ALBERT

Description: Defects in cell death signalling might be responsible for treatment failure and relapse in acute leukaemia. In a study on paediatric acute lymphoblastic leukaemia (ALL) we recently reported the importance of intact apoptosis signalling for favourable outcome. Here we addressed this issue in 45 paediatric acute myeloid leukaemia (AML) patient samples. Two key apoptogenic events: cytochrome c release and caspase-3 activation were analysed by flowcytometry in the leukaemia cells after induction of apoptosis by factor deprivation. Release of cytochrome c and consecutive apoptosome formation leads to activation of downstream effector caspases such as caspase-3. Both events were closely correlated to each other in good responding patients (less than 5% blasts in bone marrow or minimal residual disease negativity) and in patients achieving remission. No correlation was found in patients with poor treatment response or patients not reaching remission. Caspases other than caspase-3 acting upstream of mitochondria (e.g. caspase-8) are also involved in initiating or amplifying this complex. By incubation in presence or absence of the pan-caspase inhibitor zVADfmk cytochrome c release was observed to be dependent or independent on upstream caspases. Of note, only cytochrome c released in dependence on amplifying caspases was observed to correlate with activated caspase-3 in patients with good response to treatment or patients without relapse. This indicates that an intact apoptosome function and active amplifying caspases are elementary for favourable outcome in childhood AML. The functional integrity of this important apoptogenic checkpoint is subsumed by the parameter caspase dependent cytochrome c-related activation of caspase-3 (CRACdep). Division of our cohort according to this new paramenter resulted in two groups of 38 CRACdep -positive and 7 -negative patients, the distribution of the values being unrelated to features such as FAB subtype or risk groups. 30 of 32 patients sustaining complete remission for more than one year were CRACdep -positive (Fisher exact test, P= .015). Analysis of survival (N=45, log rank P= .014) and remission duration (N=40, log rank P= .001) revealed a superior outcome for CRACdep -positive patients. This was also observed when considering the high risk patients only. Notably, standard risk stratified patients merely displayed CRACdep -positivity. Thus, the propensity to undergo apoptosis as reflected by intact signalling in leukaemia cells is an important feature for favourable treatment outcome and may serve as additional stratification tool for paediatric AML patients.

Description: Poor response to induction therapy is the major risk factor identified in ALL and is used within the ALL BFM study to identify high risk groups (e.g. prednisone poor response, PPR: more than 1000 blasts/μl peripheral blood after treatment with prednisone systemically for 8 days and MTX intrathecally once on day 1). Despite the efforts achieved by the stratification strategies the majority of relapses are recruited from the group of initially good responding patients (in ALL BFM the standard and medium risk, SR and MR groups) emphasising the need for additional independent stratification factors. In our study we transplanted primary leukaemia cells from 50 children with newly diagnosed B cell precursor ALL (BCP-ALL) into NOD/SCID mice. Time to leukaemia was determined for each patient sample transplanted as weeks from date of transplant to date of clinical manifestation of the disease. Leukaemia was verified in spleen and bone marrow by flow cytometry staining for human CD19 and CD45. Time to leukaemia of less than 10 weeks (short TLL) was observed in 6 patient samples whereas 44 leukaemia samples took more than 10 weeks until appearance of leukaemia (long TTL). A clear cut in relapse free survival (Kaplan Meier analysis, N=50, log rank: P= .0000) was found for patients whose leukaemia cell samples showed short TTL (N=6, mean survival: 12.1 months, SE: 3.9, CI: 4.6–19.6) in contrast to patients with long TTL (N=44, mean survival: 54.3 months, SE: 2.9, CI: 48.6–60.1). Of note, the same distinct difference in relapse free survival was observed considering the SR and MR groups only (N=40, log rank P= .0000, long TTL: 44.9 months; short TTL: 12.5 months). By multivariate analysis (N=50, 3-years relapse free survival) patients exhibiting short TTL in the xenotransplant model exhibited a strongly increased risk for relapse with a risk ratio of 18.31 (CI: 5.03–66.72, P= .000). Interestingly, patients in our cohort showing prednisone poor response known as important clinical risk factor revealed only a risk ratio of 6.59 (CI: 1.32–32.75, P= .021). None of the patients with long TTL encountered early relapse. These findings in 50 directly transplanted samples were confirmed transplanting different cryopreserved BCP-ALL samples. In order to further characterise the biological properties of the leukaemia cell in the two groups, gene expression profiles of samples with short or long TTL in the xenograft model were investigated using a human whole genome array (Affymetrix U133 Plus 2.0). We identified a signature of differentially expressed genes distinguishing both groups. The differential expression was confirmed for selected genes by semi-quantitative PCR. Taken together, estimation of time to leukaemia (TTL) of leukaemia samples transplanted onto NOD/SCID mice is a new promising factor in paediatric ALL. Using an expression array approach patient samples displaying short TTL can be discriminated from those with long TTL by a unique gene expression signature. This allows direct identification of patients with increased risk for relapse by this new independent risk factor avoiding transplant in the mouse model.

Description: Drug resistance and treatment failure in acute leukemia may be attributed to apoptosis resistance in leukemia cells since defects in apoptosis signal transduction are commonly acquired during malignant transformation. Expression analysis of single molecules with regard to clinical outcome could so far not identify common apoptosis defects with prognostic value in primary acute leukemias. We addressed the role of apoptosis signaling for the initial response to induction treatment in pediatric B precursor lymphoblastic leukemia by functional assays. Apoptosis signaling in response to survival factor withdrawal was assessed in 82 primary leukemia samples by measurement of the key apoptotic events, caspase-3 activation and mitochondrial cytochrome c release. Caspase-3 activation directly correlated to the initial response to treatment assessed by the percentage of leukemia cells in bone marrow on day 15 (p = 0.015). Intact apoptosis signaling was indicated by a significant correlation of caspase activation and cytochrome c release was found especially in the groups of good responding patients (rs 0.375 – 0.502; p = 0.001). Interestingly, this correlation was completely absent in all groups of poor responding patients defined by insufficient leukemia cell reduction on day 8, 15 or 33. The efficacy of apoptosis signal induction in the individual leukemia is expressed by the parameter CRAC (Cytochrome c Related Activation of Caspase-3), relating the extent of caspase activation to cytochrome c release in a single patient sample. Ten of twelve patients with prednisone poor response (chi square, p= 0.031) and all patients not achieving remission on day 33 (4 of 4) had negative CRAC values, indicating deficient cytochrome c related caspase activation. Furthermore, the CRAC negative group revealed significant higher percentages of leukemia cells in bone marrow on day 15 (n=37, mean 22.8) than the CRAC positive patients (n=30, mean 6.2, p = 0.004). In addition all four relapse patients showed negative CRAC values indicating a prognostic value beyond initial treatment response. The data show that the intact relation of cytochrome c release and caspase activation indicating an intact apoptosome function is important for efficient remission induction treatment in childhood B precursor ALL. Assessment and quantification of this relation for individual patients by the parameter CRAC may serve as potential factor for treatment stratification.