Impact of Age and Treatment Group in Childhood High Hyperdiploid Low Risk B-Cell Acute Lymphoblastic Leukemia (ALL): Results of the CLG-EORTC 58951 Study

Background: Since the last decades, pediatric ALL classification has integrated new cytogenetic and genomic data, and several genetic risk factors are identified for treatment stratification. Hyperdiploidy, which is the most common cytogenetic abnormality pattern of pediatric B-lineage ALL, is known since the early 80s and is found in 25-30% of cases. It has been recognized as an isolated good prognostic factor, especially High Hyperdiploidy (HeH), defined by a modal chromosome number higher than 50. In addition, treatment descalations were performed based on NCI criteria for low risk ALL (low leucocytosis < 10 x 109/L and/or age from 1 to ²10 years old) and/or lack of high risk features. Aim: The aim of this study was to assess whether age, under or over 10 years old, has a prognostic impact on the outcome in children presenting B-cell ALL with low risk criteria, and whether therapeutic intensification might improve the outcome within each age group. Methods: Among 2039 patients treated in EORTC 58951 (BFM backbone) from 1998 to 2008, 370 children with B-cell ALL and low risk criteria such as HeH (with classical profile of chromosome gains), low leucocytosis (< 10x109/L), a lack of CNS or gonadal involvement and a lack of very high features (poor response to prephase) were included in this study. Hyperdiploidy was determined either by cytogenetics or flow cytometry. Patients were stratified into 2 risk groups: very low risk (VLR) group or standard risk (AR1) group. VLR group was defined by the criteria indicated above. It led to a less intensive treatment, particularly lower number of injections of anthracyclines and alkylating agents. AR1 group included children with surreptitious or hemorrhagic CNS involvement, and/or HeH with mismatch between modal chromosome number and DNA index. The upper limit of age in the EORTC 58951 was less than 18 years. Main endpoints were Event-Free Survival (EFS) and Overall Survival (OS). Results: Overall patients with low risk hyperdiploid B-cell ALL features treated according to a VLR or AR1 protocol had 6-year EFS and OS rates of 90.3% and 96.2% respectively. These results were comparable to previous published studies on hyperdiploidy in childhood B-lineage ALL. All patients but one reached complete remission after the induction phase. There were 315 children aged less than 10 years old and 55 patients aged 10 to 17 years old. Among children aged 10 years and older treated in VLR or AR1 groups, 6-year EFS and OS rates were respectively 86.7% and 96.4%. In comparison, among less than 10 year old childrentreated in VLR or AR1 groups, 6-year EFS and OS rates were respectively 89.8% (p=0.42) and 95.9% (p=0.93). Age did not appear as a significant prognostic factor in the outcome. Furthermore, among children aged 10 years and older, those treated in the standard risk group (AR1) had 6-year EFS and OS rates of 90% and 100% respectively. This seemed better than for children treated in the very low risk group, with respectively 84% and 92% rates. However, no significant gain was found for children who received standard risk treatment (for EFS and OS rates: p=0.47 and p=0.12 respectively). Relapse and treatment toxicity rates were comparable in both groups. Concerning long-term cardiac toxicity, despite the fact that no significant difference was found between the two risk groups of treatment, standard treatment leads to higher dose of anthracyclines and an increased theoretical risk of cardiac toxicity. Conclusion: In conclusion, age did not appear as a significant prognostic factor in outcome of children treated for a low risk B-cell ALL. Moreover, among children aged 10 years and older, therapeutic intensification in standard risk group did not lead to a significant gain in outcome. However, results are not significant, partly due to a low number of patients, and larger studies should be performed to evaluate whether these children could benefit from a less intensive treatment. Disclosures No relevant conflicts of interest to declare.

Outcome of treatment in children with hypodiploid acute lymphoblastic leukemia

One-hundred thirty-nine patients with acute lymphoblastic leukemia (ALL) and hypodiploidy (fewer than 45 chromosomes) were collected from 10 different national ALL study groups and single institutions. Patients were stratified by modal chromosome number into 4 groups: 24 to 29 (N = 46); 33 to 39 (N = 26); 40 to 43 (N = 13); and 44 (N = 54) chromosomes. Nine patients were Philadelphia chromosome (Ph) positive (4 cases: 44 chromosomes; 5 cases: 40-43 chromosomes) and were not considered further. Event-free survival (EFS) and overall survival (OS) of the remaining 130 patients were 38.5% ± 4.4% and 49.8% ± 4.2% at 8 years, respectively. There were no significant differences in outcome between patients with 24 to 29, 33 to 39, or 40 to 43 chromosomes. Compared with patients with fewer than 44 chromosomes, patients with 44 chromosomes had a significantly better EFS (P = .01; 8-year estimate, 52.2% vs 30.1%) and OS (P = .017; 69% vs 37.5%). For patients with 44 chromosomes, monosomy 7, the presence of a dicentric chromosome, or both predicted a worse EFS but similar OS. Doubling of the hypodiploid clone occurred in 32 patients (24-29 chromosomes [n = 25] and 33-39 chromosomes [n = 7]) and had no prognostic implication. Children and adolescents with ALL and hypodiploidy with fewer than 44 chromosomes have a poor outcome despite contemporary therapy.

Bone Marrow Fibrosis Evaluation in Childhood Acute Lymphoblastic Leukaemia: Correlation to Biological Factors and Treatment Response.

Fibrosis may complicate bone marrow (BM) disease and in its advanced stage negatively affect the outcome of the patients due to BM failure, particularly in myeloproliferative disorders. Studies of the potential importance of BM fibrosis are rare in childhood acute lymphoblastic leukemia (ALL). We have previously shown a prognostic impact of BM fibrosis measured as reticulin fibre density (RFD) at diagnosis in childhood ALL. To further investigate the consequence of BM fibrosis in childhood ALL in relation to immunophenotype, cytogenetic findings and minimal residual disease (MRD) we retrospectively evaluated the RFD in 139 diagnostic BM biopsies from patients with a total mean follow up time of five years and eight months from two childhood oncology centers in Sweden. Patients with pre-B-ALL showed a higher mean RFD (19.1%) compared to patients with T-ALL (11.4%, p < 0.001). This was true also when comparing high-risk (HR) pre-B-ALL patients (18.1%) with the T-ALL patients (p = 0.002). RFD correlated inversely with the white blood cell count in the HR group (r = −0.41, p = 0.009), probably reflecting our finding of low degree of fibrosis in T-ALL. The cytogenetic analysis revealed that for patients with a hyperdiploid ALL (modal chromosome number: 51–61, n: 41) in the low-risk (LR) group, mean RFD was higher for relapsed patients (22.6%) compared to patients in continuous complete remission (17.2%, p = 0.019). The probability of disease free survival for the same group using RFD cutoff of 21.1%, representing the upper third of the material, was 85%±8% for patients with hyperdiploid ALL and low RFD compared to 51%±14% for patients with hyperdiploid ALL and high RFD (p = 0.01, Figure 1). Data for 32 of the patients demonstrated an association between high RFD at diagnosis and high minimal residual disease (MRD) on day 29 after start of treatment. Patients with FACS-MRD > 10−3 day 29 displayed higher mean RFD at diagnosis (21.2%) compared to patients with FACS-MRD < 10−3 (16.7%, p = 0.027, Figure 2). When analyzing the PCR-MRD data day 29 the findings were similar. We conclude that RFD is higher in pre-B-ALL compared to T-ALL, that RFD has prognostic impact in LR patients with hyperdiploid ALL and that high RFD at diagnosis is associated to high MRD on treatment day 29. All these findings are to our knowledge novel, suggesting that evaluation of BM fibrosis at diagnosis is a potentially new therapy stratifying factor and support the need of further research on BM fibrosis in childhood ALL and expanded use of BM biopsy at diagnosis. Figure 1 Figure 1. Figure 2 Figure 2.

Prognostic Impact of Trisomies of Chromosomes 10, 17, and 5 Among Children With Acute Lymphoblastic Leukemia and High Hyperdiploidy (> 50 Chromosomes)

PURPOSE: Children with acute lymphoblastic leukemia (ALL) and high hyperdiploidy (> 50 chromosomes) have improved outcome compared with other ALL patients. We sought to identify cytogenetic features that would predict differences in outcome within this low-risk subset of ALL patients. MATERIALS AND METHODS: High-hyperdiploid ALL patients (N = 480) were enrolled between 1988 and 1995 on Children’s Cancer Group (CCG) trials. Karyotypes were determined by conventional banding. Treatment outcome was analyzed by life-table methods. RESULTS: Patients with 54 to 58 chromosomes had better outcome than patients with 51 to 53 or 59 to 68 chromosomes (P = .0002). Patients with a trisomy of chromosome 10 (P < .0001), chromosome 17 (P = .0002), or chromosome 18 (P = .004) had significantly improved outcome compared with their counterparts who lacked the given trisomy. Patients with a trisomy of chromosome 5 had worse outcome than patients lacking this trisomy (P = .02). Patients with trisomies of both chromosomes 10 and 17 had better outcome than those with a trisomy of chromosome 10 (P = .09), a trisomy of chromosome 17 (P = .01), or neither trisomy (P < .0001). Multivariate analysis indicated that trisomy of chromosome 10 (P = .001) was the most significant prognostic factor for high-hyperdiploid patients, yet trisomy of chromosome 17 (P = .02) or chromosome 5 (P = .01) and modal chromosome number (P = .02) also had significant multivariate effects. CONCLUSION: Trisomy of chromosomes 10 and 17 as well as modal chromosome number 54 to 58 identify subgroups of patients with high-hyperdiploid ALL who have a better outcome than high-hyperdiploid patients who lack these cytogenetic features. Trisomy of chromosome 5 confers poorer outcome among high-hyperdiploid patients.

Flow Cytometric DNA Index and Karyotype in Childhood Lymphoblastic Leukemia

Flow cytometric DNA-index (DIFCM) and karyotype were analysed in 82 consecutive children with acute lymphoblastic leukemia (ALL) during a 10 year period. A statistically significant correlation existed between modal chromosome number and DIFCM(p= 0.009). DIFCMcould reliably identify leukemias with >51 chromosomes, whereas only three out of 12 cases with modal chromosome numbers between 47–51 were classified as aneuploid by DIFCM. In the pseudodiploid group only one out of 20 leukemias had a DIFCM>1.0. Five leukemias with a diploid karyotype showed an aneuploid DIFCMand in three patients the flow cytometric measurement revealed biclonality undetected by karyotyping. During treatment aneuploid clones could be detected by DIFCMin a substantial number of cases where the cytogenetic analysis was normal, and the opposite was also demonstrated in one case. DIFCMgave prognostic information, showing that cases with a DI >1.12 (corresponding to 51 chromosomes) had a superior outcome with treatment protocols today in use.

The chromosomes Of Platynereis dumerilii (Polychaeta: Nereidae)

The chromosomes of Platynereis dumerilii (Audouin & Milne-Edwards) are described here for the first time. A modal chromosome number of 2n=28 was recorded, based on counts conducted on metaphase spreads prepared from 24-h-old larvae. The karyotype comprises seven pairs, each of relatively large median (arm ratio, p/q=l.00–0.59) and submedian (arm ratio, 0.59–0.33) chromosomes. Attempts were also made to band the chromosomes using C-banding and silver staining methods. C-band-positive regions were localized on four chromosome pairs (three median, one submedian). A further two chromosome pairs (both median) were observed to have terminal nucleolar organizer regions (NORs). To our knowledge, this is the first time that these banding methods have been successfully applied to any polychaete species. These results are discussed in relation to the karyotypic variation within the class Polychaeta, and the family Nereidae in particular. A stable karyotype consisting of a moderate number of large and morphologically well-differentiated chromosomes, coupled with the ease of culture under laboratory conditions and short generation time, suggests that P. dumerilii is a potentially suitable model for evaluating marine contaminants for genotoxic activity.

Characterization of a new megakaryocytic cell line: the Dami cell

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryoblastic leukemia. The Dami cells grow primarily in suspension with a doubling time of 24 to 30 hours. By light and electron microscopy, the Dami cells range in size from 12 to 120 micron in diameter and have lobulated nuclei characteristic of megakaryocytes. At least 89% of the cells react with monoclonal antibodies against platelet glycoproteins (GP) Ib and IIB/IIIa, and glycophorin. The cells do not react with antibodies against lymphoid, monocyte, granulocyte, or macrophage antigens. Thirteen percent of the cells become polyploid, spontaneously achieving greater than 4N DNA ploidy levels. In response to phorbol myristate acetate (PMA), the proportion of cells with ploidy levels greater than 4N increased threefold and could be separated into discrete ploidy groups. PMA also increased the expression of GPIb, the GPIIb/GPIIIa complex,l and von Willebrand factor. Cytogenetic analysis revealed a human male hyperdiploid karyotype with a modal chromosome number of 54 to 64 and several consistent clonal chromosomal abnormalities. These included a partial deletion of chromosome 5 and a translocation involving chromosome 3. In contrast to other megakaryocytic cell lines in which only a small portion of the cells express the megakaryocytic phenotype, nearly all of the Dami cells express platelet glycoproteins. Thus, the Dami cells provide a superior model in which to study human megakaryocyte biochemistry and differentiation.

Characterization of a new megakaryocytic cell line: the Dami cell

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryoblastic leukemia. The Dami cells grow primarily in suspension with a doubling time of 24 to 30 hours. By light and electron microscopy, the Dami cells range in size from 12 to 120 micron in diameter and have lobulated nuclei characteristic of megakaryocytes. At least 89% of the cells react with monoclonal antibodies against platelet glycoproteins (GP) Ib and IIB/IIIa, and glycophorin. The cells do not react with antibodies against lymphoid, monocyte, granulocyte, or macrophage antigens. Thirteen percent of the cells become polyploid, spontaneously achieving greater than 4N DNA ploidy levels. In response to phorbol myristate acetate (PMA), the proportion of cells with ploidy levels greater than 4N increased threefold and could be separated into discrete ploidy groups. PMA also increased the expression of GPIb, the GPIIb/GPIIIa complex,l and von Willebrand factor. Cytogenetic analysis revealed a human male hyperdiploid karyotype with a modal chromosome number of 54 to 64 and several consistent clonal chromosomal abnormalities. These included a partial deletion of chromosome 5 and a translocation involving chromosome 3. In contrast to other megakaryocytic cell lines in which only a small portion of the cells express the megakaryocytic phenotype, nearly all of the Dami cells express platelet glycoproteins. Thus, the Dami cells provide a superior model in which to study human megakaryocyte biochemistry and differentiation.