Abstract A82: Prognostic relevance and genomic profile of circulating tumor cells in multiple myeloma
Authors: Mishima Yuji , Paiva Bruno , Shi Jiantao , Massoud Mira , Manier Salomon , Perilla-Glen Adriana , Aljawai Yosra , Takagi Satoshi , Huynh Daisy , Huynh Daisy , Roccaro Aldo , Sacco Antonio , Alignani Diego , Mateos Maria-Victoria , Blade Joan , Lahuerta Juan-Jose , Richardson Paul , Laubach Jacob , Schlossman Robert , Anderson Kenneth , Munshi Nikhil , Prosper Felipe , San Miguel Jesus , Michor Franziska , Ghobrial Irene M. 
Published: May 2016

Abstract
Introduction: Genomic sequencing of tumor cells obtained from the bone marrow (BM) of patients with multiple myeloma (MM) has demonstrated significant clonal heterogeneity. However, it could be envisioned that such clonal diversity may be even higher since the pattern of BM infiltration in MM is typically patchy. In addition, BM biopsies cannot be repeated multiple times during the course of therapy, indicating a need for less invasive methods to genomically characterize MM patients. In this study, we aimed to determine the overall applicability of performing genomic characterization of MM patients non-invasively using circulating tumor cells (CTC).
Methods: We performed CTC enumeration using multi-parameter 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 sequencing studies, we obtained 8 samples of newly-diagnosed untreated patients. We sequenced 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 sequenced up to 100x for each duplicate. Only single nucleotide variants (SNVs) shared in both parallel WGA libraries were used.
Results: 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. Nineteen of the 40 newly-diagnosed cases displaying PB CTCs had relapsed (median TTP of 31 months); by contrast, only 1 of the 10 patients with undetectable CTCs has relapsed (median TTP not reached; P = .08). Afterward, increasing CTC counts in sequential PB samples from patients with relapsed disease or in remission/on maintenance therapy 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 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 non-synonymous 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. 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 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.
Citation Format: Yuji Mishima, Bruno Paiva, Jiantao Shi, Mira Massoud, Salomon Manier, Adriana Perilla-Glen, Yosra Aljawai, Satoshi Takagi, Daisy Huynh, Daisy Huynh, Aldo Roccaro, Antonio Sacco, Diego Alignani, Maria-Victoria Mateos, Joan Blade, Juan-Jose Lahuerta, Paul Richardson, Jacob Laubach, Robert Schlossman, Kenneth Anderson, Nikhil Munshi, Felipe Prosper, Jesus San Miguel, Franziska Michor, Irene M. Ghobrial. Prognostic relevance and genomic profile of circulating tumor cells in multiple myeloma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A82.