ALBERT

Description: Introduction: Reduced intensity conditioning (RIC) now accounts for approximately one fourth of all allogeneic stem cell transplantations (SCT) worldwide (IBMTR database 2002). A reduction of transplant-related mortality has been observed in uncontrolled trials of patients with MDS, CLL, NHL and M. Hodgkin, but at the same time some of these trials revealed an increased relapse rate. Long term follow-up of acute leukemias after RIC has not been reported so far. Own results have demonstrated continuous complete remissions in patients with ALL transplanted early in the course of disease (Arnold 2002). Patients and Methods: Between August 1998 and December 2003, 40 pts with high-risk ALL (n = 11) or AML (n = 29) received allogeneic SCT after RIC with fludarabine, busulfan and ATG because of contraindications against standard high-dose conditioning (age 〉 50 years, prior allogeneic SCT, fungal infections, low performance status). 8/40 had already relapsed prior standard SCT. 13 pts were in CR-1, 7 were in subsequent CR and 20 had uncontrolled disease. Median age was 48 years (range 19 – 67), 24 were male and 16 were female. All but one patient with 1 HLA-class I mismatch were transplanted from HLA-identical related (n = 23) or unrelated (n = 17) donors. Stem cell sources were mobilized peripheral blood in 37 and bone marrow in 3 patients. Patients received 5.0 x 10E6 CD34+ cells/kg bw in median (range 2.1 – 19.8). GvHD-prophylaxis was performed with cyclosporine A (CSA) alone (n = 23) or CSA + mycophenolate mofetil (n = 17). Results: 37/40 pts reconstituted and reached CR including 17/20 patients with uncontrolled disease before SCT. 1/40 had a primary graft failure and was successfully retransplanted; two patients had refractory disease. After a median follow-up of 14 months (range 2 – 64 months), cumulative transplant-related mortality is 8 % ( 3/40 patients). 9/40 patients had developed acute GvHD III–IV and 11/24 patients had chronic extensive disease. 19 patients relapsed (including 2 refractory patients and 7/8 patients with salvage transplantation). Probability of disease-free survival at 3 years is 49 %. Overall survival at 3-year is 37 %. Median survival for patients with ALL is 13 months (95% C.I., 1 – 26) and for AML 14 months (95% C.I. 4 – 24) (n.s.). Among 18 survivors in CR, 8 were transplanted in CR-1 and 10 with advanced disease. Discussion: RIC results in sustained complete remissions in patients with high-risk acute leukemias. Even advanced diseases can be cured by allogeneic SCT after RIC. The high relapse rate is worrisome and calls for effective post-transplant strategies like prophylactic DLI or maintenance therapy to reduce relapse risk. TRM is comparably low in this group despite intensive pre-treatment and high rates of GvHD.

Description: Stem cells reside in a physical niche where a balance of signals controls their growth, differentiation and death. Niche components have generally been defined in terms of cells and positive effects on stem cell maintenance or expansion. Here we define a role for a matrix glycoprotein that provides a constraining function in the hematopoietic stem cell niche. Osteopontin (OPN) is an abundant glycoprotein in bone that can function as either cytokine or cell adhesion mediator. It is known to be produced by multiple cells types including osteoblasts, cells recently defined to be a regulatory component of the hematopoietic stem cell niche. Using studies combining OPN deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell numbers and function. In OPN deficient mice, increased primitive cell numbers were observed in vivo associated with reduced progenitors and reduced primitive cell apoptotic fraction. To determine whether the effect of OPN deficiency was stroma dependent, we performed in vitro stem cell assays on OPN−/− stroma and observed greater LTC-IC supportive capacity compared with wild type stroma. Furthermore, OPN−/− recipients showed a significantly higher proportion of hematopoietic stem cells after transplantation of OPN+/+ bone marrow in comparison to wild-type recipients, indicating that the OPN null microenvironment was sufficient to increase stem cell number. A reduction in apoptotic fraction was seen in primitive cells in the OPN−/− recipient marrows. A role for OPN in apoptosis was confirmed by exogenous OPN in in-vitro studies. Hypothesizing that OPN may serve as a physiologic constraint on stem cell pool size, we compared OPN−/− with wild type animals following parathyroid hormone activation of the stem cell niche. The expansion of stem cells by PTH was superphysiologic in the absence of OPN. Therefore, OPN is a restricting element of the stem cell niche, limiting the number of stem cells produced by niche activation. Extracellular matrix components such as OPN may serve as modulable, regulatory participants in the stem cell niche.

Description: Introduction: The outcome of salvage chemotherapy in refractory or relapsed adult acute lymphoblastic leukemia (ALL) is poor. In this situation allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy, but it is not clear whether reinduction chemotherapy prior to HSCT is required to achieve long-term remissions. Methods: Here, we present a retrospective analysis of 65 patients (median age 29 years, 17–54) who received allogeneic HSCT for refractory or relapsed ALL from 1995 until 2006 (1st relapse n=50, 2nd or higher relapse n=7, primary refractory n=8, B-lineage ALL n=50, T-lineage ALL n=15). 22/65 patients were transplanted without reinduction chemotherapy prior to HSCT (median blasts in the bone marrow 30%, 7–90), whereas 43/65 patients received reinduction. 23 out of these 43 patients achieved CR2, but 21/43 patients were refractory (median blasts 70%, 10–95). Patients received standard high-dose (n=61) or reduced intensity conditioning (RIC) (n=4) and HLA-matched (n=58) or -mismatched (n=7) HSCT from related (n=26) or unrelated (n=39) donors. Results: Median follow-up of the surviving patients was 53 months (2–130). Overall survival (OS) of the whole cohort at 6 months, 1, 3 and 5 years was 61%, 40%, 27% and 24%, respectively. Deaths were due to relapse (n=29), GVHD (n=8), infections (n=7) or other causes (n=4). Chronic GVHD (cGVHD) occurred in 28/53 patients (53%) who survived more than 100 days after HSCT. Patients who developed cGVHD had significantly better outcome than patients without cGVHD (p

Description: Karyotypes like Philadelphia-chromosome (Ph+) and t(4;11) adversely influence clinical outcome in adult patients with acute lymphoblastic leukemia (ALL) after chemotherapy. Here, we analyze the potential impact of karyotype on outcome after allogeneic hematopoietic stem cell therapy (HSCT) in ALL. We present a retrospective analysis of 135 adult ALL patients treated unicentrically within the German ALL (GMALL) protocol using allogeneic HSCT. All patients were high-risk patients in CR1 according to GMALL definitions (i.e., WBC 〉 30.000/μl, pro-B-ALL, t(4;11), t(9;22), early T- or mature T-ALL, no CR after first induction) or were beyond CR1. Median age of all patients was 29 years (range 16 – 55), 99 B-lineage and 36 T-lineage ALLs were transplanted. Normal karyotype was present in 57 patients, Ph+ in 36 patients, complex chromosomal anomalies in six patients, t(4;11) in five patients, other aberrations in 22 patients and no growth/no cytogenetic data were available for 9 patients. Patients were transplanted in complete remission (CR) CR1 (60), CR2 (23), CR3 (7), first relapse (30), second relapse (6), third relapse (1) and eight patients had primary induction failure. 41 patients received bone marrow and 94 patients received peripheral blood stem cell transplants. Patients received standard high-dose (n=125) or reduced intensity conditioning (RIC) (n=10) HSCT from related (n=58) or unrelated (n=77) donors. Overall, after a median follow-up of 11 months (range 1–120), 74 (55%) patients died and 61 (45%) are alive. Median follow-up of the living is 29 months (range 1–120). Deaths were due to treatment-related mortality (TRM; n=33; 24%) or relapse (n=41; 31%). Leukemia-free survival (LFS), overall survival, and TRM were not significantly different between the karyotype subgroups studied. LFS at 6 months, 1 year, 3 years, and five years was 62%, 60%, 50%, and 41% for patients with a normal karyotype; 63%, 47%, 40%, and 28% for patients with Ph+; and 61%, 40%, 27%, and 27% for patients with other aberrations. 4/5 patients with t(4;11) and 4/6 patients with complex chromosomal abnormalities are alive in CR. In conclusion, there is no adverse impact of classical poor risk karyotypes on outcome within this high-risk group of ALL patients after allogeneic stem cell transplantation. In contrast, there was a trend that patients with t(4;11) and complex chromosomal anomalies did better (4/5 alive, 1/5 TRM and 4/6 alive, 1/6 TRM, 1/6 relapse, respectively) than patients with other aberrations (8/22 alive, 8/22 death due to relapse, 6/22 TRM). High-risk ALL patients with poor risk cytogenetics are candidates for allogeneic stem cell transplantation which can be curative. Whether other conditioning regimens or adoptive immunotherapy can reduce relapse rate in this patient group is a matter of ongoing clinical research.

Description: Purpose: 115 patients with acute lymphoblastic (n = 56) and myeloid leukemia (n = 59) underwent allogenic stem cell transplantation (SCT) from HLA-matched related or unrelated donors between 1998 and 2003. 52/115 patients (45 %) received donor lymphocyte infusions (DLI) for prophylaxis or treatment of relapse after SCT. Subject of this study was to assess efficacy and toxicity of DLI in these patients. Methods: 83/115 patients were transplanted after standard high-dose radio- and chemotherapy (standard SCT) and 32/115 after reduced intensity conditioning (RIC) with fludarabine, busulfan and ATG. 47/115 patients were in CR-1, 22/115 were in CR-2 and 46/115 patients had advanced stages at the time of transplant. DLI were given to 52 patients, 17/52 patients after RIC and 35/52 patients after standard SCT. Indications for DLI were 1. prophylaxis of relapse in 40/52 patients either after RIC (n = 14) or in high-risk leukemias after standard SCT (n = 26; Ph+ ALL in CR-1, AML and ALL 〉 CR-1) and 2. treatment of hematologic relapse in 12/52 patients. DLI were given in escalating doses every 4 weeks. Chimerism analysis of leukocyte subpopulations were performed before and after DLI by multiplex PCR. Patients with refractory leukemia or those who died before day 60 after SCT were not included in this analysis. Results: 52/115 patients received 103 DLI with a median of 2.7 x 107 CD3+ cells/kg. 29/52 patients (56 %) are alive with a median follow-up of 22 months (range 3 – 69 months); 24 (46 %) are in CR and 5 (10 %) are alive in relapse. The 3 year-probability of overall survival (OS) for all patients treated with DLI is 53 % versus 66 % for patients not treated with DLI (not significant). Patients with ALL treated with DLI have a 3 year probability of overall survival (OS) of 68 % versus 46 % for ALL-patients, who did not receive DLI (p=0.03). For patients with AML, 3 year probability of OS is 41% after DLI versus 78% for patients not treated with DLI (p=0.03). The differences in patients with AML can probably be attributed to the fact that 55 % of those receiving DLI were transplanted in relapse compared to only 30 % of those not receiving DLI. In ALL patients equal number of patients were transplanted in relapse (DLI: 43%; no DLI: 38%). 35/49 patients with available chimerism analysis had mixed chimerism before 1. DLI. After DLI 16/30 patients (53 %) with available analysis had converted to full donor chimerism. All patients who received DLI for leukemic relapse died due to disease progression. 34/52 patients (65 %) developed acute GVHD after administration of DLI, with 7/52 patients (13 %) having grade III/IV. Conclusions: In retrospective analysis DLI seem to be an effective treatment strategy with acceptable toxicity to prevent relapse in high-risk leukemias. To monitor the effect of prophylactic DLI Chimerism analysis is a valuable tool. Patients transplanted with advanced disease have a very high relapse risk independent of the administration of DLI. DLI are not suited to treat leukemic relapse.

Description: Abstract LBA-5 Intra-clonal, mutational complexity is a hallmark feature of cancer and provides the substrate for sub-clonal selection, progression and therapeutic resistance. There is limited insight however into detailed sub-clonal, genetic architecture in cancers and their propagating ’stem’ cells. Childhood acute lymphoblastic leukaemia (ALL) genotypes are characterized by chimeric fusion genes (or hyperdiploidy) coupled with recurrent, copy number alterations (CNA), primarily in genes regulating cell cycle or differentiation. For ETV6-RUNX1-positive cases of B precursor ALL, the temporal sequence of events is known with the fusion gene usually arising as a pre-natal, initiating event. Recurrent CNA, presumed to be functional or ’driver’ mutations, are secondary to gene fusion and probably post-natal in origin. We have interrogated clonal architecture by multiplexed FISH analysis of single cells using probes labelled with three or four distinct fluorochromes. We pre-selected diagnostic samples from 30 ETV6-RUNX1-positive cases that we found to have deletion of ETV6 and CDKN2A or PAX5 or combinations of these. The application of multiple FISH probes allowed us to score all cells (200 per patient sample) for up to six genetic lesions: ETV6-RUNX1 fusion (and multiple copies of the fusion), deletion of the unrearranged ETV6 allele, extra copies of chromosome 21q (via RUNX1 signals), and one or two copy deletions of PAX5 and CDKN2A. The genetic classification of individual cells using this method allowed a designation of sub-clones and the assembly of putative ancestral, or evolutionary, trees. Although sub-clones were previously known to exist at diagnosis of ALL, the extent of sub-clonal diversity revealed by this study was unanticipated and very marked. Sub-clones, of varying sizes (1–90% of total cells), numbered 3 to 14 per case. This must be an under-estimate of genetic complexity as only selected mutations were included in the screen. The common CNA (ETV6, CDKN2A and PAX5 deletions, chromosome 21q gains) arose independently and recurrently in sub-clones and in no preferential order. Matched relapse and diagnosis pairs were available on 5 patients. In all of these, the clones at relapse could be matched to individual sub-clones at diagnosis, with relapse originating from a major or minor subclone at diagnosis. Importantly, the relapse clone often diversified further, reiterating the evolution pathway of the primary clones. These data indicate that sub-clonal diversification does not arise via linear, clonal succession but rather has a complex, branching architecture reminiscent of Charles Darwin’s 1837 evolutionary speciation model. The ancestral trees so revealed in ALL are single time point snapshots and therefore disguise temporal or sequential dynamics. We show that sub-clonal architecture changes in the months leading up to a diagnosis of ALL and is re-ordered by treatment and subsequent relapse. An important prediction derived from this pattern of sub-clonal architecture is that leukaemia propagating or ’stem’ cells in ALL (and other cancers) should themselves be genetically diverse. Preliminary transplantation experiments in NOD/SCID/IL2Rgamma(null) mice show that this is the case, since multiple, genetically distinct sub-clones regenerated in vivo (Figure1). The complexity of genetic architecture in ALL has substantial implications for the cancer stem cell concept and for the efficacy of generic or targeted treatments. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.