ALBERT

Description: Among patients with adult acute lymphoblastic leukemia (ALL) the t(4;11) translocation (resulting in the MLL-AF4 fusion gene) is a recurrent chromosomal abnormality occurring in approximately 10–15% of cases. It is associated with poor outcome and in the majority of cases stem cell transplantation is the only possible cure. The MLL-AF4 cases show a more immature phenotype than other subtypes frequently lacking Ig light chain and/or TCR gene rearrangements (Brumpt et al; Blood, 2000; 96:2254). We have previously observed that in adult ALL there is a bias towards the use of JH-proximal VH genes and in particular an over-usage of the VH6 gene segment, the most JH-proximal VH element. This is also common in MLL-AF4 patients while rarer in ALL carrying other chromosomal abnormalities (Mortuza et al, 2001; Blood, 97: 2716). VH6 gene usage is also predominant (8–12%) in adult ALL when compared to the mathematical expected VH6 gene usage (0.8%) or the usage observed in normal PB or in other B-cell malignancies, such as CLL (3.6%) and B cell lymphomas (0%). Finally, mutations of FLT3 are detected in a large proportion of acute myeloid leukemias and in a small proportion of childhood ALL carrying the MLL-AF4 fusion gene while it has seldom been observed in adult ALL with t(4;11). The aims of our study were twofold: 1) to expand on the initial observation that the VH6 gene rearrangement may be more frequent in adult patients carrying the t(4;11) translocation; 2). To assess the incidence of FLT3 mutations in an homogenous cohort of adult ALL patients carrying t(4;11) abnormality. Thirty-two cases of MLL-AF4 fusion positive adult ALL patients (17M/15F) were analysed in addition to the RS4;11 cell line. Pro-B phenotype (n=11)predominated. Age ranged between 22–55 yrs(median: 32 yrs). Patients had cytogenetic or molecular evidence of t(4;11). The WBC count at presentation was high (60–550x109/l; median: 220). Patients were tested for VH gene rearrangement using FRI and consensus-JH primers. Internal tandem duplication and codon 835–836 kinase mutations of the FLT3 were screened by DNA PCR analysis. Twenty-seven patients (84%) of the 32 tested carried a VH6 gene rearrangement. Of 36 IgH alleles, 27 (75%) were VH6 rearrangements. VH1 in 4 (11%) rearrangements was the second most common rearrangement while 3 were VH3 (8%) and 2 (5.5%) were VH4. Compared to our previous study (Mortuza et al, 2001; Blood,97:2716) the incidence of VH6 IGH rearrangements in MLL positive cases represents a nine-fold increase (75% vs 9.4%) compared to non-MLL + cases. No Internal Tandem Duplication of the FLT3 gene was detected in the 31 cases tested, while one (3.6%) of the 28 cases analysed showed the recurrent mutation of the Asp835Tyr of the FLT3 gene. In conclusion, patients carrying the t(4;11) translocation show a very skewed pattern of IgH gene rearrangement restricted almost exclusively to the VH6 usage, in agreement with both the more immature B cell phenotype and observation in the mouse immune system. However, in t(4;11) adult ALL FLT3 is rarely found mutated and may therefore play a minor role in the poor outcome of this ALL subgroup.

Description: Key Points Efforts to understand mechanisms of disease initiation in human adult pre-B ALL are hampered by lack of appropriate animal models. Optimized xenotransplant assays show that niche-based SDF-1/CXCR4 interaction is crucial for adult non-t(4;11) pre-B ALL leukemia initiation.

Description: Introduction TP53 is the most frequently altered gene in cancer. Until recently data on its frequency and prognostic impact in ALL has been scant, particularly in the elderly. TP53 alteration in the context of telomere shortening (the hallmark of aging) results in telomere attrition and genomic instability with resultant overexpression of telomerase to overcome the genomic crisis. Objective To determine TP53 alteration frequency and its association with cytogenetic subgroups, key B cell differentiation/cell cycle genes abnormalities and induction therapy response in ALL patients aged ≥60 and its impact on leukemic telomere state Methods Patients enrolled in the UK NCRI UKALL14 and UKALL60+ trials aged ≥60 years were assessed at diagnosis for 17p deletions by cytogenetics, TP53 alterations by FISH and/or direct sequencing of hot spots exons 5-8. Data were correlated with IKZF1, CDKN2A/B, PAR1, BTG, RB1, ETV6, EBF and PAX5 gene copy number status, assessed by MLPA (P335-B1). MYC-rearrangement was an exclusion criterion. Minimal residual disease (MRD) was assessed by quantitative PCR (qPCR) for Ig/TCR rearrangements or BCR-ABL1, where applicable. Leukemic DNA telomere length relative to that of remission DNA was assessed by monochrome multiplex (MM) qPCR and expressed as a log2 ratio. Using expectation maximization mixture model a log2 ratio 〉3.18 defined gain (reflecting telomerase overexpression) and values below this were defined as loss. Results The cohort included 63 patients; characteristics are presented in table 1. Median age was 63 years (range 60–83). TP53 state by cytogenetics and/or sequencing was available in all. Eleven had TP53 alteration (17%); TP53 mutation only, n=3; TP53 deletion only, n=6; TP53 deletion and mutation, n=2. Cytogenetics was available in 60 patients. Nine patients had HoTr (14%). Seven of 9 patients with HoTr had TP53 alteration (78%) versus 4/51 without HoTr (4%); p

Description: Introduction and aims. Molecular monitoring of minimal residual disease (MRD) using Immunoglobulin (IG) and T cell receptor (TCR) targets has provided an independent and prognostically significant parameter of outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to evaluate the impact of molecular tests carried out during the first 20 to 24 weeks of chemotherapy for MRD in a standard risk group of adult B cell ALL patients. Patients and Methodology. We evaluated MRD tests in 63 patients with B cell ALL (37M/26F). Median age of our cohort was 24 yrs (range: 15.5–54.6 yrs) while median WBC was 9.9 (range: 1.1–553 × 109l). All were negative for the t(9;22) or t(4;11) translocation and received standard induction chemotherapy or auto stem cell transplant (A-SCT) only. All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4 and all tests were carried out at time of morphological remission. End-points were relapse or continuous clinical remission (CCR) with follow up ≥12 months. Time point for molecular evaluation were post induction phase 1, TP1 (up to 1.8 mo), post induction phase 2 (1.8–3.5 mo), post intensification (3.5–5.7 mo). Median follow up was 92 months for patients in CCR (range 12–141 mo) and 15.5 months for patients who relapsed (range: 2.4–49 mo). Results. Thirty-five patients in complete morphological remission were tested at TP1. Relapse free survival (RFS) tests showed a statistically significant correlation (p=0.02) between MRD positive tests and relapse (n=19 pts) and MRD negative tests (n=16 pts) and relapse free survival. In addition, pts with resistant disease at TP1 (n=10; not included above) and MRD positive pts were indistinguishable as far as relapse FS and both faired extremely poorly (Figure A). Thirty patients were analysed at TP2. RFS confirmed the significant association between MRD positivity and poor outcome (n=16 pts) and MRD negativity and CCR (n=14 pts) (p=0.03) (Figure B). MRD data were also available for 43 patients at TP3. Association of MRD positive tests (n=15 pts) and MRD negative tests (n=28 pts) with poor and good outcome, respectively (p=0.0006) (Figure C) was strongest at this time point. Outcome correlated with level of MRD at all time points, with poorer outcome in patients with MRD 〉1E3. Conclusions. Molecular monitoring during induction and intensification for standard risk B cell ALL patients treated with UKALL12 protocol provides a prognostically significant parameter for the management of adult ALL in otherwise morphological remission and may in the future be used for patients’ stratification. Figure Figure

Description: Analysis of immunoglobulin gene rearrangement for minimal residual disease as a prognostic parameter is a well established test in a variety of leukaemias such as ALL and CLL. The aim of this study was: 1) to analyse VH gene usage, occurrence of in-frame (IF) and out-of-frame (OF) sequences and rate of mutation; 2) to correlate these parameters with overall clinical outcome in adult ALL patients, as this may identify a novel prognostic marker in ALL, especially in the absence of other prognostic indicators. Patients and methods: DNA from 82 de novo adult ALL patients (31F/51M) was obtained from PB or BM diagnostic samples. Using primers for the FR1 and JH segments, 102 clonal rearrangements were identified. Median WBC was 13.5 × 109/I (range 1.1–550 × 109/I). Sixty-three patients are, to date, in complete clinical remission (median follow up: 7 months; range: 3 weeks–82.5 months), 15 patients have relapsed (median relapse 5.7 mo; range: 2.1–22 mo) and 2 patients had resistant disease. Patients carrying the t(9;22) translocation and patients who received an allogeneic stem cell transplantation were excluded when calculating relapse free survival to avoid bias towards bad and good performance, respectively. Results: A heterogeneous representation of VH gene usage was demonstrated. Recurrent rearrangements showed preferential use of VH3-66 (4 cases), VH1-2 (7 cases) and VH6 (8 cases; predominant in MLL-AF4 cases), when compared to the non-leukaemic controls (Mortuza et al, 2002; JCO, 20:1094). JH4 and JH6 were found in approximately equal proportions both in control and leukaemic groups, while JH5 (32 cases) was favoured in the leukaemic group. A similar trend was observed with D2-2 (23 cases) when compared to the other D segments in the latter. There was a predominance of OF sequences (66 alleles; 64%; from 53 patients), although no statistical difference in relapse free survival between IF and OF was observed. Mutations 〉1% were observed in 18/82 (22%) patients (18/102 alleles; 18%). These were twice as common in the IF sequences (11/18 patients; 61%) compared to OF sequences (in 6/17 pts, 35.3%). However mutations had no impact on overall survival (p=NS) and neither did immunophenotype (pre B Vs common ALL) (p=NS). Conclusion: Unique to ALL patients, a considerable proportion of cases carry out-of-frame immunoglobulin rearrangements while a smaller proportion are in-frame, without any apparent clinical impact. In contrast to observations made in other leukemias, such as CLL, occurrence of mutation does not appear to affect clinical outcome in our data set, although a longer follow-up period may be required for this prognostic factor to be fully evaluated.

Description: Molecular monitoring of minimal residual disease (MRD) has become an independent and prognostically significant parameter in assessing outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to evaluate the impact of MRD in a standard risk group of adult B cell ALL patients who had achieved morphological remission following induction phase 1 therapy as part of the MRC UKALL12 protocol. Patients and Methodology. MRD tests were evaluated in fourty-seven patients with adult B cell ALL. They were negative for the t(9;22) or t(4;11) translocation and had received chemotherapy based treatment or auto stem cell transplant (A-SCT) only. Median age was 23 yrs (range 15.5–54.6 yrs); median WBC was 9.6 (range 1.1–163×109/l) with a predominance of common (28 pts) and pre B ALL (12 pts). All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4. End points were either clinical relapse or disease free survival in complete remission with follow up ≥12 months. Time point for molecular evaluation were post induction phase 1, TP1 (median 0.9 mo; range: 0.6–1.8 mo) and post induction phase 2 (median 2.79 mo; range 1.8–3.5 mo). Results. Thirty three pts were tested at TP1 following morphological remission. Relapse free survival (RFS) analysis showed a statistically significant association between MRD positive tests (in 16 pts) and relapse, and MRD negative tests (in 17 pts) and CCR (p=0.03) (Figure 1, left diagram). Twenty five pts were analysed at TP2 following their early morphological remission. At this time point we observed the strongest association between MRD negative tests (in 13 pts) and CCR (in 10 pts) and MRD positive (12 pts) and relapse (in 11 pts) (p=0.01)(Figure 1, right diagram). Conclusions. Molecular monitoring of MRD shows that even among early morphological remitters a group of MRD positive patients can be identified that have poor overall outcome and may benefit from tailored therapies. Molecular assessment of residual disease should be used to stratify treatment in future adult ALL trials. Figure Figure

Description: Introduction and aims. Molecular monitoring of minimal residual disease has provided an independent and prognostically significant parameter in evaluating outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to assess the impact of MRD measured in bone marrow samples collected at time of harvest or prior to harvest in adult B cell receiving autologous stem cell transplant and correlate MRD with overall clinical outcome. Patients and Methodology. Patients were selected as de novo adult ALL of B cells. All patients were negative for the t(9;22) or (4;11) and had received chemotherapy followed by an autologous SCT (A-SCT). All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4. End points were either clinical relapse or CCR with follow up ≥12 months except for two patients who died in CR following transplant, due to infections. Time point for molecular evaluation was a test preceding A-SCT or harvested BM. Nineteen patients were evaluable prior to/or at time of harvest. Nine were females and nine were males. Median age was 25.2 yrs (range: 15.2–52 yrs), total WCC was 7.1 (range: 2.3–68.9×109/l) at time of diagnosis with common ALL (14 pts) as the predominant phenotype. Patients received an A-SCT at a median period of 6 months from diagnosis (range: 5–18 months). Eight patients relapsed (median period to relapse: 22 mo; range: 8–35 mo) and 8 were in continuous clinical remission (CCR) (median follow up 33 mo; range: 8–139 mo). Median interval between Auto-SCT and relapse was 13.7 mo (range: 3–29 mo). Results. In seven patients residual disease was demonstrated in the BM prior to A-SCT and 6 of them relapsed. In 12 patients no residual disease was detected at time of harvest or prior to transplant and 10 are at present in CCR. The association between MRD positivity and relapse and MRD negativity and CCR was statistically significant in this cohort of patients (p=0.002)(Figure 1). Conclusions. Molecular monitoring of MRD can provide a useful tool for the monitoring of residual disease in BM harvest prior to SCT and correlates with outcome. It is therefore important that all BM harvests are tested for residual disease in future clinical trial that may use MRD for patients’ stratification. MRD tests prior to Auto-SCT and relapse FS MRD tests prior to Auto-SCT and relapse FS