ALBERT

Description: Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Description: Background Diagnosis and prognosis of patients with established MDS/CMML is currently based on WHO criteria and the IPSS prognostic Index. High variation of survival in subgroups warrants the search for additional criteria. A comparison of CFU-C cultures with WHO criteria and IPSS score has not yet been done in a large patient group. Patients and methods We analyzed in a single center retrospective cohort study 93 untreated consecutive patients (55 male/ 38 female; median age: 66 years; range: 13 – 88 years) admitted between July 1992 and June 2002 and diagnosed as MDS/CMML (RA/RARS(4), MDS-U (2), RCMD (26), RAEB I/II (44) and CMML I/II (17). All patients had an unequivocal diagnosis of MDS or CMML according to WHO criteria. Simultaneous examinations of blood, bone marrow (cytology and biopsy), BM-cytogenetics and BM-and PB cultures for CFU-GM and BFU-E were done. Culture results were scored blindly and classified either as “Low risk CFU-C“ including normal growth (N=2), no colony growth (N=6) or reduced growth of normal colonies (N=19) or as „High risk CFU-C’s“ including excess normal growth in PB termed “MPS pattern” (N=5), discrete leukemic cluster growth (N=22), abundant leukemic cluster growth (N=14) or the „CMML pattern“ defined as giant“ pseudonormal“ CFU-GM and strongly reduced BFU-E (N=23). Culture score was compared with the WHO diagnosis and with the IPSS score in all 93 patients and survival was assesed in 82 patients treated with conventional therapy (12 allografted patients were excluded) after minimal observation time of 3 years in July 2005. Results Comparison of WHO diagnosis with culture score Low risk CFU-C score High risk CFU-C score RA/RARS/RCMD/MDS-U 15 16 RAEBI/II 12 30 CMML-I/II 0 17 The typical CMML pattern was observed in 13 of 17 patients with CMML (Specificity 94%). Comparison of IPSS Score with culture score low risk CFU-C score low risk CFU-C score High risk CFU-C score High risk CFU-C score IPSS Score alive/dead mean survival(d) alive/dead mean survival(d) Low and Int-1 4/4 2022+/−543 8/39 965+/−143 Int-2 and High 1/1 1024+/−701 1/26 565+/−92 Mean survival time for patients with a low/intermediate-1 IPSS score was less than half if they had a high risk CFU-score (p

Description: Abstract 182 Chromosomal alterations are a hallmark of acute lymphoblastic leukemia (ALL), but many cases lack a recurring cytogenetic abnormality. To identify novel alterations contributing to leukemogenesis, we previously performed genome-wide profiling of genetic alterations in pediatric ALL using single nucleotide polymorphism (SNP) microarrays. This identified a novel focal deletion involving the pseudoautosomal region (PAR1) of Xp/Yp in 15 B-progenitor ALL cases lacking sentinel chromosomal abnormalities, including six of eight cases of ALL associated with Down syndrome (DS-ALL). The deletion involved hematopoietic cytokine receptor genes, including IL3RA and CSF2RA, but due to poor array coverage, it was not possible to define the limits of deletion using SNP array data alone. To characterize this abnormality, we examined an expanded cohort of 329 B-ALL cases, including 22 B-progenitor DS-ALL cases. Strikingly, 12 (55%) DS-ALL cases harbored the PAR1 deletion. Mapping using high density CGH arrays showed the deletion to be identical in each case, and involved a 320kb region extending from intron 1 of the purinergic receptor gene P2RY8 to the promoter of CRLF2 (encoding cytokine receptor like factor 2, or thymic stromal lymphopoietin receptor). The deletion resulted in a novel fusion of the first, non-coding exon of P2RY8 to the entire coding region of CRLF2 in each case. The P2RY8-CRLF2 fusion resulted in elevated expression of CRLF2 detectable by quantitative RT-PCR, and flow cytometric analysis of leukemic cells. One DS-ALL case with elevated CRLF2 expression lacked the PAR1 deletion, but had an IGH@-CRLF2 translocation detected by fluorescence in situ hybridization (FISH). CRLF2 alteration was associated with gain of chromosome X (which was shown by FISH to result in duplication of the PAR1 deletion), deletion of 9p, and the presence of Janus kinase (JAK1 and JAK2) mutations. Ten (53%) of patients with CRLF2 alteration had JAK mutations, compared with two patients lacking CRLF2 abnormalities (P

Description: Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Advances in therapy, in particular stratification of patients to appropriate treatment according to risk factors improved long term survival attaining cure rates of up to 80 %. Despite the efforts achieved by the stratification strategies the majority of relapse patients are recruited from the low risk groups emphasizing the need for additional independent risk factors. In a recent study we transplanted primary leukemia samples obtained from pediatric patients with newly diagnosed B cell precursor ALL (BCP-ALL) into NOD/SCID mice. Time to leukemia (TTL) was analyzed for each patient sample transplanted from date of transplant to date of leukemia manifestation in the recipients. We demonstrated that patients whose leukemia cells engrafted rapidly leading to manifestation of the disease within 10 weeks (TTLshort) showed a clearly inferior relapse free survival in contrast to patient samples with prolonged in vivo growth (TTLlong). Interestingly, the same distinct difference in relapse free survival was observed in the low risk groups only. Multivariate analysis showed an almost 45- fold increased risk for relapse in TTLshort patients. In order to further characterize the biological properties of the leukemia cells in the two groups, gene expression profiles of samples with short versus long TTL in the xenograft model were analyzed using a human whole genome array (Affymetrix U133 Plus 2.0). Here, we used quantitative traits analysis (QTA) correlating gene expression values (relative expression) to the time from transplant to manifestation of leukemia in the NOD/SCID mice (TTL, in weeks). 14 different xenograft samples (TTLshort n= 7, mean TTL 8.14 weeks; TTLlong n= 7, mean TTL 19.71 weeks; T-test P = .03) isolated from leukemia bearing mice were investigated. All 14 patients included were stratified in low (standard or intermediate) risk groups. All patients were negative for TEL/AML1- fusion, BCR/ABL- fusion or MLL rearrangement. By QTA we identified 5 genes that were significantly correlated (Spearman correlation, for all 5 genes P 〈 .0001) to time from transplant to leukemia manifestation in the recipient mice (TTL). Analysis of the 14 xenograft samples using the 5 genes identified showed clustering of all but one sample in one group. The 5 genes were than explored for their power to predict relapse in an independent cohort of pediatric ALL patients. For these patients expression profiles were analyzed in leukemia samples obtained at diagnosis. All patients in this cohort were also stratified in low risk groups and negative for the presence of TEL/AML1-, BCR/ABL- or MLL- fusion transcripts. 8 of the 38 patients encountered relapse, 5 at early and 3 at late time points (〈 or 〉 24 months after diagnosis). Clustering according to the 5 genes identified by QTA lead to separation of the patients into two groups. Interestingly, all relapsed patients except one clustered in the group associated with the signature characteristic for TTLshort. Most importantly, all 5 patients with early relapse gathered in this cluster. Taken together, we used a novel approach directly correlating gene expression values to the continuous variable time to leukemia (TTL) which might be reflected more accurately by this strategy than comparing two groups. We applied this gene signature characteristic for TTLshort, thereby identifying poor overall survival in a set of independent pediatric ALL patients and found clustering of all early relapse patients in one group. This new independent risk factor might identify patients with high risk for early relapse avoiding xenografting in the mouse model.

Description: Practice guidelines for use of an erythropoietic agent (EpA) have been previously developed for patients with all cancers, but these have not specifically addressed non-myeloid hematological malignancies. Given issues of benefit, cost, access to treatment and practice variation for these patients, the Cancer Care Ontario Program in Evidence - Based Care (CCO PEBC) conducted a systematic review and has developed a practice guideline. Entries to MEDLINE, CANCERLIT, EMBASE, the Cochrane Library databases, and abstracts of the American Societies of Clinical Oncology (ASCO) and Hematology (ASH) were searched. A hierarchy of outcomes was developed that included survival, quality of life (QoL), transfusion requirements and improvements in hemoglobin concentration/hematocrit. To validate conclusions and recommendations, the practice guideline was sent to Ontario practitioners in July 2006; responses are now being collated. Eighteen reports (14 articles, 4 abstracts) of randomized trials met eligibility criteria. None of the 3 trials reporting survival outcomes detected a benefit with use of an EpA. Of 7 trials evaluating QoL, 6 reported superior outcomes in patients receiving an EpA. None of those trials sufficiently reported methodologic parameters required by proposed guidelines for analyzing, interpreting and reporting QoL measures. Use of an EpA significantly reduced the proportion of patients transfused in 5 trials evaluating this outcome; reductions ranged from 15% to 40%. The absolute risk reduction ranged from 15% to 24%; the number needed to treat to prevent a transfusion ranged from 4 to 6. Use of an EpA was not associated with a reduction in the mean/median number of units transfused. In 10 studies, either a statistically significant increase in the hemoglobin concentration/hematocrit or in the proportion of patients with an increase in the hemoglobin concentration/hematocrit was seen. For this practice guideline, we interpreted the evidence as providing insufficient data to justify use of an EpA in order to improve survival or QoL. However, there are compelling data showing that use of an EpA reduces the risk of requiring a transfusion. Initiatives such as the Commission of Inquiry on the Blood System in Canada (“the Krever Commission”), state that alternatives to transfusion be offered to patients because of associated known and potentially unknown adverse consequences of blood products. Thus, the use of an EpA is recommended as an alternative to transfusion. However, the decision to use an EpA to reduce transfusion requirements should primarily consider individual patient values and the likelihood that a patient will require a transfusion so that patients are not exposed to unnecessary treatment and the health care system to additional costs.

Description: Leukaemias with MLL gene rearrangement are usually considered prognostically unfavourable and the clinical symptoms typically follow the translocation formation rapidly. MLL rearrangement is thus thought to be a major hit in leukaemogenesis that is either sufficient to cause the disease or it is a very strong and rapid inducer of the subsequent hit(s) required for the malignant transformation. We report an unusual presentation of secondary acute lymphoblastic leukaemia (sALL) with MLL rearrangement. Our patient was diagnosed originally with acute myeloid leukaemia (AML-M3) characterised by PML/RARα fusion and an internal tandem duplication of FLT3 (FLT3/ITD). After 30 months of complete remission of AML, she developed sALL with MLL/FOXO3A fusion gene. Bone marrow (BM) samples taken during AML therapy were analysed for the presence of these aberrations. Both the PML/RARα fusion and FLT3/ITD disappeared shortly after AML onset and did not reappear. However, FISH and quantitative RT-PCR showed the presence of the MLL/FOXO3A fusion 20 months before the diagnosis of sALL, present in 10–90% of BM cells. Morphological examination showed no blast infiltration of the BM at this time. Experiments combining FISH and morphology confirmed the presence of an MLL rearrangement in myeloid as well as lymphoid cells, indicating that the fusion arose in a multipotent progenitor. In order to identify potential secondary genetic events precipitating sALL in this patient, we used Affymetrix 50K single nucleotide polymorphism (SNP) array analysis on DNA from the diagnostic sALL sample versus the "preleukaemic" (remission AML) sample taken 16 months before. This analysis revealed a 10 Mb amplification on 19q13.32 in the sALL sample, not present in the preleukaemic sample: this was confirmed by FISH with a BAC from the amplified region. A difference between the pre-leukaemic and leukaemic cells is also demonstrated by the incomplete rearrangement of IgH gene (DH1/JH) present only at the diagnosis of sALL. There are about 450 genes in the amplified region on 19q and several of them might be involved in deregulation of the preleukaemic cell if overrepresented (e.g. FLT3 ligand, interleukin 11, Ras interacting protein 1, Stem cell growth factor, Aurora C). The long latency period prior to the onset of the secondary leukaemia in our case resembles the mouse model of MLL/FOXO3A. However, in contrast to the animal model and also to the previous reports of MLL/FOXO3A patients (2 cases described so far, both secondary AMLs after Hodgkin’s disease), our child developed leukaemia from the lymphoid lineage. Taken together, these results indicate that the MLL/FOXO3A fusion alone is not sufficient to cause leukaemia and that second hit is required to the onset of the disease. A responsible gene is possibly located on the telomeric part of the 19q.

Description: Background: The EXtended CLinical prophylaxis in Acutely Ill Medical patients (EXCLAIM) trial was a randomized, double-blind, placebo-controlled, multicenter, international study that demonstrated a 38% relative risk reduction (RRR) for venous thromboembolism (VTE) with extended-duration enoxaparin prophylaxis compared with placebo (2.5% vs 4.0%; absolute difference [AD], −1.5% 95.8% CI −2.5 to −0.5%; P=0.002). Major bleeding occurred in 0.7% (20/2975) and 0.2% (7/2988) of patients who received enoxaparin and placebo, respectively (AD, 0.4% [CI 0.1% to 0.8%]; P=0.012). As age is a known independent risk factor for VTE, we conducted a pre-specified sub-analysis of the EXCLAIM trial to compare the efficacy and safety of extended-duration enoxaparin prophylaxis in patients 〉75 years old with patients ≤75 years old. Methods: EXCLAIM eligibility required a recent (≤3 days) reduction in mobility due to acute medical illness, an anticipated level 1 (total bed rest/sedentary) or level 2 (level 1 with bathroom privileges) decreased mobility, and age ≥40 years. During the latter part of the study, a protocol amendment required patients with level 2 mobility to have ≥1 of 3 additional pre-specified risk factors (i.e., active or prior cancer, history of VTE, age 〉75 years). Of the 7500 patients enrolled, 7415 received open-label enoxaparin 40 mg SC od for 10±4 days. Of these, 6085 were randomized to double-blind therapy (enoxaparin 40mg SC od or placebo) of 28±4 additional days duration. The incidence of VTE, the primary efficacy end point, was defined as the composite of symptomatic or asymptomatic proximal deep vein thrombosis (DVT), symptomatic pulmonary embolism (PE), or fatal PE during the double-blind treatment period. Patients were screened for DVT with bilateral proximal lower extremity compression ultrasound at the end of randomized therapy. The incidence of major bleeding, the primary safety end point, was assessed through 48 hours after the last dose of study treatment. Results: Of the 5963 randomized patients that received at least one dose of double-blind therapy, 29.9% (1781) were 〉75 years of age (mean age 81.5 years) and 70.1% (4182) were ≤75 years old (mean age 61.8 years). In patients 〉75 years old, the incidence of VTE was 2.5% (18/725) in the enoxaparin group compared with 6.7% (50/743) in the placebo group (AD −4.2% [95.8% CI −6.5 to −2.0%]; P75 years old (0.6% vs 0.2%; AD 0.3%, 95% CI −0.2 to 0.9%; P=0.282) and significantly higher in patients ≤75 years old (0.7% vs 0.2%; AD 0.5%, 95%CI 0.1 to 0.9%; P=0.041). Though the older group had a higher death rate compared to the younger group (3.2% vs 1.6%), the survival between the treatment groups was similar within the two age groups. Without extended prophylaxis (i.e., placebo group), patients 〉75 years old had a significantly higher risk of VTE than those 75 years of age.

Description: Abstract 2060 Poster Board II-37 Introduction: The Flt3-internal tandem duplication can be found in up to 30% of all acute myeloid leukemia (AML) patients and confers a poor risk status characterized by an increased relapse rate and poor overall survival. Moreover, Flt3-ITD-positive AML patients relapsing after allogenic stem cell transplantation (SCT) have very limited therapeutic options. Sorafenib is a multikinase inhibitor that is approved for the treatment of metastatic renal cell and hepatocellular carcinoma. Besides targeting Raf, the platelet derived growth factor receptor (PDGFR) and the vascular endothelial growth factor receptor (VEGFR) it has also significant inhibitory activity against the Flt3 receptor tyrosine kinase, and, specifically the mutated variant of Flt3, Flt3-ITD. It has previously been shown that sorafenib monotherapy may have considerable activity in relapsed Flt3-ITD positive AML. Nevertheless, clinical experience is still limited. Here we report compassionate use experience on 18 relapsed or refractory Flt3-ITD positive AML patients treated with sorafenib monotherapy. Methods: A questionnaire was developed and sent to 28 centers in Germany in order to obtain more insight into the clinical efficacy and tolerablilty of sorafenib monotherapy in Flt3-ITD positive AML. Forms were returned from 13 centers, reporting 26 patients. Among them, eight had to be excluded from further analysis. Five of them were Flt3-ITD mutation negative and three received contemporary chemotherapy. Available patient information included age, FAB-classification, karyotype, type and response to prior therapy, sorafenib dosing, tolerability, treatment duration, and response. Results: Of the 18 patients (12 male, 6 female), five were primary refractory to induction chemotherapy and 13 received sorafenib in first (n=11) or second (n=2) relapse. Eight of 18 patients relapsed after SCT and were treated with sorafenib. One patient was treated for steadily increasing Flt3-ITD copy numbers, that is, in molecular relapse after SCT. Patients received between 200mg and 800mg sorafenib p.o. daily. The median treatment duration was 98 days (range, 16-425 days). All patients achieved a hematological response (HR) characterized by complete (n=17) or near complete peripheral blast clearance (n=2). Of the 18 patients the documented best response to sorafenib were: HR in 9 cases, bone marrow response (HR and blast reduction in marrow) in 4 cases, complete remission (normalization of peripheral blood counts and bone marrow blasts 〈 5%) in one case and complete molecular remission (molecular negativity for Flt3-ITD) in 4 patients. After a median treatment duration of 180 days (range, 82-270 days) 7 of the 18 (39%) patients developed clinical sorafenib resistance: two of eight (25%) of the SCT-group and 5 of 10 (50%) of the non-SCT group. Sorafenib was generally well tolerated. Pancytopenia or thrombocytopenia grade III and IV were the most significant side effects, observed in 13 patients. Other reported side effects such as diarrhea, exanthema were documented from the centers as being minor. Conclusion: Sorafenib monotherapy has significant clinical activity in Flt3-ITD positive relapsed and refractory AML and may be particularly effective in the context of allo-immunotherapy where 3 CMR could be seen. Disclosures: Enghofer: Bayer Schering Pharma: Employment. Off Label Use: sorafenib, used to treat Flt3-ITD positive AML patients.

Description: Introduction. The NCIC CTG is conducting phase II testing to establish the tolerability and estimate the efficacy of combining lenalidomide (L) + melphalan (M) over multiple cycles for previously untreated patients with multiple myeloma who are ineligible for stem cell transplantation. We report the data from the preliminary safety phase of this trial. Methods. The MY.11 trial was initially designed as a randomized phase II test of M 9 mg/m2 given on days 1–4 (M9) plus either L 10 mg (L10) or 20 mg given on days 1–21 of a 28 day cycle. Primary prophylaxis with G-CSF was not included, but use of G-CSF for established neutropenia was permitted; our intent was to determine dose levels that could be given independently of G-CSF. A dose attenuation schedule was developed for neutropenia and/or thrombocytopenia that was severe and occurred during the administration of L or was persistent and caused a delay in the next treatment cycle. A hematologic dose limiting toxicity (H-DLT) was defined as the need for 2 dose attenuations of L or 1 dose attenuation of M. Prior to the randomized phase of the trial, a safety run-in phase testing M9+L10 in 6 patients was planned; closure of this arm was to occur if, during the first 3 treatment cycles, there were ≥ 3 H-DLTs or any non-hematologic serious adverse event (SAE) judged to be treatment related. Four of the planned 6 patients were entered; during their first 3 treatment cycles, each experienced a H-DLT and one died from sepsis. The study was therefore amended with the intent to conduct a randomized phase II test of M 4 mg/m2 days 1–4 plus L 15 mg days 1–21 (M4L15) and M 6 mg/m2 days 1–4 plus L 10 mg days 1–21 (M6L10). Prior to the randomized phase of testing, each regimen was tested in non-randomized safety run-ins that included 6 patients per arm. The same parameters as above for study arm closure were applied. If the M6L10 arm was closed, the trial would proceed to test M 5 mg/m2 days 1–4 and L 10 mg days 1–21 (M5L10). Results. Six patients were enrolled to M4L15: during their first 3 treatment cycles, 4 patients experienced H-DLTs related to severe or persistent grade 3–4 neutropenia and one other patient experienced an SAE related to hallucinations and multiple constitutional complaints. This arm was therefore closed to accrual. Six patients were enrolled to M6L10: during their first 3 treatment cycles, 3 patients experienced H-DLTs related to severe or persistent grade 3–4 neutropenia. One of these patients was hospitalized with pulmonary edema and another with febrile neutropenia. This arm was therefore closed and the MY.11 trial amended to proceed as a single-arm phase II testing M5L10. Conclusion. Although M + L holds promise as an effective combination for previously untreated myeloma patients, the regimen is associated with considerable myelosuppression. The efficacy of the combination when given at tolerable doses remains to be established.

Description: Abstract 5040 Introduction Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and patients with RAS mutations benefit most from high-dose cytarabine as postremission therapy (Neubauer et al., J Clin Oncol 2008). The molecular reason for this phenomenon is not well understood. Methods We used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. In addition, we used primary human inversion 16 positive AML samples with or without oncogenic RAS mutations to corroborate our findings. Results There was no difference in cell cycle kinetics, apoptosis or cellular senescence in MLL-ENL cell expressing control vector vs. MLL-ENL cells expressing oncogenic RAS in response to cytarabine treatment. However, we observed an increased activation of DNA damage checkpoints in MLL-ENL-cells expressing oncogenic RAS after incubation with chemotherapeutic agents. This resulted in an Atm/r as well as p53-dependent genetic program causing dramatically reduction of clonogenicity in MLL-ENL cells expressing oncogenic RAS due to induction of myeloid differentiation. Co-expression of dominant – negative p53 completely abolished this differentiation resulting in the rescue of the clonogic potential. Activation of p53 as a result of inhibition of Mdm2-mediated degradation of p53 further enhances this myeloid differentiation. Of note, in primary AML cases, oncogenic RAS also was associated with a more differentiated phenotype. Conclusions The data can explain the beneficial effects observed in AML patients with oncogenic RAS mutations treated with high dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy since stronger acitvation of p53 by oncogenic RAS and chemotherapeutic agents cause the differentiation of hematopoietic stem cells to more differentiated progenitors. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells. Disclosures No relevant conflicts of interest to declare.