Abstract: Myelodysplastic Syndromes (MDS) are hematopoietic stem cell disorders, which are characterized by marrow failure and a substantial risk of developing Acute Myeloid Leukemia (AML). While some patients gradually progress to more advanced MDS subtypes, others experience an apparently immediate AML onset without any transition period. In order to get a better understanding of these different types of MDS progression, we retrospectively analyzed the data of 3213 patients included into the MDS registry Düsseldorf. As assessed by bone marrow examination a disease progression to AML or to advanced MDS subtype was observed in 24,5% of the patients. The progression rate was lowest in the unilineage dysplasia group (RA/RARS: 9%) as compared to 5q- (26%), multilineage dysplasia (16%), RAEB I (26%) and RAEB II (37%). The progression rate in CMML I was 17%, in CMML II 31%, in RARS-T 9% and in the former RAEB-T group 57%. We then evaluated the survival-time of the progressive patients. In the entire group, patients who progressed had a median survival of 17 months compared to 30 months in those with stable disease (p=0.0005). In the group of patients with less than 5% of medullary blasts at time of diagnosis, those who progressed had a median survival of 28 months compared to 48 months in those who did not progress (p=0.00005). Interestingly, in this group of patients disease progression into an advanced MDS subtype did not affect survival (4% progression in advanced subtype, 46 vs. 48 months, p= n.s.), whereas disease progression into AML was associated with a shorter survival (11% progression into AML, 23 vs. 48 months, p=0,0005) In the group of patients who had & gt;5% of medullary blasts at diagnosis, the progression did not influence survival substantially (14 vs.15 months, p=0.01). The cumulative risk of AML evolution at 2 and 5 years after initial diagnosis was lowest in unilineage dysplasia (4% and 8%), multilineage dysplasia (11% and 19%), 5q- (10% and 18%), RAEB I (26% and 44%), RAEB II (50% and 74%), CMML I (14% and 24%), CMML II (33% and 74%), RARS-T (5% and 5%) and RAEB –T (70% and 77%). In the following we investigated the course of MDS progression in those patients who did not develop AML. Fifty-three % of the patients in the unilineage group (RA/RARS) progressed to RAEB I or RAEB II, 10% to RCMD and 3% developed a 5q- syndrome. In the multilineage group 41% of the patients transformed into RAEB I or II, while 40% of the patients with 5q- progressed to RAEB I or RAEB II. In the RAEB I group, 28% of the patients developed RAEB II and 18% of the CMML I patients ended up as CMML II. Finally, we analyzed the effect of progression within the IPSS groups with regard to survival. Patients within the low risk group who progressed (22%) had a median survival of only 46 months, compared to 88 months among those who did not progress (p=0.00005). This correlation was also significant among patients within the intermediate I group (29% progressive patients, 26 vs. 36 months, p=0.0004). In both, the intermediate II and the high risk group progression was not associated with a shorter survival. Conclusions: About 25% of the patients progress to a more advanced MDS type or to AML. A substantial part of the patients with 5q- Syndrome as well as patients with multilineage dysplasia and RAEB types show disease progression. Progression is only associated with a shorter survival among patients within the IPSS low and Intermediate I risk group. In patients with less than 5% of marrow blasts at time of diagnosis only progression into AML is associated with a shorter survival.

Abstract: Background: Using real-time quantitative (RQ) PCR we recently (Haematologica89,2004) identified a prognostic cut-off level of residual clonotypic cells in the bone marrow of patients with multiple myeloma before high-dose therapy (HDT) and autologous peripheral blood stem cell transplantation (PBSCT). In this study we validate this report with a larger number of patients. Patients and Methods: Bone marrow samples of 68 patients with stage II/III multiple myeloma and heavy chain disease were obtained at the time of diagnosis and after induction therapy and stem cell collection but before single HDT and autologous PBSCT. Sequencing of the patient specific immunoglobulin heavy chain (IgH) locus was successful in 51 patients (75%). For 49 patients (72%) RQ-PCR using allele-specific oligonucleotide (ASO) Taqman probes together with LightCycler technology could be established with a sensitivity of 10−4 to 10−6 and linear amplification conditions. The proportion of clonotypic cells was assessed as IgH / 2 beta-actin ratio in percent. Patients were divided in two prognostic groups by a threshold level of 0.03% clonotypic cells. Results: The median level of residual tumor cells in bone marrow of all patients at the time before transplantation was 0.05% (range: 0–21%). Time to progression (TTP) from the time of diagnosis of patients in the ¨good¨ prognostic group (n = 21) was 51 months and significantly (p = 0.002) longer in comparison to 20 months of patients with a pre-transplantation minimal residual disease level of more than 0.03% in BM (n = 28). Overall survival (OS) of patients within the ¨good¨ prognostic group was also significantly prolonged (median OS: not reached versus 46 months, p = 0.03). Univariate analysis also revealed kind of maintenance / consolidation therapy (thalidomide, interferon, reduced intensity conditioning (RIC) allogeneic transplant) and cytogenetic banding analysis as prognostic markers for TTP. For OS kind of maintenance therapy, cytogenetic abnormalities, ISS stage, CRP and LDH levels were of prognostic relevance. In multivariat analysis grouping by pre-transplantation MRD level was an independent prognostic factor for either TTP and OS. Conclusion: Quantitative molecular assessment of pre-transplantation tumor level in the bone marrow is an independent prognostic parameter for TTP and OS of patients with multiple myeloma. This finding has two controversial implications. One conclusion could be, that induction therapy should be continued and intensified e.g. with novel agents until a low MRD level is achieved. An alternative conclusion is, that a low tumor burden after induction therapy may be a surrogate parameter for chemosensitive disease, which makes patients more susceptible for high-dose chemotherapy. Therefore, further MRD studies are needed to answer this important question.

Abstract: Multiple myeloma (MM) is a clonal plasma cell disorder frequently accompanied by hematopoietic impairment. We show that hematopoietic stem and progenitor cells (HSPCs), in particular megakaryocyte-erythrocyte progenitors, are diminished in the BM of MM patients. Genomic profiling of HSPC subsets revealed deregulations of signaling cascades, most notably TGFβ signaling, and pathways involved in cytoskeletal organization, migration, adhesion, and cell-cycle regulation in the patients. Functionally, proliferation, colony formation, and long-term self-renewal were impaired as a consequence of activated TGFβ signaling. In accordance, TGFβ levels in the BM extracellular fluid were elevated and mesenchymal stromal cells (MSCs) had a reduced capacity to support long-term hematopoiesis of HSPCs that completely recovered on blockade of TGFβ signaling. Furthermore, we found defective actin assembly and down-regulation of the adhesion receptor CD44 in MM HSPCs functionally reflected by impaired migration and adhesion. Still, transplantation into myeloma-free NOG mice revealed even enhanced engraftment and normal differentiation capacities of MM HSPCs, which underlines that functional impairment of HSPCs depends on MM-related microenvironmental cues and is reversible. Taken together, these data implicate that hematopoietic suppression in MM emerges from the HSPCs as a result of MM-related microenvironmental alterations.

Abstract: Purpose: Therapeutic options for patients with high-risk myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) who relapse after allogeneic stem cell transplantation (SCT) are limited and prognosis is dismal. If applicable, transfusion of donor lymphocytes (DLI) with or without chemotherapy is the current standard therapy. But, in contrast to chronic myelogenous leukemia (CML), response rates after sole DLI in patients with relapsing MDS or AML after allogeneic SCT are poor. Addition of chemotherapy, usually low-dose cytarabine 100mg/m2 as continuous infusion for 7 days, can increase response rates only marginally. Also important, duration of response is short and long term survival even rare. The demethylating agent 5-aza-2′-deoxycytidine (5-Aza) has been shown to be effective in the treatment of MDS and AML. In addition to a direct cytotoxic effect, treatment with this demethylating drug also results in a rapid and stable transcription and cell surface expression of formerly unexpressed killer Ig-like receptors (KIRs) in natural killer cells (NK cells), thereby possibly enhancing the GvL effect of DLI. Patients & treatment: In an intent-to-treat approach we treated 6 patients with high risk MDS or AML who relapsed after allogeneic SCT with 5-Aza plus DLI. Patients’ median age was 47.5 years (range 32–71 years). Before allogeneic SCT 4 patients had active disease, and 2 were in complete remission (CR). Two had family donors, 4 had unrelated donors. Median time for relapse after SCT was day +99 (range day +84 to day +300). Once relapse was diagnosed patients received 100mg/m2 5-Aza for five days via subcutaneous injection in two to four weeks intervals. If practical, patients received 1×106 CD3+ cells/kg bodyweight following the first course of 5-Aza, and in the absence of graft-versus host disease, this was followed by additional 5×106 CD3+ cells/kg bodyweight after 3 months. Results: Five out of 6 patients responded to treatment with 5-Aza and DLI. Three patients achieved a complete remission (CR), two a partial remission (PR) and one patient died early due to progressive disease. Two patients developed extensive graft-versus host disease (GvHD), while, so far, four patients did not show any signs of GvHD. Side effects were manageable and limited to the hematopoietic system. Of the three patients achieving CR, two patients relapsed again at extramedullary sites (heart and CNS). Two of these three patients achieving initial CR are alive, one in CR, one in PR, while one patient died due to CNS disease. One of the patients achieving a PR died due to progressive disease, and the other died after a second allogeneic SCT with progressive disease. Median survival of all patients was 125 days (range 39–397 days). Conclusion: Overall, induction of CR after treatment with 5-Aza, and consolidation of CR via DLI followed the 5-Aza treatment is promising.

Abstract: Introduction. The prognosis of patients with follicular lymphoma (FL) has improved during recent years following the introduction of immuno-chemotherapy and Rituximab maintenance. Nevertheless, some patients still relapse early and have a poor prognosis. Several prognostic scoring systems have been developed using clinical, laboratory as well as molecular data, while the early identification of high-risk patients remains a challenge. In this context, the relevance of circulating bcl2/IgH levels for patient stratification is not clear. We could show that high circulating bcl2/IgH levels in the peripheral blood (PB) before therapy were an independent adverse prognostic factor for progression free survival (PFS) in patients receiving R-CHOP or Bendamustine-Rituximab (B-R) in the NHL1 study of the German StiL group (Zohren et al, Blood 2015). Methods. Using a sensitive quantitative PCR method as previously described (Zohren et al, Blood 2015), a total of 2,491 circulating bcl-2/IgH level analyses were performed on PB samples before (n=415) and after (n=305) 6 cycles first-line immuno-chemotherapy and during follow-up (n=1,771). Results of these molecular studies were correlated with clinical outcome. We first present a 10-year update of the 107 bcl2/IgHpositive patients from the StiL-NHL1-trial. Secondly, we report the results from the StiL-NHL7-trial including bcl2/IgH analyses of 308 bcl2/IgHpositive patients who received B-R and Rituximab maintenance. Results. With a median follow-up of 10 years in the 107 bcl2/IgHpositive patients from the StiL-NHL1-trial, high PB bcl-2/IgH levels (bcl-2/IgH to reference gene (tPA) ratio 〉 1) before treatment as compared to low (ratio 〈 1) levels remained a major independent prognostic factor for PFS (median 22 vs 71 months, HR 2.27, 95% CI 1.37-3.75; p=0.001). We also confirm that patients who were still bcl-2/IgHpositive after six cycles of immuno-chemotherapy had significantly inferior PFS (13 vs 79 months, Hazard Ratio (HR) 2.97, 95% CI 1.53-5.78; p=0.001) and overall survival (OS, 128 months vs not reached , HR 3.90, 95% CI 1.39-11.00; p=0.010). In contrast, among the 308 bcl-2/IgHpositive patients of the StiL-NHL7-trial, who all received B-R and Rituximab maintenance, PB bcl-2/IgH levels (ratio 〉 1 vs 〈 1) before therapy were no longer prognostic for PFS (99 months vs not reached, HR 1.06, 95% CI 0.66 - 1.69; p=0.814) or OS. On the other hand, being bcl-2/IgHpositive after 6x B-R remained a poor prognostic factor for PFS (43 months vs not reached, HR 2.44, 95% CI 1.18-5.04; p=0.016 ) and OS (72 months vs not reached, HR 4.03, 95% CI 1.82-8.96; p=0.001) despite Rituximab maintenance. When comparing StiL-NHL1 and StiL-NHL7 patients with respect to bcl-2/IgH levels and the effect of Rituximab maintenance, we found that Rituximab maintenance led to a significantly better PFS. In patients with low (ratio 〈 1) bcl-2/IgH levels before therapy the hazard ratio of 1.7 was modest (71 months vs not reached, HR 1.70, 95% CI 1.16-2.50; p=0.006) in comparison to 3.46 as observed in patients with high (ratio 〉 1) bcl-2/IgH levels (22 vs 99 months, HR 3.46, 95% CI 1.93-6.20; p 〈 0.000). These findings suggest that patients with high bcl-2/IgH levels before therapy have a greater benefit from Rituximab maintenance therapy. There was no difference with regard to OS between StiL-NHL1 and StiL-NHL7 patients who were still bcl-2/IgHpositive after 6 cycles of immuno-chemotherapy implying that these patients may not benefit from Rituximab maintenance. Conclusion. High circulating bcl-2/IgH levels in the PB before first line therapy identify a subgroup of patients with advanced FL who have significantly shorter PFS after standard immuno-chemotherapy. These patients greatly benefit from the addition of Rituximab maintenance, because pre-treatment bcl-2/IgH levels lose their predictive value with Rituximab maintenance therapy. On the other hand, patients who remain bcl-2/IgHpositive after standard immuno-chemotherapy have short PFS and OS despite treatment with Rituximab maintenance and therefore are candidates for experimental treatment approaches. Disclosures Kobbe: Amgen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding. Zohren:Pfizer Inc.: Employment. Buske:Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Bayer: Research Funding. Germing:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Research Funding. Greil:Sandoz: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES. Schroeder:Celgene: Consultancy, Honoraria, Research Funding. Rummel:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Astellas: Honoraria; Eisai: Honoraria; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Symbio: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: Abstract 656 Background: Relapse after allo-SCT is a major cause of treatment failure in patients (pts) with myeloid malignancies and is associated with a poor prognosis. As therapeutic options are limited, treatment of these pts is challenging. Indeed, there is a need for novel strategies which ideally target the leukemic clone and direct the immune system towards an enhanced GvL effect without aggravating GvHD. The hypomethylating agent Aza might provide these properties, and results from retrospective studies investigating Aza+/−DLI in pts with AML/MDS relapsing after allo-SCT were encouraging. Design/Methods: We here report the final results from a prospective single-arm EBMT multicenter phase II trial which aimed to investigate the efficacy and safety of a combination of Aza and DLI as 1st salvage therapy in pts with AML or MDS with hematological relapse after allo-SCT. Treatment schedule contained up to 8 cycles Aza (100 mg/m2/d d1-5, every 28 d) followed by DLI with increasing dosages (1-5×106–1-5×108cells/kg) after every 2ndAza cycle. Results: A total of 30 pts (19 f/11 m, median age 56 years, range 29–71) from 6 German transplant centres were included between January 2009 and May 2010. The majority of pts (n=28, 93%) suffered from AML, while 2 pts (8%) had MDS or MDS/MPN, respectively. At transplant, 16 pts (53%) had active disease (6 induction failure, 7 relapse I/II, 3 untreated) and 14 pts (47%) were in remission (12 CR1, 2 CR2). Following standard-dose (n=4, 13%) or dose-reduced conditioning (n=26, 87%), 10 pts (33%) received a graft from MSD and 20 pts (67%) from MUD. PBSC were used in 29 pts (97%), while 1 pt (3%) received BM. Acute GvHD occurred in 13 pts (46%) and 4 pts (13%) had chronic GvHD prior inclusion. None of the pts had active GvHD at the time of relapse, but 6 pts were still on immunosuppressive therapy. All pts had hematological relapse (median BM blasts: 34%, median chimerism: 67%) at a median time of 160 days (range 19–1699) following allo-SCT. A median of 3 courses Aza (range 1–8) were administered, and 22 of 30 pts (73%) received DLI (median: 1, range: 1–4, median CD3 dose 5×106/kg/DLI, range: 1–100×106). Overall response rate was 47%. Seven pts (23%) achieved CR or CRi, 2 pts (7%) PR, and 5 pts (17%) had stable disease (SD). Median time and median number of Aza cycles to best response were 79 days (range 28–299) and 3 cycles (range 1–8) respectively. Of the 7 pts who achieved a CR, 5 pts continue in CR for a median of 605 days (range 307–763) without any additional treatment, while 1 pt relapsed after 490 days and 1 pt died from GvHD. By July 2011 median follow-up of surviving pts is 645 days (range 564–857) and 5 of 30 pts (17%) are currently alive. Twelve pts have died due to progressive disease (PD), while 7 pts died during (n=3, 2 infection, 1 bleeding) or after the end of therapy (n= 4, 1 GvHD, 2 infection, 1 bleeding). All 5 pts who underwent 2nd allo-SCT died. Median overall survival (OS) of all pts is 117 days (95% CI 66–168 days). Patients with CR/CRi had a significant longer OS than pts not reaching CR/CRi (not reached vs. 83 days, p 〈 .001). Eleven pts (37%) developed aGvHD, while cGvHD was observed in 5 pts (17%). Any type of GvHD was seen in 78% of pts with CR/PR as opposed to 33% in pts with SD/PD (p=.03). Cytopenias grade III/IV occurred in all pts, but were considered to be drug-related in only 11 pts (37%). The most common drug-related grade III/IV non-hematological toxicities were infection and bleeding. Conclusion: Aza and DLI as first salvage therapy is a safe and active approach for pts with AML or MDS who relapse after allo-SCT, and induces durable remissions in a subgroup of pts. Further research to better define target patient groups and combination partners for AZA+DLI is needed. Disclosures: Schroeder: Celgene: Financial travel support. Bug:Celgene: Lecture fees, Membership on an entity's Board of Directors or advisory committees. Luft:Celgene: Research Funding. Kobbe:Celgene: Financial travel support, Research Funding.