ALBERT

Description: Precursor T-cell acute lymphoblastic leukemia (T-ALL) in children represents a clinical challenge, because relapses are usually fatal. It is thus necessary to identify high-risk patients as early as possible to effectively individualize treatment. We aimed to define novel molecular risk markers in T-ALL and performed array-based comparative genomic hybridization (array-CGH) and expression analyses in 73 patients. We show that DNA copy-number changes are common in T-ALL and affect 70 of 73 (96%) patients. Notably, genomic imbalances predicted to down-regulate the TGF-β or up-regulate the PI3K-AKT pathways are identified in 25 of 73 (34%) and 21 of 73 (29%) patients, suggesting that these pathways play key roles in T-ALL leukemogenesis. Furthermore, we identified a deletion at 6q15-16.1 in 9 of 73 (12%) of the patients, which predicts poor early treatment response. This deletion includes the CASP8AP2 gene, whose expression is shown to be down-regulated. The interaction of CASP8AP2 with CASP8 plays a crucial role in apoptotic regulation, suggesting a functional link between the clinical effect of the deletion and the molecular mode of action. The data presented here implicate the TGF-β and PI3K-AKT pathways in T-ALL leukemogenesis and identify a subgroup of patients with CASP8AP2 deletions and poor early treatment response.

Description: Abstract 2773 Introduction: For patients with high-risk myelodysplastic syndromes an epigenetic therapy with hypomethylating agents is considered standard of care. Intensive chemotherapy can be offered to a subset of patients; however, data about the long-term outcome of MDS patients receiving intensive chemotherapy are scarce. Methods: For this evaluation, 104 adult patients with IPSS intermediate-2 or high-risk MDS with at least 10% bone marrow blasts of all age groups treated within the AMLCG1999 trial were included. Patients were randomized upfront to receive 1. double induction therapy with either standard-dose containing TAD - versus high-dose containing HAM–HAM, 2. TAD consolidation therapy followed by either a monthly maintenance therapy for 3 years after achievement of CR or an autologous stem cell transplantation (patients aged ≥ 60 years were all assigned to maintenance therapy), and 3. blast priming with filgastrim starting on day -1 of chemotherapy in selected centers. Results: Fifty-four patients had IPSS Score intermediate-2 and 50 patients were IPSS high risk. Median bone marrow blast count at diagnosis was 15%. The median age was 63.5 years (range: 27–76 years), 39 patients (37.5 %) were female. Median lactate dehydrogenase (LDH) serum level was 296 U/l, median leukocyte count at diagnosis was 5,950 per μl. The cytogenetic risk groups were as follows: favorable 3, intermediate 57, unfavourable 37, missing 7. Among 38 patients with normal karyotype, NPM1/FLT3 mutational status was available for 22 with 5 patients having the combination NPM1 mutated/FLT3 wildtype. Comparison with 2051 patients with de novo AML within the same trial revealed the following significant differences: patients with MDS were older, had a higher male to female ratio, a lower LDH serum level at diagnosis, a lower leukocyte count at diagnosis and were more likely to have adverse cytogenetic risk. Compared to 636 patients with secondary AML after MDS, cytotoxic therapy or irradiation, the cohort of patients with MDS did not display any significant differences except the sex distribution. Patients with MDS displayed a CR rate of 48% (50/104 patients), which was significantly lower than de novo AML patients (67%) and not different to secondary AML patients (47%). Median overall survival in MDS patients was 320 (95% CI: 236 to 505) days with a 2-year and 5-year survival of 33.4% (95% CI: 23.6% to 43.2%) and 22.7% (95% CI: 13.5% to 31.9%), respective, which was significantly (p=0.03) lower than in patients with de novo AML (median 484, 95% CI 435 to 541 days) and comparable to patients with secondary AML (median 282, 95% CI 224 to 311 days, p=0.13). Median relapse-free survival in responding MDS patients was 536 (95% CI: 264 to 1299) days with no significant differences of RFS compared to de novo or secondary AML patients. In multivariate analyses, the diagnosis of MDS remained an independent prognostic factor for CR probability but had no independent influence on survival compared with de novo AML patients. Nine patients proceeded to allogeneic stem cell transplantation in first complete remission of whom six remain in first complete remission between 1354 and 1911 days after achievement of CR. In addition, 16 patients remained in CR for more than one year without allogeneic transplantation. Discussion: Taken together, outcome of patients with intermediate-2 or high-risk MDS after intensive chemotherapy is comparable to the outcome of patients with secondary AML. Adjustment for known risk factors such as age, cytogenetic risk and LDH revealed that inferior outcome of MDS patients compared to patients with de novo AML is attributable to the higher incidence of adverse risk factors. CR-rates appear to be higher compared to hypomethylating therapy and a fraction of MDS patients experiences long-term survival by intensive chemotherapy. Allogeneic transplantation can improve long-term survival for patients achieving remission. Disclosures: Krug: MedA Pharma: Honoraria; Novartis: Honoraria; Alexion: Honoraria; Boehringer Ingelheim: Research Funding; Sunesis: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: The molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9; 22) and t(4; 11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12; 21)(p13; q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12; 21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12; 21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre–B immunophenotype. Among the B-lineage subgroup, the t(12; 21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (≥1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children were investigated for the presence of TEL/AML1 fusion gene and 99 cases (28.9%) were positive. The patients expressing the TEL/AML1 fusion mRNA appeared to have a better event-free survival (EFS) than the patients who lacked this chimeric product. Whereas three of the TEL/AML1 positive cases (3.0%) have relapsed to date, 27 patients without TEL/AML1 rearrangement (11.1%) suffered from relapse. To date, the only subset of B-lineage ALL with a favorable prognosis has been the hyperdiploid group (DNA index ≥1.16

Description: Dose density during early induction has been demonstrated to be one of the prime determinants for antileukemic efficacy. The German AML-CG therefore pilots a dose dense induction regimen S-HAM (sequential HD-AraC [3g/m2/12h d1,2,8,9] and Mitoxantrone 10mg/m2 [d3,4,10,11] followed by pegfilgrastim) in which two induction cycles are applied over 11 – 12 days as compared to conventional double induction, in which two cycles are applied over 25 – 29 days - thereby increasing dose density ca. two-fold in the critical first weeks of treatment. In the past 2,5 years 168 patients with de-novo AML (excluding APL) have been recruited into the trial with a median age of 53 years (range 18 – 78). Of 136 patients evaluable for response the following results were achieved: CR 62%, CRi 22%, PL 7%, ED 9% - resulting in an overall response rate (ORR) of 84%. The early death rate (ED) of 9% and the toxicity profile compared favourably with a historical control group of the AML-CG 1999 study (de-novo AML, 〈 60 years, HAM-HAM double induction) which demonstrated an ED rate of 14% (ORR 68%, persistent leukemia (PL) 18%). The high antileukemic efficacy of S-HAM was also demonstrated by the fact that 89% of patients had a blast count of 〈 10% one week after therapy as compared to less than 48% of patients of the HAM-HAM double induction group. Whereas even for patients with unfavourable cytogenetics (including complex aberrations) a median overall survival of 13,5 months was reached (23% at 2 years), for patients with favourable karyotypes overall survival at 2 years was 81%and for patients with intermediate karyotypes 74% after S-HAM treatment. Importantly the compression of the two induction cycles into the first 11 – 12 days of treatment seems actually beneficial for normal hematopoesis as demonstrated by a significantly shortened duration of critical neutropenia of 30 days as compared to 45 days after conventionally timed double induction. This shortening of critical neutropenia by more than 2 weeks was highly relevant for the duration of hospital stay and hospital costs. In conclusion S-HAM with pegfilgrastim support is a highly effective regimen in primary de-novo AML with a very favourable safety profile and significantly shortened duration of neutropenia. This regimen will therefore constitute the (dose-dense) experimental arm for a randomized comparison with standard double induction in the next generation of the German AML-CG studies.

Description: A female, 48-year old patient was referred to an emergency ward with dyspnoea, haemoptysis and suspected respiratory infection that had been refractory to prior antimicrobial therapy. The onset of dyspnoea dated back to a febrile episode three months prior to presentation. The patient continued to work in spite of severe shortness of breath. Her history revealed a cervical cancer which had been treated by neoadjuvant radiochemotherapy and extended hysterectomy in 2005. Upon admission, extensive spontaneous haematomas were noted on the upper extremities. Following a diagnostic puncture of the radial artery, severe haemorrhage and compartmentalization developed, and the patient was transferred to our hospital for a surgical emergency intervention and haemostaseological counselling. A decreased factor VIII activity was noted, with a prolonged partial thromboplastin time, a normal von Willebrand Factor activity and concentration, a prolonged bleeding time and a low-titer inhibitor to factor VIII. Additionally, a profound hyponatremia (117 mmol/l) awaited explanation. Haemorrhage was treated with recombinant Factor VIIa, allowing for a successful surgical de-compartmentalization. With regard to the consistent feature of dyspnoea and partial respiratory insufficiency, bronchoscopic examination revealed only minor hemorrhagic suffusion of the bronchial epithelium, but no sign of infection or major pulmonary bleeding. Chest X-ray and laboratory findings showed right ventricular enlargement, bilateral infiltrates and an excessively high NT-pro-BNP concentration in plasma (13027 ng/l). Chest X-ray radiographs performed two months previously for suspected respiratory infection showed pulmonary infiltrates of comparable size, albeit no right ventricular enlargement. Computerized tomography of the chest showed a complete obstruction of the right pulmonary artery and of the lower branch of the left pulmonary artery. Pulmonary arterial pressure was measured at 70 mm Hg. Immunosuppressive therapy was initiated, with concomitant aPTT-adjusted anticoagulation. Upon clinical deterioration with right-ventricular failure, a catheter-based disruption of the emboli was attempted, albeit with no success. A cardio-surgical intervention with thrombendectomy was performed successfully; however, the patient succumbed to sepsis and multiorgan failure ten days later. The autopsy revealed multiple thrombi, a bilateral pulmonary haemorrhagic infarction and a disseminated lymphangiosis and hemangiosis carcinomatosa of the lungs. We conclude that the acquired inhibitor to factor VIII was of paraneoplastic origin and, although detected at a low titre, clinically relevant. Based on previous radiographic findings, a massive pulmonary embolism must be assumed to have occurred months prior to presentation. The anticoagulatory effect of the acquired inhibitor to Factor VIII may have averted the timely detection of the pulmonary embolism.

Description: In childhood acute lymphoblastic leukemia (ALL), early response to therapy is of crucial prognostic significance. In the frontline ALL-BFM (Berlin-Frankfurt-Münster) trial, treatment stratification is based on blast count estimation in peripheral blood at day 8 of induction prephase with prednisone and one dose of intrathecal methotrexate at day 1. To approach yet unknown mechanisms of therapy resistance and to characterize cells persisting under therapy on molecular level, we investigated gene expression profiles of leukemic blasts at day 8 of therapy (“day 8” cells) and their changes as compared with blast cells at initial diagnosis (“day 0” cells). To this end, an experimental procedure has been established including flow sorting of leukemic cells by their leukemia-associated immunophenotype and preparation of cRNA, starting from a small number of cells and using an additional amplification step. Experiments have shown that flow sorting procedure does not affect RNA quality, and this experimental approach facilitates investigation of patient samples with blast cell counts as low as 50 blast cells/μl. Blast cells from ten patients with B-cell precursor ALL were investigated using Affymetrix HG U133A microarrays, and gene expression data were processed by normalization procedure on probe level by variance stabilization. Genes commonly up- or downregulated in blast cells under therapy were identified in matched pairs of day 8 and day 0 samples using Significance Analysis of Microarrays (SAM) and a filtering criterion of at least two-fold mean change. By this procedure a group of 84 genes with a false discovery rate of less than 10 % was identified. In this group, 24 genes, reportedly involved at different levels of the cell cycle regulation, including cell cycle progression (CDC2, cyclin B2), DNA replication (e.g. thymidylate synthetase TYMS, ribonucleotide reductase RRM2, proteins MCM 4 and 6) and execution of mitosis (e.g. cell cycle checkpoint kinases CHEK1 and BUB1B, kinesin-like proteins 1 and 7, and MAD2L1), were found to be downregulated in the day 8 cells. In contrast, genes (n=7) encoding for proteins involved in survival signaling (e.g. IL-4/IL-13 common receptor chain, membrane-spanning 4-domains MS4A1 and CD20) showed increased expression in day 8 blasts. Taken together, the described experimental approach enabled gene expression analysis of ALL cells persisting under therapy and pointed to increased survival signaling in day 8 blasts and their preferential positioning in the G1 cell cycle phase.

Description: Myeloid sarcoma or chloroma, an extramedullary myeloid tumor, is observed in a minority of patients with acute non-lymphocytic leukaemia. Very few cases may present without bone marrow involvement (i.e., primary myeloid sarcoma). Among the various sites of disease manifestation reported, solitary bone lesions may occur. Diagnosis and, even more so, assessment of response to systemic therapy is strikingly more difficult than in systemic disease, i.e. acute leukaemia. We report the case of a patient diagnosed with primary myeloid sarcoma involving thoracic vertebral bones and associated with spinal cord compression. Here, positron emission tomography/computer tomography imaging allowed assessment of the initial extent of the disease as well as the response to a combined radiochemotherapeutic approach. A male, 60 years old patient presented with pain in the flanks extending to the left leg. MRI imaging revealed an intraspinal mass in close proximity to the eleventh thoracic vertebral bone. Surgical resection of the mass including biopsy of the vertebral bone allowed the histopathological diagnosis of a myeloid sarcoma. Upon presentation, the assessment of bone marrow and meningeal involvement yielded negative results. MRI imaging revealed changes suggestive of residual manifestations albeit remaining inconclusive with regard to an enhancement secondary to the surgical intervention. PET/CT imaging revealed a distinct signal located to the eleventh vertebral bone as a result of enhanced metabolic turnover (SUV 8.8) that was interpreted as active myelosarcoma tissue. Induction chemotherapy was initiated consisting of high-dose Ara C and Mitoxantrone (HAM). Following induction therapy, PET/CT assessment showed no change in metabolic activity. Extended field radiation therapy was performed, involving the two adjacent upper and lower vertebral bones, with a total dose of 30 Gy. This time, response assessment via PET/CT showed only a slight enhancement in metabolic turnover, suggestive of remission. As a consolidation, high dose Ara C therapy was initiated. Until now the patient is fine and shows no sign of residual myeloid sarcoma. PET/CT in this case was the only method which reliably allowed the assessment of tumor location, extent and activity in a previously operated vertebral bone and, as a tool for the assessment of response, was guiding therapeutic decisions. In our opinion, the exposure to substantial radiation exposure due to PET/CT imaging is outweighed by the obvious diagnostic benefit.

Description: In vivo response to initial therapy, as assessed by determination of minimal residual disease after five and 12 weeks of treatment, has evolved as one of strong prognostic factors in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. It is currently not known if the individual treatment response might be influenced by copy number alterations (CNA) leading to altered gene expression. We compared leukemic genomic profiles of 25 treatment sensitive (MRD-SR) and 25 resistant (MRD-HR) childhood ALL patients by means of high-resolution array-CGH. CNA were found in 46 patients (92%) of both treatment response groups. Microscopic alterations affecting the whole or nearly whole chromosome arm were frequently found, e.g. gain of 21 in 11/50, loss of 9p in 5/50, loss of 8p in 3/50, loss of 20q in 3/50 and loss of 7p in 2/50 or gain of 1q in 2/50. The most significant difference was a gain of chromosome 1q23-qter due to an unbalanced t(1;19), found in 10/25 MRD-SR patients, but in none of the MRD-HR patients (p

Description: The lymphocyte surface glycoprotein CD26 is involved in an array of diverse signalling pathways and costimulatory events. CD26positive cells possess dipeptidylpeptidase IV (DPP IV) activity, a serine protease known to inactivate SDF-1/CXCL12, a key mediator of stem cell homing and engraftment. Furthermore, CD26 modulates surface expression of CTLA-4 and contributes to T-cell migration through endothelial layers. CD26 has been attributed with a central role in alloantigen-mediated immune pathways and memory T-cell responses. The purpose of this study was to evaluate the levels of a distinct memory T-cell subset, CD26bright/CD45RO-positive, in autologous hematopoietic progenitor-cell transplant (HPCT) recipients. Between 2003 and 2006 we enrolled 42 patients scheduled to undergo high-dose chemotherapy and autologous HPCT (multiple myeloma, n=31; Hodgkin’s Disease, n=3; NHL, n=6; PNET, n=1; AML, n=1). Levels of memory and naïve CD26, CD34, CD4, CD8, as well as co-expression of CD4 or CD8 among CD26bright/CD45RO-positive cells were analyzed before autologous HPCT. In addition, the number of memory and naïve CD26-positive T cells transfused per kg body weight in the progenitor cell harvest were determined. The subsets of CD26-positive cells were correlated with kinetics of engraftment and with the occurrence of disease progression or relapse after autologous transplantation. With regard to kinetics of engraftment, only the number of CD34-positive cells transfused was associated with rapid engraftment (P=0.001). However, CD26-positive cells were of predicitve value for the occurrence of disease progression or relapse. Pre-transplant CD26-positive T-cell levels correlated with progression-free survival (PFS) (P=0.022). Specifically, the number of CD26bright/CD45RO-positive memory T-cells in the autograft correlated with PFS and, in regression analyses, emerged as the only variable predictive for the occurrence of diease progression or relapse (P=0.006). The prognostic effects of pre-transplant CD26bright/CD45RO-positive memory T-cells were independent of the type of disease and of the conditioning regimen applied. The analysis of antigens CD4 and CD8, respecively, on CD26bright/CD45RO-positive T-cells yielded no additional information. Our results suggest that pre-transplant levels of CD26-positive T cells, specifically a memory cell subset of CD26bright cells coexpressing CD45RO, may yield information with regard to outcome in autologous HPCT recipients. These observations may contribute to a prospective identification of those patients at higher risk of relapse, based on their immune status.