Original Article

Leukemia (2007) 21, 143–150 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE 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 NC Gutie´rrez1,13, MV Castellanos1,13, ML Martıń2, MV Mateos1, JM Hernańdez1, M Fernańdez2, D Carrera3, L Rosinõl4, JM Ribera5, JM Ojanguren6, L Palomera7, S Gardella8, L Escoda9, JC Hernańdez-Boluda10, JL Bello11, J de la Rubia12, JJ Lahuerta2 and JF San Miguel1, on behalf of GEM/PETHEMA Spanish Group 1Servicios de Hematologıá: Hospital Universitario de Salamanca and Centro de Investigacioń del Cańcer (CIC), Universidad de Salamanca-CSIC, Spain; 2Hospital 12 de Octubre, Madrid, Spain; 3Hospital Central de Asturias, Oviedo, Spain; 4Hospital Clıńic Universitari, IDIBAPS, Barcelona, Spain; 5Hospital German Trias i Pujol, Badalona, Spain; 6Hospital de Galdakao, Bilbao, Spain; 7Hospital Clıńico, Zaragoza, Spain; 8Hospital Dr Josep Trueta, Gerona, Spain; 9Hospital Joan XXIII, Tarragona, Spain; 10Hospital Clıńico Universitario, Valencia, Spain; 11Hospital Universitario, Santiago de Compostela and 12Hospital Universitario La Fe, Valencia, Spain Fluorescence in situ hybridization (FISH) has become a power- most important in order to obtain individualized information ful technique for prognostic assessment in multiple myeloma about disease outcome and to design risk-adapted therapeutic (MM). However, the existence of associations between cytoge- strategies. The cytogenetic status have emerged as the most netic abnormalities compels us to re-assess the value of each 2–5 abnormality. A total of 260 patients with MM at the time of relevant prognostic factor in MM. However, the low diagnosis, enrolled in the GEM-2000 Spanish transplant proto- proliferative activity of PC, as well as the limited extent of bone col, have been analyzed by FISH in order to ascertain the marrow (BM) involvement, reduce the number of analyzable independent influence on myeloma prognosis of IGH transloca- metaphases and hamper cytogenetic studies. In addition, the tions, as well as RB and P53 deletions. Survival analyses resolution of conventional cytogenetics makes it impossible to showed that patients with t(4;14), RB or P53 deletions had a recognize cryptic translocations.6–8 These factors are leading to significantly shorter survival than patients without these abnormalities. However, patients with RB deletions without the replacement of classical cytogenetics by interphase fluores- other abnormalities in FISH analysis, displayed a similar cence in situ hybridization (FISH) technology, which allows a outcome to those patients without genetic changes by FISH rapid and reproducible identification of specific target regions (46 vs 54 months, P ¼ 0.3). In the multivariate analysis the frequently affected in MM and with prognostic influence. presence of t(4;14), RB deletion associated with other abnorm- Several groups, including our own, have investigated by alities, age 460 years, high proportion of S-phase cells and FISH the cytogenetic abnormalities most frequently involved in advanced stage of the disease according to the International 3,4,9,10 Staging System retained their independent prognostic influ- MM. Immunoglobulin heavy-chain (IGH ) translocations, ence. In summary, RB deletion as a sole abnormality does not as well as retinoblastoma (RB) and P53 deletions represent lead to a shortening in the survival of MM patients, whereas chromosomal abnormalities with a widely recognized prognos- t(4;14) confers the worst prognosis in MM patients treated with tic impact. Although RB deletions have been considered as a high-dose chemotherapy. powerful adverse prognostic factor consistently reported in Leukemia (2007) 21, 143–150. doi:10.1038/sj.leu.2404413; large series,3–5,11,12 the coexistence of RB deletions and IGH published online 5 October 2006 translocations raises the question of whether the adverse Keywords: multiple myeloma; genetic abnormalities; FISH; RB deletion prognosis of each abnormality may be influenced by the other. In order to ascertain the individual contribution of each abnormality as well as the influence of associations between abnormalities in MM outcome, we have systematically analyzed by FISH RB and P53 deletions, and IGH translocations in 260 Introduction patients uniformly treated according to the GEM 2000 protocol, which includes an induction phase with VBCMP/VBAD Multiple myeloma (MM) is a clonal plasma cell (PC) disorder followed by autologous cell transplantation (ASCT). In addition, that remains as an incurable disease. Nevertheless, the survival we have explored whether or not these cytogenetic subgroups of myeloma patients is highly variable, ranging from a few display distinct clinical and biological disease characteristics. months to more than 10 years. This heterogeneity relates mainly to prognostic factors associated with specific characteristics of 1 both the tumor itself and the host. The identification of those Patients and methods characteristics associated with either a good or poor prognosis is Patients Correspondence: Professor JF San Miguel, Servicio de Hematologıá, Patients under the age of 70 years, with newly diagnosed MM, Hospital Universitario de Salamanca, Paseo San Vicente 58-182, enrolled in the GEM 2000 Spanish protocol (six alternating Salamanca 37007, Spain. cycles of VBCMP/VBAD followed by high-dose therapy – E-mail: [email protected] 2 13These authors contributed equally to this work melphalan 200 mg/m supported by ASCT) were included in Received 5 July 2006; revised 25 August 2006; accepted 30 August the study. The study was approved by the research ethics 2006; published online 5 October 2006 committees of all participating centers and written informed Genetic abnormalities in multiple myeloma NC Gutie´rrez et al 144 Table 1 Clinical and biological characteristics of MM patients analyzed using the scoring criteria recommended by the manufacturer. Based on the results using these probes in 25 Characteristics All patients healthy controls, the cutoff point for the identification of alteration was set at more than 8% cells with abnormal signal. Sex Male 55% Female 45% Statistical analysis Statistical analysis were performed using SPSS statistical soft- ISS 2 I 28% ware version 11.5 (SPSS Inc., Chicago, IL, USA). The w and the II 48% Fisher’s exact test were used to test associations between III 24% chromosomal abnormalities as well as between genomic changes and other categoric variables. For continuous variables, Ig subtype the Wilcoxon rank sum test and t-test were used. OS was IgG 52% calculated from the start of the initial treatment to the date of IgA 28% IgD 0.4% death or last visit. Time to progression (TTP) was estimated from Bence Jones protein 18% the day of initiation of treatment to the date of relapse or disease Non-secretory 1.2% progression. Survival curves were plotted by means of the Kaplan–Meier method and the difference in survival curves was ASCT (%) tested for statistical significance using the log-rank test. P-values Yes 82% below 0.05 were considered to reflect statistical significance. No 18% Multivariate analysis of survival was performed using the Cox Age (years) 60 (39–70) proportional hazards model (stepwise regression approach). b2-microglobulin (mg/l) 3.6 (0.2–28.7) Factors were retained in the model if they were statistically Hemoglobin (g/dl) 10.4 (4–15.5) significant at Pp0.05. Creatinine (mg/dl) 1.1 (0.3–13.3) Calcium (mg/dl) 9.6 (6.7–16.1) Albumin (mg/dl) 3.5 (1–5.2) Results C-reactive protein (mg/dl) 1.07 (0–25.5) S-phase plasma cells (%) 1.5 (0.2–14.6) Frequency of chromosomal abnormalities Abbreviations: ASCT, autologous stem cell transplantation; Ig, Chromosomal abnormalities explored by FISH were identified in immunoglobulin; ISS, International staging system; MM, multiple 151 (58%) of the 260 MM patients. IGH translocations and RB myeloma. Values are expressed as median (range). deletions were observed in 95 (36%) and 109 (42%) out of the 260 patients, respectively; whereas P53 deletions were present in 8.5% (22/260) of patients. The distribution of IGH translocations according to 14q32 partners were: t(11;14) in 13% (34/260 consent was obtained from all patients. In order to interpret patients), t(4;14) in 11% (29/260 patients), t(14;16) in 3% (7/260 accurately FISH analysis only those patients with BM PC patients) and IGH rearrangements with other unknown partners infiltration by flow cytometry above 10% were eligible for this in 10% (25/260 patients). study (n ¼ 260). The main clinical and laboratory characteristics of these patients are shown in Table 1. Forty-seven of 260 patients (18%) did not undergo ASCT because of comorbidity Correlations between chromosomal abnormalities (20 cases), progression of the disease (13 cases), failure in A significant association between t(4;14) and RB deletions mobilization (11 cases) or withdrawal of informed consent was observed. Thus, 79% of patients with t(4;14) had RB (three cases). The median overall survival (OS) for the whole deletions vs 37% of patients without t(4;14) (Po0.001). group was 43 months (95% confidence interval, 36–49), and the However, no correlation was found between this translocation median follow-up for survivors was 34 months. At the time of and P53 deletions (Table 2). In contrast, t(11;14) was signifi- study, 102 patients remained alive. cantly associated with P53 deletions but not with RB deletions (Table 2). Translocations involving 16q and other unknown IGH partners did not correlate with RB or P53 deletions. Finally, a FISH analysis significant association was observed between P53 and RB Interphase FISH studies for the detection of IGH rearrangements deletions (P ¼ 0.009) (Table 2). were carried out by means of LSI IGH dual color, break apart rearrangement probe (Vysis, Downers Grove, IL, USA).

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