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Multiparameter Flow Cytometry Evaluation of Plasma Cell DNA Content and Proliferation in 595 Transplant-Eligible Patients with Myeloma Included in the Spanish GEM2000 and GEM2005<65y Trials

2012; Elsevier BV; Volume: 181; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2012.07.020

ISSN

1525-2191

Autores

Bruno Paiva, María‐Belén Vidriales, María-Ángeles Montalbán, José J. Pérez, Norma C. Gutiérrez, Laura Rosiñol, Joaquin Martínez‐López, María‐Victoria Mateos, Lourdes Cordón, Albert Oriol, María José Terol, M Asuncion Echeveste, Raquel de Paz, Felipe de Arriba, Luis Palomera, Javier de la Rubia, Joaquín Diaz‐Mediavilla, Anna Sureda, Ana Gorosquieta, Adrián Alegre, Alejandro Martı́n, Juan José Lahuerta, Joan Bladé, Alberto Órfão, Jesús F. San Miguel,

Tópico(s)

Peptidase Inhibition and Analysis

Resumo

The incorporation of high-dose therapy/autologous stem cell transplantation (HDT/ASCT) and novel agents has significantly improved survival in patients with multiple myeloma (MM), but whether this improvement also benefits patients harboring poor prognostic features, such as nonhyperdiploid MM (NH-MM) and a high proliferation index, remains largely unknown. We analyzed the DNA content and proliferation index of bone marrow plasma cells (PCs) by multiparameter flow cytometry in 595 newly diagnosed transplant-eligible patients with MM included in two consecutive PETHEMA/GEM trials: GEM2000 [VBMCP/VBAD (vincristine, carmustine, melphalan, cyclophosphamide, prednisone/vincristine, bischloroethylnitrosourea, adriamycin, and dexamethasone) followed by HDT/ASCT; n = 319] and GEM2005<65y (randomized induction with VBMCP/VBAD/bortezomib or thalidomide/dexamethasone or bortezomib/thalidomide/dexamethasone followed by HDT/ASCT; n = 276). Of the 595 patients, 295 were classified as NH-MM (49.6%) and 336 (56.5%) as high-proliferative MM (≥1% PCs in S-phase). Detection of NH-MM DNA content and ≥1% PCs in S-phase were of independent prognostic value for overall survival. Treatment with bortezomib-based regimens abrogated the inferior overall survival of patients with ≥1% PCs in S-phase but not of patients with NH-MM. Finally, a comparative analysis of PC proliferation index at diagnosis versus disease progression showed a twofold increase at relapse in 44 of 52 patients (85%) analyzed at both time points. NH-MM and a high proliferation index assessed by multiparameter flow cytometry remain as independent prognostic factors in MM, but the latter may be overcome by incorporating novel agents in the HDT/ASCT setting. The incorporation of high-dose therapy/autologous stem cell transplantation (HDT/ASCT) and novel agents has significantly improved survival in patients with multiple myeloma (MM), but whether this improvement also benefits patients harboring poor prognostic features, such as nonhyperdiploid MM (NH-MM) and a high proliferation index, remains largely unknown. We analyzed the DNA content and proliferation index of bone marrow plasma cells (PCs) by multiparameter flow cytometry in 595 newly diagnosed transplant-eligible patients with MM included in two consecutive PETHEMA/GEM trials: GEM2000 [VBMCP/VBAD (vincristine, carmustine, melphalan, cyclophosphamide, prednisone/vincristine, bischloroethylnitrosourea, adriamycin, and dexamethasone) followed by HDT/ASCT; n = 319] and GEM2005<65y (randomized induction with VBMCP/VBAD/bortezomib or thalidomide/dexamethasone or bortezomib/thalidomide/dexamethasone followed by HDT/ASCT; n = 276). Of the 595 patients, 295 were classified as NH-MM (49.6%) and 336 (56.5%) as high-proliferative MM (≥1% PCs in S-phase). Detection of NH-MM DNA content and ≥1% PCs in S-phase were of independent prognostic value for overall survival. Treatment with bortezomib-based regimens abrogated the inferior overall survival of patients with ≥1% PCs in S-phase but not of patients with NH-MM. Finally, a comparative analysis of PC proliferation index at diagnosis versus disease progression showed a twofold increase at relapse in 44 of 52 patients (85%) analyzed at both time points. NH-MM and a high proliferation index assessed by multiparameter flow cytometry remain as independent prognostic factors in MM, but the latter may be overcome by incorporating novel agents in the HDT/ASCT setting. The incorporation of high-dose therapy/autologous stem cell transplantation (HDT/ASCT) and novel therapeutic agents1Palumbo A. Anderson K. 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Dumontet C. Fuzibet J. Azais I. Dorvaux V. Zandecki M. Bataille R. Minvielle S. Harousseau J. Facon T. Mathiot C. Genetic abnormalities and survival in multiple myeloma: the experience of the Intergroupe Francophone du Myélome.Blood. 2007; 109: 3489-3495Crossref PubMed Scopus (816) Google Scholar, 6Avet-Loiseau H. Li C. Magrangeas F. Gouraud W. Charbonnel C. Harousseau J. Attal M. Marit G. Mathiot C. Facon T. Moreau P. Anderson K. Campion L. Munshi N. Minvielle S. Prognostic significance of copy-number alterations in multiple myeloma.J Clin Oncol. 2009; 27: 4585-4590Crossref PubMed Scopus (247) Google Scholar, 7Fonseca R. Bergsagel P.L. Drach J. Shaughnessy J. Gutierrez N. Stewart K. Morgan G. Van N.B. Chesi M. Minvielle S. Neri A. Barlogie B. Kuehl W.M. Liebisch P. Davies F. Chen-Kiang S. Durie B.G. Carrasco R. Sezer O. Reiman T. Pilarski L. vet-Loiseau H. 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Prognostic and biological implications of genetic abnormalities in multiple myeloma undergoing autologous stem cell transplantation: t(4;14) is the most relevant adverse prognostic factor, whereas RB deletion as a unique abnormality is not associated with adverse prognosis.Leukemia. 2007; 21: 143-150Crossref PubMed Scopus (163) Google Scholar have markedly changed the landscape of multiple myeloma (MM). Overall, MM is mainly divided into two major categories: hyperdiploid (H-MM; usually harboring multiple numerical chromosomal alterations) and nonhyperdiploid (NH-MM; typically enriched for IgH translocations),7Fonseca R. Bergsagel P.L. Drach J. Shaughnessy J. Gutierrez N. Stewart K. Morgan G. Van N.B. Chesi M. Minvielle S. Neri A. Barlogie B. Kuehl W.M. Liebisch P. Davies F. Chen-Kiang S. Durie B.G. Carrasco R. Sezer O. Reiman T. Pilarski L. vet-Loiseau H. International Myeloma Working Group molecular classification of multiple myeloma: spotlight review.Leukemia. 2009; 23: 2210-2221Crossref PubMed Scopus (738) Google Scholar the latter being associated with inferior survival.10Chng W.J. Santana-Davila R. Van Wier S.A. Ahmann G.J. Jalal S.M. Bergsagel P.L. Chesi M. Trendle M.C. Jacobus S. Blood E. Oken M.M. Henderson K. Kyle R.A. Gertz M.A. Lacy M.Q. Dispenzieri A. Greipp P.R. Fonseca R. Prognostic factors for hyperdiploid-myeloma: effects of chromosome 13 deletions and IgH translocations.Leukemia. 2006; 20: 807-813Crossref PubMed Scopus (120) Google Scholar, 11Decaux O. Lode L. Magrangeas F. Charbonnel C. Gouraud W. Jezequel P. Attal M. Harousseau J.L. Moreau P. Bataille R. Campion L. Avet-Loiseau H. Minvielle S. Prediction of survival in multiple myeloma based on gene expression profiles reveals cell cycle and chromosomal instability signatures in high-risk patients and hyperdiploid signatures in low-risk patients: a study of the Intergroupe Francophone du Myelome.J Clin Oncol. 2008; 26: 4798-4805Crossref PubMed Scopus (307) Google Scholar, 12Nair B. Shaughnessy Jr, J.D. Zhou Y. Astrid-Cartron M. Qu P. van Rhee F. Anaissie E. Alsayed Y. Waheed S. Hollmig K. Szymonifka J. Petty N. Hoering A. Barlogie B. Gene expression profiling of plasma cells at myeloma relapse from tandem transplantation trial Total Therapy 2 predicts subsequent survival.Blood. 2009; 113: 6572-6575Crossref PubMed Scopus (19) Google Scholar, 13Smadja N.V. Bastard C. Brigaudeau C. Leroux D. Fruchart C. Hypodiploidy is a major prognostic factor in multiple myeloma.Blood. 2001; 98: 2229-2238Crossref PubMed Scopus (349) Google Scholar However, in-depth analysis through microarray gene expression profiling (GEP) has shed significant light on the molecular heterogeneity of MM plasma cells (PCs), with up to 10 patient subgroups being identified.14Broyl A. Hose D. Lokhorst H. de Knegt Y. Peeters J. Jauch A. Bertsch U. Buijs A. Stevens-Kroef M. Beverloo H. Vellenga E. Zweegman S. Kersten M. van der Holt B. el Jarari L. Mulligan G. Goldschmidt H. van Duin M. Sonneveld P. Gene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients.Blood. 2010; 116: 2543-2553Crossref PubMed Scopus (241) Google Scholar, 15Zhan F. Huang Y. Colla S. Stewart J. Hanamura I. Gupta S. Epstein J. Yaccoby S. Sawyer J. Burington B. Anaissie E. Hollmig K. Pineda-Roman M. Tricot G. van Rhee F. Walker R. Zangari M. Crowley J. Barlogie B. Shaughnessy J.J. The molecular classification of multiple myeloma.Blood. 2006; 108: 2020-2028Crossref PubMed Scopus (893) Google Scholar Among these, the proliferation, MMSET, and MAF/MAFB subgroups encompass patients with high-risk disease and a dismal outcome.15Zhan F. Huang Y. Colla S. Stewart J. Hanamura I. Gupta S. Epstein J. Yaccoby S. Sawyer J. Burington B. Anaissie E. Hollmig K. Pineda-Roman M. Tricot G. van Rhee F. Walker R. Zangari M. Crowley J. Barlogie B. Shaughnessy J.J. The molecular classification of multiple myeloma.Blood. 2006; 108: 2020-2028Crossref PubMed Scopus (893) Google Scholar, 16Shaughnessy Jr, J.D. Zhan F. Burington B.E. Huang Y. Colla S. Hanamura I. Stewart J.P. Kordsmeier B. Randolph C. Williams D.R. Xiao Y. Xu H. Epstein J. Anaissie E. Krishna S.G. Cottler-Fox M. Hollmig K. Mohiuddin A. Pineda-Roman M. Tricot G. van Rhee F. Sawyer J. Alsayed Y. Walker R. Zangari M. Crowley J. Barlogie B. A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome 1.Blood. 2007; 109: 2276-2284Crossref PubMed Scopus (752) Google Scholar Since the MMSET molecular subgroup is intrinsically associated with t(4;14) and this abnormality can be systematically evaluated by fluorescence in situ hybridization (FISH) analysis, important data are emerging suggesting that the poor outcome of patients harboring t(4;14) may be partially overcome by some regimens.17Avet-Loiseau H. Leleu X. Roussel M. Moreau P. Guerin-Charbonnel C. Caillot D. Marit G. Benboubker L. Voillat L. Mathiot C. Kolb B. Macro M. Campion L. Wetterwald M. Stoppa A.M. Hulin C. Facon T. Attal M. Minvielle S. Harousseau J.L. Bortezomib plus dexamethasone induction improves outcome of patients with t(4;14) myeloma but not outcome of patients with del(17p).J Clin Oncol. 2011; 28: 4630-4634Crossref Scopus (0) Google Scholar, 18Cavo M. Tacchetti P. Patriarca F. Petrucci M.T. Pantani L. Galli M. Di Raimondo F. Crippa C. Zamagni E. Palumbo A. Offidani M. Corradini P. Narni F. Spadano A. Pescosta N. Deliliers G.L. Ledda A. Cellini C. Caravita T. Tosi P. Baccarani M. Bortezomib with thalidomide plus dexamethasone compared with thalidomide plus dexamethasone as induction therapy before, and consolidation therapy after, double autologous stem-cell transplantation in newly diagnosed multiple myeloma: a randomised phase 3 study.Lancet. 2011; 376: 2075-2085Abstract Full Text Full Text PDF Scopus (741) Google Scholar In turn, information on the remaining high-risk subgroups is limited. Assessment of the proliferation of the tumor cells through the PC labeling index (PCLI) is an important prognostic factor in MM,19Kumar S.K. Mikhael J.R. Buadi F.K. Dingli D. Dispenzieri A. Fonseca R. Gertz M.A. Greipp P.R. Hayman S.R. Kyle R.A. Lacy M.Q. Lust J.A. Reeder C.B. Roy V. Russell S.J. Short K.E. Stewart A.K. Witzig T.E. Zeldenrust S.R. Dalton R.J. Rajkumar S.V. Bergsagel P.L. Management of newly diagnosed symptomatic multiple myeloma: updated Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) consensus guidelines.Mayo Clin Proc. 2009; 84: 1095-1110Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar but its routine use has been hampered by the labor-intensive fluorescent microscope slide–based method required for its application.20Larsen J.T. Chee C.E. Lust J.A. Greipp P.R. Rajkumar S.V. Reduction in plasma cell proliferation after initial therapy in newly diagnosed multiple myeloma measures treatment response and predicts improved survival.Blood. 2011; 118: 2702-2707Crossref PubMed Scopus (18) Google Scholar Multiparameter flow cytometry (MFC) immunophenotyping is a widely available tool in routine laboratories. It provides not only rapid measurement of PC proliferation but also simultaneous assessment of PC total DNA content.21Kumar S. Kimlinger T. Morice W. Immunophenotyping in multiple myeloma and related plasma cell disorders.Best Pract Res Clin Haematol. 2010; 23: 433-451Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 22Paiva B. Almeida J. Perez-Andres M. Mateo G. Lopez A. Rasillo A. Vidriales M.B. Lopez-Berges M.C. Miguel J.F. Orfao A. Utility of flow cytometry immunophenotyping in multiple myeloma and other clonal plasma cell-related disorders.Cytometry B Clin Cytom. 2010; 78: 239-252PubMed Google Scholar Importantly, we recently observed in elderly patients with MM receiving novel agents during induction and maintenance that the poor prognosis of NH-MM was not abrogated,23Mateos M.V. Gutierrez N.C. Martin-Ramos M.L. Paiva B. Montalban M.A. Oriol A. Martinez-Lopez J. Teruel A.I. Bengoechea E. Martin A. Diaz-Mediavilla J. de Arriba F. Palomera L. Hernandez J.M. Sureda A. Bargay J. Penalver F.J. Ribera J.M. Martin-Mateos M.L. Fernandez M. Garcia-Sanz R. Vidriales M.B. Blade J. Lahuerta J.J. San Miguel J.F. Outcome according to cytogenetic abnormalities and DNA ploidy in myeloma patients receiving short induction with weekly bortezomib followed by maintenance.Blood. 2011; 118: 4547-4553Crossref PubMed Scopus (49) Google Scholar but whether survival of transplant-eligible patients with NH-MM and high PC proliferation is improved by the incorporation of novel agents up front remains largely unknown.24Kapoor P. Kumar S. Mandrekar S.J. Laumann K.M. Dispenzieri A. Lacy M.Q. Dingli D. Gertz M.A. Kyle R.A. Greipp P.R. Rajkumar S.V. Witzig T.E. Efficacy of thalidomide- or lenalidomide-based therapy in proliferative multiple myeloma.Leukemia. 2011; 25: 1195-1197Crossref PubMed Scopus (8) Google Scholar, 25Hose D. Rème T. Hielscher T. Moreaux J. Messner T. Seckinger A. Benner A. Shaughnessy J.D. Barlogie B. Zhou Y. Hillengass J. Bertsch U. Neben K. Möhler T. Rossi J.F. Jauch A. Klein B. Goldschmidt H. Proliferation is a central independent prognostic factor and target for personalized and risk-adapted treatment in multiple myeloma.Haematologica. 2011; 96: 87-95Crossref PubMed Scopus (157) Google Scholar In the present study, we applied MFC to analyze the DNA content and proliferation index of myelomatous PCs from a series of 595 newly diagnosed transplant-eligible patients with MM. These results show that the PC DNA content and proliferation status are independent prognostic factors in MM and that novel agents–based regimens cannot overcome the poor prognosis of NH-MM but rather prolong survival of patients with MM and high proliferation indices. In addition, we investigated which of the commonly assessed cytogenetic abnormalities drives PC proliferation. Finally, we assessed whether there is a difference in individual patients in the proliferative rate of PCs between diagnosis and disease progression. The study included 595 patients with MM diagnosed according to the International Myeloma Working Group criteria.26Kyle R.A. Rajkumar S.V. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma.Leukemia. 2009; 23: 3-9Crossref PubMed Scopus (952) Google Scholar Patients were included in two consecutive PETHEMA (Programa para el Estudio de la Terapéutica en Hemopatías Malignas)/GEM (Grupo Español de MM) trials: GEM2000 [VBMCP/VBAD (vincristine, carmustine, melphalan, cyclophosphamide, prednisone/vincristine, bischloroethylnitrosourea, adriamycin, and dexamethasone) followed by HDT/ASCT and 2 years of maintenance with interferon plus prednisone; n = 319] and GEM2005 65 years, levels of serum calcium >14 mg/dL and/or serum creatinine >2 mg/dL were excluded from the analysis to avoid confounding survival bias. Median follow-up was 38 months (range, 1 to 123 months). All the samples were collected after informed consent was given, and the study was approved by the clinical research ethical committee. Simultaneous staining for DNA (with propidium iodide) and PC surface antigens (CD38 and CD138) was performed as described elsewhere.27Orfao A. Garcia-Sanz R. Lopez-Berges M.C. Belen Vidriales M. Gonzalez M. Caballero M.D. San Miguel J.F. A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique.Cytometry. 1994; 17: 332-339Crossref PubMed Scopus (74) Google Scholar A minimum of 2 × 106 nucleated bone marrow (BM) cells per case were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) using FACSDiva software version 6.0 (BD Biosciences). Total PC DNA content was calculated by dividing the median fluorescence channel of the G0/G1 peak of CD38/CD138hi myelomatous PCs by the median fluorescence channel of G0/G1 residual normal BM cells present in the sample. Patients were considered to be hyperdiploid (H-MM) when the PC DNA content ranged from 1.06 to 1.74; nonhyperdiploid (NH-MM) cases included patients with a PC DNA content 1.74 (tetraploid/near-tetraploid), and ranging from 0.95 to 1.05 (diploid).23Mateos M.V. Gutierrez N.C. Martin-Ramos M.L. Paiva B. Montalban M.A. Oriol A. Martinez-Lopez J. Teruel A.I. Bengoechea E. Martin A. Diaz-Mediavilla J. de Arriba F. Palomera L. Hernandez J.M. Sureda A. Bargay J. Penalver F.J. Ribera J.M. Martin-Mateos M.L. Fernandez M. Garcia-Sanz R. Vidriales M.B. Blade J. Lahuerta J.J. San Miguel J.F. Outcome according to cytogenetic abnormalities and DNA ploidy in myeloma patients receiving short induction with weekly bortezomib followed by maintenance.Blood. 2011; 118: 4547-4553Crossref PubMed Scopus (49) Google Scholar The distribution of myelomatous PCs in the G0/G1, S-phase, and G2/M cell-cycle phases was assessed according to well-established methods28San Miguel J.F. Garcia-Sanz R. Gonzalez M. Moro M.J. Hernandez J.M. Ortega F. Borrego D. Carnero M. Casanova F. Jimenez R. A new staging system for multiple myeloma based on the number of S-phase plasma cells.Blood. 1995; 85: 448-455Crossref PubMed Google Scholar after specifically selecting CD38/CD138hi PCs and excluding debris and cell doublets. The PC proliferation index was calculated as the percentage of PCs in S-phase in the whole BM PC compartment. FISH was performed at baseline in immunomagnetically enriched PCs from 325 patients. Patients harboring t(4;14), t(14;16), and/or del(17p13) were classified as having high-risk disease and all other cases as standard risk, according to the International Myeloma Working Group guidelines.7Fonseca R. Bergsagel P.L. Drach J. Shaughnessy J. Gutierrez N. Stewart K. Morgan G. Van N.B. Chesi M. Minvielle S. Neri A. Barlogie B. Kuehl W.M. Liebisch P. Davies F. Chen-Kiang S. Durie B.G. Carrasco R. Sezer O. Reiman T. Pilarski L. vet-Loiseau H. International Myeloma Working Group molecular classification of multiple myeloma: spotlight review.Leukemia. 2009; 23: 2210-2221Crossref PubMed Scopus (738) Google Scholar The Mann-Whitney U-test was used to estimate the statistical significance of differences between groups. Progression-free survival (PFS) and overall survival (OS) curves were plotted using the Kaplan-Meier method, and the log-rank test was used to estimate the statistical significance of differences observed between curves. A univariate analysis was conducted to assess the impact of various baseline prognostic factors on PFS and OS (Table 1). The Cox proportional hazards regression model (stepwise regression) was used in a multivariate analysis of PFS and OS, retaining those variables with a significant predictive value (P ≤ 0.05) in the predictive model. For all the statistical analyses, SPSS software version 15.0 (SPSS Inc., Chicago, IL) was used.Table 1Baseline Disease Features with a Significant Effect on PFS and/or OS (Univariate and Multivariate Analyses)FeatureUnivariate analysisMultivariate analysisPFSOSPFSOSMedian (months)P valueMedian (months)P valueHRP valueHRP valueISS disease stage1.20.341.90.02 I48<0.001NR<0.001 II3564 III2546Age (years)——0.60.084 10045<0.00193 3.52861LDH (IU/L)2.00.0042.20.009 Normal43<0.00180<0.001 Increased2446BM PCs (%)1.50.07—— <30480.004790.15 ≥303475BM PCs by MFC (%)1.60.021.40.14 <1547 553<0.001780.04 ≤53876% PCs in S-phase1.9<0.0012.00.003 <143<0.001930.001 ≥13466DNA ploidy status by MFC1.20.211.70.02 Hyperdiploid440.004840.005 Nonhyperdiploid3467Interphase FISH cytogenetics1.60.012.10.003 Standard risk44<0.00180<0.001 High risk⁎High-risk cytogenetics includes any t(4;14), t(14;16), and del(17p); standard-risk cytogenetics includes all other cases.2341HR, hazard ratio; ISS, International Staging System; NR, not reported; —, not tested. High-risk cytogenetics includes any t(4;14), t(14;16), and del(17p); standard-risk cytogenetics includes all other cases. Open table in a new tab HR, hazard ratio; ISS, International Staging System; NR, not reported; —, not tested. Of the 595 patients included in the present study, 295 were classified as having NH-MM (49.6%) and the remaining 300 as having H-MM (50.4%). As expected, patients with NH-MM showed an increased frequency of t(4;14) (20% versus 9%, P = 0.008), t(11;14) (28% versus 5%; P < 0.001), t(14;16) (5% versus 1%; P = 0.055), and del(13q14) (50% versus 33%, P = 0.002) but not del(17p13) (5% versus 7%, P = 0.37). Moreover, NH-MM showed more frequently advanced disease (International Staging System stage III, 21% versus 13%; P = 0.03) and higher BM PC infiltration by MFC (16% versus 9%; P = 0.008). As expected, patients with NH-MM had significantly inferior median PFS (34 versus 44 months; P = 0.004; Figure 1A) and OS (67 versus 84 months; P = 0.005; Figure 1B) than those with H-MM. We detected the presence of two different clones of myelomatous PCs (with different DNA content) in 34 of the 595 patients (5.7%) by MFC (Figure 2), and although the number is small, this subgroup had a dismal outcome compared with patients in whom only one PC clone was detected (median PFS and OS of 26 and 52 months, respectively; P ≤ 0.005). After this analysis in the overall MM population, we specifically looked at whether the outcome of NH-MM versus H-MM differed according to the trial: GEM2000 and GEM2005<65y. As occurred in the overall series, patients with NH-MM included in the GEM2000 trial had a significantly inferior PFS (34 versus 42 months, P = 0.04; Figure 3A) and a trend toward decreased OS (63 versus 79 months, P = 0.11; Figure 3B). Likewise, significant differences emerged for PFS (40 months versus not reached; P = 0.03; Figure 3C) and OS (both medians not reached; P = 0.004; Figure 3D) for patients included in the GEM2005<65y trial according to the NH-MM versus H-MM classification.Figure 2Bivariate dot plot histograms illustrating the detection of two different PC clones (with different DNA content) by MFC in the BM of three newly diagnosed patients with myeloma. A represents a case with diploid and tetraploid PC clones, whereas on B and C, two hyperdiploid PC clones coexist.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3PFS and OS of patients with symptomatic MM treated with HDT/ASCT grouped according to the presence of H-MM and NH-MM PC DNA content assessed by MFC at diagnosis. A and B: PFS and OS of patients with MM included in the GEM2000 protocol (n = 319). C and D: PFS and OS of patients with MM included in the GEM2005<65y trial (n = 276).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The median percentage of PCs in S-phase in the whole series was 1.14% (range, 0% to 13%). When we looked for a specific association between the percentage of PCs in S-phase and other baseline parameters, no significant correlations were found (data not shown), except for an inferior proliferation index in patients with NH-MM versus those with H-MM (1.00% versus 1.23%; P = 0.008). We then investigated the potential association between specific cytogenetic alterations and PC proliferation. Patients harboring t(11;14) showed a significantly decreased percentage of PCs in S-phase (0.7% versus 1.2%, P < 0.001), but no significant differences were recorded for t(4;14), t(14;16), del(13q14), and del(17p13). Accordingly, the presence or absence of high-risk cytogenetics was not associated with different PC proliferation, irrespective of whether we looked in all patients (1.12% versus 1.16%, respectively; P = 0.33) or specifically in patients with NH-MM versus H-MM (see Supplemental Table S1 at http://ajp.amjpathol.org). In accordance with the median proliferation index of the whole series (1.14%), patients were stratified using a cutoff point of ≥1% versus <1% S-phase PCs, which translated into a significantly different PFS (median of 34 versus 43 months; P < 0.001) and OS (median of 66 versus 93 months; P = 0.001). Moreover, we confirmed that the higher the percentage of S-phase PCs, the worse the associated survival. In particular, a proliferation index ≥3% identified a subgroup of patients (92 of 595, 15.5%) with high-risk disease (median PFS and OS of 22 and 45 months, respectively; P < 0.001; Figure 4). In the group of patients with ≥1% S-phase PCs (n = 336), the complete response rate was slightly higher than that in the remaining patients (45% versus 36%, P = 0.053); however, the duration of the complete response tended to be shorter (PFS at 3 years: 65% versus 80%; P = 0.057). Thereafter, we explored whether the poor prognosis of patients with a high proliferation index (≥1% PCs in S-phase) could be abrogated in the most recent GEM2005<65y trial. The results show that while for patients included in the GEM2000 trial patient stratification into ≥1% versus <1% S-phase PCs predicted for different PFS (33 versus 43 months; P = 0.003; Figure 5A) and OS (61 versus 93 months; P < 0.001; Figure 5B), patients with ≥1% S-phase PCs receiving novel agents up front still showed only slightly inferior PFS (40 months versus not reached; P = 0.09; Figure 5C) and similar OS (both medians not reached; P = 0.99; Figure 5D) compared with patients with <1% PCs in S-phase. Accordingly, it seems that treatment with novel agents overcomes the adverse prognosis of a high proliferation index (≥1% S-phase PCs); however, this applied only to patients treated with either VTD or VBMCP/VBAD/bortezomib (median PFS of 40 and 44 months, respectively) but not to patients who received TD (PFS of 23 months; P = 0.03).Figure 5PFS and OS of patients with symptomatic MM submitted to HDT/ASCT grouped according to the presence of <1% and ≥1% S-phase PCs as assessed by MFC at diagnosis. A and B: PFS and OS of patients with MM included in the GEM2000 protocol (n = 319). C and D: PFS and OS of patients with MM included in the GEM2005 5%) at baseline by MFC, and high-risk cytogenetics. For OS, all the same factors except BM PC infiltration by morphology retained their prognostic value, in addition to patient age. In the multivariate analysis (Table 1), the presence of high-risk cytogenetics, increased lactate dehydrogenase level, ≥1% S-phase PCs, >15% PCs by MFC, and >5% normal PCs in the BM PC compartment remained as inde

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