José A. Pérez-Simón

A prospective PETHEMA study of tandem autologous transplantation versus autograft followed by reduced-intensity conditioning allogeneic transplantation in newly diagnosed multiple myeloma

One hundred ten patients with multiple myeloma (MM) failing to achieve at least near-complete remission (nCR) after a first autologous stem cell transplantation (ASCT) were scheduled to receive a second ASCT (85 patients) or a reduced-intensity-conditioning allograft (allo-RIC; 25 patients), depending on the human leukocyte antigen (HLA)-identical sibling donor availability. There was a higher increase in complete remission (CR) rate (40% vs 11%, P = .001) and a trend toward a longer progression-free survival (PFS; median, 31 months vs not reached, P = .08) in favor of allo-RIC. In contrast, it was associated with a trend toward a higher transplantation-related mortality (16% vs 5%, P = .07), a 66% chance of chronic graft-versus-host disease and no statistical difference in event-free survival and overall survival. Although the PFS plateau observed with allo-RIC is very encouraging, this procedure is associated with high morbidity and mortality, and therefore it should still be considered investigational and restricted to well-designed prospective clinical trials. This trial is registered at ClinicalTrials.gov ID number NCT00560053.

Sustained Remissions of High-Risk Acute Myeloid Leukemia and Myelodysplastic Syndrome After Reduced- Intensity Conditioning Allogeneic Hematopoietic Transplantation: Chronic Graft-Versus-Host Disease Is the Strongest Factor Improving Survival

PURPOSE Reduced-intensity conditioning (RIC) for allogeneic stem-cell transplantation (allo-SCT) reduces nonrelapse mortality (NRM). This reduction makes it possible for patients who are ineligible for high-dose myeloablative conditioning allo-SCT to benefit from graft-versus-leukemia reaction. In this multicenter, prospective study of patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS), we investigated the efficacy of RIC allo-SCT from a human leukocyte antigen-identical sibling by using a regimen that uses fludarabine and busulfan. PATIENTS AND METHODS Ninety-three patients with AML (n = 59) and MDS (n = 34) were included, and the median age was of 53 years. Follow-up for survivors was 43 months (range, 3 to 89 months). The conditioning regimen consisted of fludarabine (150 mg/m(2)) and oral busulfan (8 to 10 mg/kg). All except one patient received mobilized peripheral blood stem cells. Graft-versus-host disease (GVHD) prophylaxis consisted of cyslosporine and methotrexate or mycophenolate mofetil. RESULTS The 100-day, 1-year, and 4-year incidences of NRM were 8, 16%, and 21%, respectively. The 1- and 4-year relapse cumulative incidences were 23% and 37%, respectively, and leukemia recurrence was the main cause of death. The 4-year disease-free survival (DFS) and overall survival (OS) rates were 43% and 45%, respectively. The 4-year cumulative incidence of chronic GVHD was 53% (45% extensive), and its development was the major factor associated with lower relapse incidence and improved DFS and OS. CONCLUSION Our results confirm the capacity of this RIC regimen to obtain long-term remissions in patients ineligible for a conventional allo-SCT. The results suggest an important role of the development of chronic GVHD in reducing relapse and improving DFS and OS.

The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes

Mesenchymal stem cells are multipotent non-hematopoietic progenitor cells capable of differentiating into various lineages including osteoblasts, chondrocytes and adipocytes. The findings of this study indicate that mesenchymal stem cells promote survival and inhibit proliferation and maturation of B cells, and support a role of these cells in the immune response. Background Mesenchymal stem cells are multilineage non-hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T- and B-lymphocytes. While their effect on T-lymphocytes has been extensively analyzed, data on the effect of mesenchymal stem cells on B cells are more limited. We analyzed the effects of mesenchymal stem cells on B-lymphocytes and the pathways involved in these effects. Design and Methods The effect of MSC on the proliferation and viability of B cells was evaluated using MTT assays, annexin/7-amino-actinomycin D and propidium iodide staining. The B-cell maturation pattern was established using flow cytometry based on the expression of different markers related to the differentiation of B cells, such as CD38, CD138, CD19 and CCR7, and to the expression of surface and intracellular immunoglobulins. Finally, western blot assays were used to identify the pathways involved in the effects of mesenchymal stem cells on B-lymphocytes. Results Mesenchymal stem cells increased viability and blocked the cell cycle of B-lymphocytes in the G0/G1 phase. In vitro exposure of B cells to plasmacytoid dendritic cells induced B-cell differentiation as shown by an increased number of CD38++/CD138++ cells, which also displayed higher levels of cytoplasmic immunoglobulin and lower levels of CD19, CCR7 and surface immunoglobulin. Interestingly, this maturation pattern was inhibited by adding mesenchymal stem cells to the culture. Finally, mesenchymal stem cells modified the phosphorylation pattern of the extracellular response kinase 1/2 and p38 pathways which are both involved in B-cell viability, proliferation and activation. Conclusions Mesenchymal stem cells increase B-cell viability while inhibiting proliferation, arresting B-lymphocytes in the G0/G1 phase of the cell cycle. The presence of mesenchymal stem cells blocked B-cell differentiation as assessed by flow cytometry. Finally, mesenchymal stem cells modified the activation pattern of the extracellular response kinase and the p38 mitogen-activated protein kinase pathways in B-lymphocytes.

International Myeloma Working Group Consensus Statement Regarding the Current Status of Allogeneic Stem-Cell Transplantation for Multiple Myeloma

PURPOSE To define consensus statement regarding allogeneic stem-cell transplantation (Allo-SCT) as a treatment option for multiple myeloma (MM) on behalf of International Myeloma Working Group. PATIENTS AND METHODS In this review, results from prospective and retrospective studies of Allo-SCT in MM are summarized. RESULTS Although the introduction of reduced-intensity conditioning (RIC) has lowered the high treatment-related mortality associated with myeloablative conditioning, convincing evidence is lacking that Allo-RIC improves the survival compared with autologous stem-cell transplantation. CONCLUSION New strategies are necessary to make Allo-SCT safer and more effective for patients with MM. Until this is achieved, Allo-RIC in myeloma should only be recommended in the context of clinical trials.

Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G(0) to G(1) phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.

Mesenchymal stem cells expanded in vitro with human serum for the treatment of acute and chronic graft-versus-host disease: results of a phase I/II clinical trial

This trial evaluated the feasibility and efficacy of the infusion of mesenchymal stem cells expanded using human serum for the treatment of refractory acute or chronic graft-versus-host disease. Twenty-eight expansions were started. In 22, a minimum of more than 1x106 mesenchymal stem cells/kg were obtained after a median of 26 days; this threshold was not obtained in the remaining cases. Ten patients received cells for the treatment of refractory or relapsed acute graft-versus-host disease and 8 for chronic disease. One patient treated for acute graft-versus-host disease obtained a complete response, 6 had a partial response and 3 did not respond. One of the chronic patients achieved complete remision, 3 a partial response, and 4 did not respond. The current study supports the use of this approach in less heavily treated patients for both acute and chronic graft-versus-host disease. The trial has been registered at ClinicalTrials.gov: identifier NCT00447460.

Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q-syndrome

The presence of cytogenetic aberrations on mesenchymal stem cells (MSC) from myelodysplastic syndrome (MDS) patients is controversial. The aim of the study is to characterize bone marrow (BM) derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridization (FISH) and genetic changes by array-based comparative genomic hybridization (array-CGH). In all 36 cases of untreated MDS were studied. MDS–MSC achieved confluence at a significantly slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic. These results were confirmed by FISH. To confirm that genomic changes were also present in freshly obtained MSCs they were enriched by sorting BM cells with the following phenotype: CD45−/CD73++/CD34−/CD271++. They also showed genomic changes that were confirmed by FISH. To analyze the relationship of these aberrations with clinical–biological data an unsupervized hierarchical cluster analysis was performed, two clusters were identified: the first one included the 5q− syndrome patients, whereas the other incorporated other MDS. Our results show, for the first time that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to a particular MDS subtype, the 5q− syndrome.

Influence of the intensity of the conditioning regimen on the characteristics of acute and chronic graft‐versus‐host disease after allogeneic transplantation

The graft‐versus‐host disease (GVHD) characteristics of 150 consecutive patients undergoing reduced intensity conditioning allogeneic (allo‐RIC) transplants and 88 patients undergoing myeloablative conditioning regimen were analysed. All patients received the same GVHD prophylaxis and peripheral blood stem cells from a human leucocyte antigen identical sibling. The cumulative incidence of acute GVHD (aGVHD) was 67% and 44% in the myeloablative and allo‐RIC regimen groups, respectively (P < 0·001), and was 39% vs. 29%, respectively (P = 0·043), for grades 2–4 aGVHD. Only conditioning type (myeloablative versus allo‐RIC) significantly influenced the incidence of aGVHD in multivariate analysis: Hazard ratio (HR) = 2·16 [95% confidence interval (CI): 1·52–3·07], P < 0·0001. The cumulative incidence of chronic GVHD (cGVHD) was 63% and 71% among myeloablative and allo‐RIC patients respectively (P = 0·084). This trend was because of the higher incidence of limited cGVHD, but not extensive cGVHD among allo‐RIC recipients [HR = 3·3 (95% CI: 1·42–8·08), P = 0·0017]. Moreover, among patients who developed cGVHD, the cumulative incidence of limited cGVHD was significantly lower in the myeloablative group than in the allo‐RIC group (7% vs. 25%, P = 0·007). Duration of immunosuppression was shorter among allo‐RIC patients (35·5% vs. 68·8% required systemic immunosuppression 36 months after transplant, P = 0·028). Although prospective controlled trials are required to further evaluate the effect of the conditioning regimen on GVHD, our results suggest that RIC modifies the incidence and characteristics of both acute and cGVHD after allogeneic transplantation, and decreases the immunosuppression requirements in long‐term follow up when compared with myeloablative conditioning.

Chronic but not acute graft-versus-host disease improves outcome in multiple myeloma patients after non-myeloablative allogeneic transplantation.

Summary. The outcome of 29 multiple myeloma patients receiving fludarabine and melphalan‐based non‐myeloablative allogeneic transplant (NMT) was evaluated. Event‐free survival (EFS) at 24 months was 33%, being significantly higher for patients who developed chronic graft‐versus‐host disease (cGVHD) when compared with those who did not [51%vs 0% respectively, P = 0·02; hazard rate = 3·16 (95% confidence interval = 1·09–9·15, P = 0·03)] as well as for patients transplanted in complete remission/partial response (CR/PR) or stable disease (SD), compared with those with refractory/progressive disease (43%vs 0% respectively, P = 0·02). Overall survival (OS) at 24 months was 60%[72%vs 42% for patients who did and did not develop cGVHD respectively (P = 0·1); 63%vs 41% for patients in CR/PR or SD vs refractory/progressive disease at transplant respectively (P = 0·013)]. At a median follow‐up of 366 d, 13 patients remained in CR/PR (45% overall response rate). Nine patients have died, three of them as a result of disease progression and six (21%) as a result of transplant‐related mortality (TRM). Actuarial incidence of TRM was 37% for patients who developed acute GVHD vs 13% for those who did not (log rank, P = 0·04). The present study suggests that graft‐versus‐myeloma effect is the main weapon for disease control after NMT in MM patients and the efficacy of this immune effect depends on tumour burden before transplant.

Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation

Since graft-versus-leukemia (GVL) is the main weapon for disease eradication after reduced intensity conditioning (RIC) allogeneic SCT, the availability of sensitive and specific techniques to monitor changes in tumor load after transplant are especially helpful. These minimal residual disease techniques would allow an early intervention in the event of low tumor burden, for which immunotherapy is highly effective. Some authors have found an association between persistence of MRD, mixed chimerism and risk of relapse. Nevertheless, data from the literature remain contradictory and further correlations should be established, especially in RIC transplants. In this study we have analyzed the impact of MRD and chimerism monitoring on the outcome of 34 patients undergoing RIC allogeneic SCT who were considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions. At day +100 25 (75%) patients reached complete remission (CR), there were five (15%) partial responses and three patients progressed. Incidence of grade 2–4 aGVHD and extensive cGVHD were 35% and 58%, respectively. Sixteen percent of patients developing aGVHD relapsed as compared to 47% in those without aGVHD (P = 0.03) and also 10% of patients developing cGVHD relapsed as compared to 50% relapses in those without cGHVD (P = 0.03). Four patients (12%) died due to early (n = 1) and late (n = 3) transplant-related mortality. After a median follow-up of 15 months, 24 out of the 34 patients remain alive. Projected overall survival and disease-free survival at 3 years are 68% and 63%, respectively. Early chimerism analysis showed 67% of patients with complete chimerism (CC) in bone marrow (BM), 86% in peripheral blood (PB), 89% in granulocytes and 68% in T lymphocytes. On day +100, these figures were 68%, 79%, 90% and 73%, respectively, and on day +180 there were 83% patients with CC in BM, 100% in PB, 100% in granulocytes and 100% in T lymphocytes. We observed a trend to a higher incidence of relapse in patients with mixed chimerism (MC) as compared to patients with CC. MRD monitoring by flow cytometry and/or RT-PCR analysis was performed in 23 patients. MRD assessment on days +21 to +56 after transplant allowed identification of patients at risk of relapse. In this sense, seven out of 12 patients (58.3%) who had positive MRD on days +21 to +56 relapsed as compared to none out of 11 patients who had negative MRD (P = 0.002). Of the seven patients with criteria to monitor MRD who relapsed after transplant, all but one remained MRD positive until relapse. By contrast, 10 patients remained MRD negative and all of them are in continuous CR. In nine additional patients, persistence of MRD or mixed chimerism was observed after transplant and withdrawal of cyclosporin with or without DLI was performed. Only two out of these nine patients relapsed. MRD clearance was preceded by CC and GVHD. In conclusion, in our study we found that RIC allogeneic transplantation can be used in patients considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions with both low toxicity and low transplant-related mortality. Simultaneous studies of both chimerism and MRD are a useful tool in order to predict risk of relapse in patients undergoing RIC transplants and so can be helpful for individualizing treatment strategies after transplant.