomic characterization to predict outcomes.

Disclosures
Paiva: Sanofi: Consultancy; Millennium: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMAb AG: Consultancy. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Schlossman:Millennium: Consultancy. San Miguel:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.