Multiparameter Flow Cytometry (MFC) Evaluation of Plasma Cell (PC) DNA Ploidy Status and Proliferative Rate in 595 Multiple Myeloma (MM) Patients (pts) Included in the Spanish GEM2000 and GEM2005<65years Trials: Clinical Value and Biological Insights,
Authors: Paiva Bruno , Vidriales María-Belén , Montalbán María-Angeles , Perez Jose J. , Gutierrez Norma C. , Rosiñol Laura , Martínez-López Joaquín , Mateos Maria Victoria , Courdón Lourdes , Oriol Albert , Terol María José , Etxebeste Maria Asunción , de Paz Raquel , de Arriba Felipe , Palomera Luis , De La Rubia Javier , Díaz-Mediavilla Joaquín , Sureda Anna , Gorosquieta Ana , Alegre Adrian , Martín Alejandro , Hernandez Miguel T. , Lahuerta Juan José , Blade Joan , San Miguel Jesús F 
Published: October 2019

Abstract
Abstract 3938
The incorporation of high-dose therapy/autologous stem cell transplantation (HDT/ASCT) and novel agents has significantly improved survival of MM young pts; however, the molecular heterogeneity of the disease warrants the investigation if such improvement also benefits patients harboring poor prognostic features such as non hyperdiploid (HY) karyotypes and high proliferation index.
Herein, we have analyzed by MFC the DNA ploidy status and proliferation of bone marrow (BM) PC in a total of 595 newly diagnosed MM pts included in two consecutive PETHEMA/GEM trials: GEM2000 (VBMCP/VBAD followed by HDT/ASCT; N=319) and GEM2005<65y (randomized induction with the same chemotherapy plus bortezomib in the last two cycles or thalidomide/dexamethasone or bortezomib/thalidomide/dexamethasone followed by HDT/ASCT; N=276). Baseline FISH analysis were performed on separated PC. DNA ploidy and cell cycle were assessed by MFC through simultaneous staining with CD38/CD138 and propidium iodide. Median follow-up of the whole series was 38 months.
Of the 595 pts, 295 were classified as non-HY (49.6%) and 300 as HY (50.4%). Patients with non-HY vs HY DNA content showed significantly inferior median PFS (34 vs 44 months, P=.004) and OS (67 vs 84, P=.005). Interestingly, we have detected by MFC the presence of two different PC clones (with different DNA ploidy status) in 34 of the 595 (6%) patients, and this subgroup showed a poor outcome (median PFS and OS of 26 and 52 months respectively, P≤.005). Regarding the proliferative index, the median percentage of PCs in S-phase in the whole series was of 1.14% (0%-13%). Accordingly, pts were stratified using a cutoff of ≥1% vs <1% PCs in S-phase, which translated in significantly different PFS (medians of 34 vs 43 months, P<.001) and OS (medians of 66 vs 93 months, P=.001). Of note, the detection of a proliferative rate ≥3% also identified a subgroup of patients (92 of 595, 15%) with high-risk disease (median PFS and OS of 22 and 45 months respectively, P≤.001). At the multivariate analysis including other baseline prognostic factors, the presence of high-risk cytogenetics (HR=1.7;P=.007), ≥1% PCs in S-phase (HR=2.0;P<.001), >15% PCs by MFC (HR=1.7;P=.008) and >5% normal PCs within the BM PC compartment (HR=6.6;P=.008) emerged as independent prognostic factors for PFS; in turn, for OS the presence of high-risk cytogenetics (HR=2.3;P<.001), non-HY DNA content (HR=1.7;P=.02), ≥1% PCs in S-phase (HR=2.0;P=.002) and the ISS disease stage (HR=1.7;P=.03) were selected.
After this analysis of the overall population, we investigated whether the incorporation of novel agents in the induction regimen previous to HDT/ASCT could abrogate the poor prognosis of patients classified as non-HY and with ≥1% PCs in S-phase. Patients with non-HY DNA status included in the GEM2005<65y trial do not show significantly superior PFS (40 vs 34 months, P=.8) nor OS (not reached vs 63 months, P=.5) compared to cases enrolled in the GEM2000 protocol. Focusing on pts with ≥1% PCs in S-phase, no differences were founded for PFS (40 vs 33 months, P=.9), but OS was significantly longer for patients treated with novel agents prior to HDT/ASCT (NR vs 61months, P=.004).
Finally, we wanted to gain further insight into the potential association between specific cytogenetic abnormalities and PC proliferation, as well as whether there is a difference in the proliferative rate of PCs between diagnosis and disease progression. Interestingly, pts harboring a t(11;14) showed a significantly decreased percentage of PCs in S-phase (0.7% vs 1.2%, P<.001), while no differences were recorded for t(4;14), t(14;16), del(Rb) and del(17p). To address our final question, we compared the proliferative rate of PCs from 52 pts with paired BM samples at diagnosis and at relapse. Of note, 45 of the 52 (85%) pts showed an increased percentage of PCs in S-phase at relapse, with a median 2-fold difference between the proliferative rate at diagnosis vs. relapse (P<.001).
In summary, our results show that the evaluation of PCs DNA content by MFC immunophenotyping provides valuable clinical information, as pts with a non-HY DNA status and high proliferative rate show a poor outcome, which is not yet fully abrogated by the incorporation of novel agents prior to HDT/ASCT. Moreover, pts at relapse show an increased proliferative index; therefore, the precise mechanisms leading to PC proliferation deserves further investigations.

Disclosures:
Paiva: Celgene: Honoraria; Janssen: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Mateos:Janssen: Honoraria; Celgene: Honoraria. Alegre:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria. San Miguel:Janssen-Cilag: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc: Consultancy.