To identify a correlation between metaphase cytogenetics and relapse after reduced intensity conditioning (RIC) allotransplant for patients with multiple myeloma, data on 60 patients (median age 52) who received grafts from a sibling (n=49) or unrelated donor (n=11) were analyzed. Fifty-three patients (88%) showed chromosomal abnormalities (CA) before the allotransplant, including 42 with abnormalities involving 13q (CA13). Twenty-two patients (41%) relapsed post-allotransplant at a median of 165 days. Of these, 11 patients showed abnormal cytogenetics at the time of post-allotransplant relapse at a median of 167 days. Of 54 patients who developed graft-versus-host disease, relapse occurred in 19 of 48 patients (43%) with CA present before RCI allotransplant, versus 1 of 6 without CA (17%) (P=0.06). Loss of CA before RIC allotransplant and disease status>PR after RIC allotransplant were significantly associated with a lower risk of post-allotransplant relapse with cytogenetic abnormalities; 5.2 vs 36%, and 18 vs 53%, (both P<0.05), respectively. The current data suggests that myeloma associated with persistent clonal cytogenetic abnormalities is an entity which most likely escapes the effects of a graft versus myeloma activity, maybe because of acquisition of resistance to immunologic manipulations.
Progress in allogeneic transplantation for patients with multiple myeloma (MM) has been impeded by its high transplant-related mortality.1, 2 The majority of early deaths following myeloablative preparation has been due to bacterial and fungal infections and interstitial pneumonia.3 Although the TRM seems to have decreased with the advent of effective antimicrobial agents and supportive care, a significant decrease in early mortality occurred only after the introduction of reduced intensity conditioning or nonmyeloablative preparation, omitting intense cytoreduction of myeloma with the main purpose of establishing donor engraftment for a graft versus myeloma effect.4, 5, 6, 7 The 1-year TRM now is <20%, with a consequent increase in a 2-year overall survival to>70%.5 Despite this apparent improvement, the benefits in survival after allotransplant following reduced intensity conditioning are not fully established at present. Follow-up has been short, compared with tandem autotransplants, although the latter still has a high-relapse rate.8, 9 The decrease in TRM following nonmyeloablative allotransplantation, although significantly improved, should be compared with 1–3% of the TRM after autotransplants.1, 8, 10 Relapse can occur even in the presence of chronic graft versus host disease (GVHD), and survival depends not only on development of graft versus myeloma effect, but also on pre-allotransplant characteristics and lack of sufficient tumor control post allotransplantation following a reduced intensity conditioning (RIC) regimen.4, 5, 6 Although some relapsing patients in our study responded to further donor lymphocyte infusions or salvage therapy, the responses were not durable and all patients showed disease progression subsequently. Furthermore, some patients demonstrated progressive cytogenetic changes coinciding with relapse. In this study, we investigated cases of relapse in relation to cytogenetic findings and other well-known prognostic factors to assess the impact of metaphase cytogenetics on disease control after allotransplantation.
Patients and methods
Patients and preparatory regimens
The analysis was performed on 60 consecutive patients with symptomatic multiple myeloma, who completed an allotransplant study following nonmyeloablative conditioning between June 1998 and March 2004 at the Myeloma Institute for Research and Therapy, the University of Arkansas for Medical Sciences. The Institutional Review Board of the University of Arkansas for Medical Sciences approved the treatment protocol and an informed consent was obtained from all patients and donors. The outcome data of the 45 patients, and study design have previously been reported.6, 11 Melphalan 100 mg/m2 was employed for conditioning for sibling allotransplants, and the same dose of melphalan with total body irradiation 200 cGy and fludarabine 60 mg/m2 for unrelated donor transplants.
Antibacterial, antifungal, and antiviral prophylaxes were administered according to institutional protocols, including sulfamethoxazole-trimethoprim and acyclovir up to 6 months post transplant. Itraconazole 400 mg/day was started before the transplant and continued until the CD4+ count exceeded 400/μl. Patients at high risk of fungal infection and those unable to tolerate oral itraconazole received intravenous liposomal amphotericin-B 1 mg/kg/day. Cytomegalovirus antigenemia was assessed weekly, and ganciclovir/foscarnet was used for those with a positive test (⩾2 positive cells/slide) as a pre-emptive therapy. For prevention of GVHD, intravenous cyclosporine 3 mg/kg/day was started on day –1 and subsequently changed to oral dose when tolerated to maintain whole blood trough levels at 300 ng/ml. Patients receiving unrelated allograft were given additional methylprednisolone 1 mg/kg/day, starting on day 5, with subsequent tapering and discontinuation by day 29 if there was no evidence of GVHD. Combination of cyclosporine with corticosteroid was given for initial therapy of acute GVHD of grade⩾II and chronic GVHD.
Patients were evaluated for cytogenetic findings, response, relapse and death. Estimation of survival, criteria for response, relapse, death, GVHD and TRM were previously described.6 After allotransplant, random bone marrow biopsies and aspirations were followed every 1–3 months for donor chimerism and cytogenetics by conventional G-banding technique and multicolor spectral karyotyping.12 At least 20 cells were evaluated and chromosomal changes were designated as clonal if complete deletion of particular chromosome(s) was seen in ⩾3 cells, and all other changes in ⩾2 cells. Pre-RIC allotransplant factors analyzed included relapse, presence of CA, achievement of ⩾PR, and loss of CA before RIC allotransplant; disease status of CR+nCR (near complete remission: normal bone marrow findings and positive only for serum or urine immunofixation electrophoresis) and development of acute and chronic GVHD for post-RIC allotransplant factors. Categorical variables were analyzed by the frequency table with Fisher's exact or Pearson's χ2 test. Cumulative incidence curves were generated to calculate the probability of GVHD, TRM, responses and relapse.13 Survival curves were estimated using the product limit method of Kaplan–Meier method and the log-rank test was applied for comparison.14 Cox proportional hazards regression was used to assess the influence of pre- and post-RIC allotransplant factors on relapse.15 For multivariate analyses, variables with a P of <0.1 in univariate analysis were entered into a stepwise multivariate regression by Cox proportional hazard model.
Patient and disease characteristics (Table 1)
Forty-nine patients received a sibling, and 11 an unrelated donor graft. All patients had at least one preceding autotransplant. Fifty-three patients (88%) had unfavorable CA, consisting of CA13 in 70% and hypodiploidy in 42%. Twenty-six patients (43%) underwent an RIC allotransplant without preceding relapse but with high-risk myeloma based on cytogenetics (three CR, four nCR, 10 PR, nine improvement), and 34 (57%) had ⩾one relapse(s) before the RIC allotransplant. Twenty-seven patients (45%) were at least in partial remission at the time of RIC allotransplant, including 12 patients in CR (n=5) and nCR (n=7) before RIC allotransplant, and 33 (55%) in less than PR.
Responses and survival
Seven patients died of transplant-related causes before day 100. Of 53 evaluable patients for response, 36 patients (60%) achieved CR (n=18) and nCR (n=18) following an RIC allotransplant, including 12 patients already in CR (n=5) or nCR (n=7) before the allotransplant. Eleven patients (18%) attained a PR, and six (10%) had progressive disease. The majority of responses occurred within 1 year post transplant, with a median time to maximal response of 132 days for patients not in CR before the allotransplant (range, 14–427 days).
Median overall survival (OS) of all patients from RIC allotransplant was 9 months and the estimated 5-year OS probability was 34% (Figure 1a). The median follow-up time for surviving patients was 45 months. Median event-free survival (EFS) was 8 months for all patients, with an estimated 5-year EFS probability of 26%. Nineteen patients (34%) died of transplant-related causes at a median of 119 days (range, 19–351 days). The causes of death included GVHD (n=7), invasive fungal infections (n=5), other infections (n=4), suicide (n=1), heart failure (n=1), and idiopathic pneumonia (n=1).
Twenty-two patients relapsed, with a 5-year cumulative incidence of 41% (Figure 1b). Most relapses occurred within 2 years, with a median time to relapse of 168 days (range, 37–789 days). Eight patients received DLI for relapse; six in combination with chemotherapy, and two without additional chemotherapy. Although 5 of these patients achieved ⩾PR at a median of 57 days (range, 22–143 days) after DLI, all patients subsequently showed progressive disease at a median of 65 days (range, 29–540 days).
Of 53 patients evaluable for relapse, five (20%) of the 25 patients who received RIC allotransplant as a planned consolidation therapy after a single autotransplant relapsed, when compared with 17 (61%) of the 28 who had ⩾one relapse before RIC allotransplant (P<0.001). Patients with <PR before RIC allotransplant more frequently relapsed: 16 (61%) of 26 versus six (22%) of 27 (P<0.005). Ten (29%) of 36 patients who were in CR+nCR, after RIC allotransplant relapsed when compared with 12 (71%) of 17 in ⩽PR post RIC allotransplant (P=0.001). Of 50 evaluable patients, 39 (78%) developed chronic GVHD, with 12 (31%) having limited and 29 (48%) extensive disease. Presence of chronic GVHD decreased the probability of relapse, but this did not reach statistical significance; 16 (41%) of 39 versus six (55%) of 11 without chronic GVHD (P=0.07). In this group of patients with chronic GVHD, the number of those who relapsed was evenly divided by CA (n=8) versus no CA (n=8). In multivariate analysis for relapse, both the absence of relapse before RIC allotransplant and achievement of CR+nCR post-RIC allotransplant were found to be significant, with a hazard ratio (P value) of 1.2 (0.03), and 1.1 (0.02), respectively (Table 2).
Follow-up cytogenetic evaluations
Of the 53 patients initially presenting with CA, 21 patients had normal cytogenetics on a random marrow just before RIC allotransplant, and the remaining 32 continued to show cytogenetic abnormalities. Of these, 51 (96%) patients achieved full donor chimerism at a median of 74 days (range 28–156 days). At 1 month post-RIC allotransplant, when 31 patients (56%) achieved full donor chimerism, all patients evaluated showed normal cytogenetic findings on bone marrow evaluations, except for two. In this group, 46 patients were evaluable for relapse, excluding seven who died early of nonrelapse causes. Eleven patients subsequently developed CA with additional abnormalities at relapse, at a median of 167 days (range, 37–350 days). Of the 18 evaluable patients whose CA disappeared before RIC allotransplant, only one (5.2%) relapsed with CA, compared to 10 (36%) of the 28 evaluable patients with persistent CA (P<0.05) (Figure 2). Relapse without CA occurred later (median 221; range, 62–789 days) (P=0.08).
Except for the normalization of the cytogenetic abnormalities before RIC allotransplant, none of the pre-allotransplant factors were significant for the presence of cytogenetic abnormalities at post-allotransplantation relapse. Of the 33 patients in <PR before RIC allotransplant, relapse with CA was seen in seven, compared to nine without CA (P=0.32). Nine of the 34 patients who had ⩾ one relapse before RIC allotransplant relapsed with CA, whereas 8 relapsed without CA (P=0.5). The majority of patients relapsing post allotransplant with CA (n=9; 53%) were those who did not achieve a CR or nCR post-RIC allotransplant, with only three (18%) of these relapsing without CA (P<0.05).
The mechanisms of relapse of myeloma patients after allotransplant have not been well recognized. Although initial results following allotransplant show that survival benefits have been associated with the graft-versus-myeloma effects, most commonly associated with development of GVHD, the current study suggests that GVHD for high-risk patients especially those with cytogenetic abnormalities may not be as beneficial for prevention of relapse as initially anticipated.4, 5, 6, 16, 17 We hypothesize that introduction of an allograft by reduced intensity conditioning alone may not provide sufficient disease control in patients with persistent cytogenetic abnormalities before allotransplantation. In our study, relapse occurred earlier when cytogenetic abnormalities were present before allotransplantation while the loss of such abnormalities before allotransplant was associated with a delay in relapse. Further additional cytogenetic changes emerging at the time of relapse suggest that there may be clonal escape from chemo-/immunotherapy of these myeloma cells. Among other potential mechanisms, lack of graft versus myeloma effect might be related to resistance of the myeloma cells to immune-mediated apoptosis associated with pathways involving Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).18, 19
Chromosomal abnormalities by metaphase analysis are not uncommon in patients with myeloma, and hypodiploidy and chromosome 13 abnormalities in particular have been associated with a high risk of relapse and poor survival.9, 20, 21, 22, 23 In an analysis of the 1000 patients treated with tandem autotransplants, chromosome 13 abnormalities, present in 16% of all patients, reduced the 5-year event-free survival from 20–0% and overall survival from 44–16% (both P<0.0001).21 In a study of 208 newly diagnosed patients, hypodiploidy was associated with a short median overall survival of <13 months compared with 34 months of those without such abnormalities.22 The worst survival was noted in a group of patients with both chromosome 13 abnormalities and a hypodiploid karyotype, with the median event-free and overall survival of 12 and 17 months following tandem autotransplants, respectively.23 Similarly, in a retrospective multicenter study on 68 patients who underwent RIC allotransplant, patients with FISH deletion of chromosome 13 had a 2-year event-free survival of 18%, compared with 42% of those without such deletion mainly due to a higher relapse rate (77 vs 44%).24 The high rate of relapse in these studies and the current study indicate that we are yet unable to overcome the adverse effect of CA by either tandem autotransplant or by an autotransplant followed by an RIC allograft. Although further investigations are necessary to confirm the current findings as the present study is limited due to its small size and retrospective nature of analysis, and the use of metaphase cytogenetics, these patients may either require novel conditioning regimens focusing on specific molecular events associated with abnormal chromosomes or a more intense conditioning regimen before allotransplantation, combined with manipulations, allowing more rapid recovery of the immune system to provide killing of resistant myeloma cells by cytotoxic allogeneic effector cells.25
Bensinger W, Maloney D, Storb R . Allogeneic hematopoietic cell transplantation for multiple myeloma. Semin Hematol 2001; 38: 243–249.
Barlogie B, Shaughnessy J, Tricot G, Jacobson J, Zangari M, Anaissie E et al. Treatment of multiple myeloma. Blood 2004; 103: 20–32.
Gahrton G, Svensson H, Cavo M, Apperly J, Bacigalupo A, Bjorkstrand B et al. Progress in allogenic bone marrow and peripheral blood stem cell transplantation for multiple myeloma: a comparison between transplants performed 1983–93 and 1994–8 at European Group for Blood and Marrow Transplantation centres. Br J Haematol 2001; 113: 209–216.
Crawley C, Lalancette M, Szydlo R, Gilleece M, Peggs K, Mackinnon S et al. Outcomes for reduced intensity allogeneic transplantation for multiple myeloma: an analysis of prognostic factors from the chronic leukemia working party of the EBMT. Blood 2005; 105: 4532–4539.
Kroger N, Perez-Simon JA, Myint H, Klingemann H, Shimoni A, Nagler A et al. Relapse to prior autograft and chronic graft-versus-host disease are the strongest prognostic factors for outcome of melphalan/fludarabine-based dose-reduced allogeneic stem cell transplantation in patients with multiple myeloma. Biol Blood Marrow Transplant 2004; 10: 698–708.
Lee CK, Badros A, Barlogie B, Morris C, Zangari M, Fassas A et al. Prognostic factors in allogeneic transplantation for patients with high-risk multiple myeloma after reduced intensity conditioning. Exp Hematol 2003; 31: 73–80.
Kroger N, Sayer HG, Schwerdtfeger R, Kiehl M, Nagler A, Renges H et al. Unrelated stem cell transplantation in multiple myeloma after a reduced-intensity conditioning with pretransplantation antithymocyte globulin is highly effective with low transplantation-related mortality. Blood 2002; 100: 3919–3924.
Attal M, Harousseau JL, Facon T, Guilhot F, Doyen C, Fuzibet JG et al. Single versus double autologous stem-cell transplantation for multiple myeloma. N Engl J Med 2003; 349: 2495–2502.
Shaughnessy J, Jacobson J, Sawyer J, McCoy J, Fassas A, Zhan F et al. Continuous absence of metaphase-defined cytogenetic abnormalities, especially of chromosome 13 and hypodiploidy, ensures long-term survival in multiple myeloma treated with Total Therapy I: interpretation in the context of global gene expression. Blood 2003; 101: 3849–3856.
Barlogie B, Jagannath S, Desikan KR, Mattox S, Vesole D, Siegel D et al. Total therapy with tandem transplants for newly diagnosed multiple myeloma. Blood 1999; 93: 55–65.
Badros A, Barlogie B, Siegel E, Cottler-Fox M, Zangari M, Fassas A et al. Improved outcome of allogeneic transplantation in high-risk multiple myeloma patients after nonmyeloablative conditioning. J Clin Oncol 2002; 20: 1295–1303.
Sawyer JR, Lukacs JL, Munshi N, Desikan KR, Singhal S, Mehta J et al. Identification of new nonrandom translocations in multiple myeloma with multicolor spectral karyotyping. Blood 1998; 92: 4269–4278.
Gooley TA, Leisenring W, Crowley J, Storer BE . Estimation of failure probabilities in the presence of competing risks: new representations of old estimators. Stat Med 1999; 18: 695–706.
Kaplan EL, Meier P . Nonparametric estimation from incomplete observation. JASA 1958; 53: 457–481.
Collett D . Modeling Survival Data in Medical Research. Chapman and Hall: London, 1994.
Lokhorst HM, Wu K, Verdonck LF, Laterveer LL, van de Donk NW, van Oers MH, et al. The occurrence of graft-versus-host disease is the major predictive factor for response to donor lymphocyte infusions in multiple myeloma. Blood 2004; 103: 4362–4364.
Alyea E, Weller E, Schlossman R, Canning C, Webb I, Doss D et al. T-cell–depleted allogeneic bone marrow transplantation followed by donor lymphocyte infusion in patients with multiple myeloma: induction of graft-versus-myeloma effect. Blood 2001; 98: 934–939.
Schmaltz C, Alpdogan O, Kappel BJ, Muriglan SJ, Rotolo JA, Ongchin J et al. T cells require TRAIL for optimal graft-versus-tumor activity. Nat Med 2002; 8: 1433–1437.
Schmaltz C, Alpdogan O, Horndasch KJ, Muriglan SJ, Kappel BJ, Teshima T et al. Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versus-host disease and graft-versus-leukemia effect. Blood 2001; 97: 2886–2895.
Fonseca R, Barlogie B, Bataille R, Bastard C, Bergsagel PL, Chesi M et al. Genetics and cytogenetics of multiple myeloma: A workshop report. Cancer Research 2004; 64: 1546–1558.
Desikan R, Barlogie B, Sawyer J, Ayers D, Tricot G, Badros A et al. Results of high-dose therapy for 1000 patients with multiple myeloma: durable complete remissions and superior survival in the absence of chromosome 13 abnormalities. Blood 2000; 95: 4008–4010.
Smadja NV, Bastard C, Brigaudeau C, Leroux D, Fruchart C . Hypodiploidy is a major prognostic factor in multiple myeloma. Blood 2001; 98: 2229–2238.
Fassas AB, Spencer T, Sawyer J, Zangari M, Lee CK, Anaissie E et al. Both hypodiploidy and deletion of chromosome 13 independently confer poor prognosis in multiple myeloma. Br J Haematol 2002; 118: 1041–1047.
Kroger N, Schilling G, Einsele H, Liebisch P, Shimoni A, Nagler A et al. Deletion of chromosome band 13q14 detected by fluorescence in situ hybridization is a prognostic factor in patients with multiple myeloma who are receiving allogeneic dose reduced stem cell transplantation. Blood 2004; 103: 4056–4061.
Mitsiades N, Mitsiades CS, Richardson PG, McMullan C, Poulaki V, Fanourakis G et al. Molecular sequelae of histone deacetylase inhibition in human malignant B cells. Blood 2003; 101: 4055–4062.
About this article
American Journal of Hematology (2010)
Journal of Immunotherapy (2007)
Modified conditioning regimen \busulfan-cyclophosphamide followed by allogeneic stem cell transplantation in patients with multiple myeloma
Chinese Medical Journal (2007)
Robertsonian translocations in stem cell recipients as a possible indication for cytogenetic analysis of family donors
Cancer Genetics and Cytogenetics (2007)
Hematological Oncology (2006)