Prospective multicentric molecular study for poor prognosis fusion transcripts at diagnosis in adult B-lineage ALL patients: the LALA 94 experience.
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Adolescent↗
Biomedical subjects
J M Cayuela
Publications and source records attributed to J M Cayuela.
Allogeneic stem cell transplantation improves the outcome of adults with t(1;19)/E2A-PBX1 and t(4;11)/MLL-AF4 positive B-cell acute lymphoblastic leukemia: results of the prospective multicenter LALA-94 study.
Adult patients with acute lymphoblastic leukemia (ALL) and t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4 have a poor outcome. We have evaluated the impact of an intensified post-remission therapy using a high-dose chemotherapy course followed by allogeneic or autologous SCT on the outcome of 58 patients with t(1;19)/E2A-PBX1 (E2A group, n=24) or t(4;11)/MLL-AF4 (MLL group, n=34) treated in the LALA-94 multicenter prospective study. Patients in the MLL group had higher WBC counts and more frequent DIC. CR rates achieved by MLL and E2A groups were similar to other B-cell ALL (87, 82 and 86% respectively). While in CR, patients with a donor were assigned to alloSCT (n=22), the remaining patients with were randomized between autoSCT (n=15) or chemotherapy (n=8). Five-year overall survival was 31 and 45% for E2A and MLL groups, respectively. In both groups, DFS was higher in the alloSCT arm as compared to autoSCT and chemotherapy arms. The results of this study show that chemotherapy intensification did not overcome the poor prognosis of adults with t(1;19)/E2A-PBX1. Allogeneic SCT should thus be offered in first CR to patients with t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4. New therapeutic approaches are needed for patients without donor.
Adolescent↗
Cyclin D2 dysregulation by chromosomal translocations to TCR loci in T-cell acute lymphoblastic leukemias.
Strong expression of at least one of the three D-type cyclins is common in human cancers. While the cyclin D1 and D3 genes (CCND1 and CCND3) are recurrently involved in genomic rearrangements, especially in B-cell lymphoid neoplasias, no clear involvement of the cyclin D2 gene (CCND2) has been reported to date. Here, we identified chromosomal translocations targeting the CCND2 locus at 12p13, and the T-cell receptor beta (TCRB) or the TCRA/D loci in T-cell acute lymphoblastic leukemias (T-ALLs). Expression analysis demonstrated dramatic cyclin D2 overexpression in the translocated cases (n=3) compared to other T-ALLs (total, n=89). In order to evaluate dysregulation in T-ALL with respect to normal T-cell differentiation, we analyzed CCND2 expression in normal purified human thymic subpopulations. CCND2 levels were downregulated through progression from the early stages of human T-cell differentiation, further suggesting that the massive and sustained expression in the CCND2-rearranged T-ALL cases was oncogenic. Association with other oncogene expression (TAL1, HOXAs, or TLX3/HOX11L2), NOTCH1 activating mutations, and/or CDKN2A/p16/ARF deletion, showed that cyclin D2 dysregulation could contribute to multi-event oncogenesis in various T-ALL groups. This report is the first clear evidence of a direct involvement of cyclin D2 in human cancer due to recurrent somatic genetic alterations.
Adult↗
Identification of a rare e8a2 BCR-ABL fusion gene in three novel chronic myeloid leukemia patients treated with imatinib.
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Adult↗
Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.
Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytic leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.
Biomarkers, Tumor↗
Prognostic significance of FLT3 internal tandem repeat in patients with de novo acute myeloid leukemia treated with reinforced courses of chemotherapy.
FLT3 internal tandem duplications (FLT3-ITDs) are present in nearly 25% of patients with AML and have been associated with poor response to conventional therapy and poor outcome. We retrospectively evaluated the effect of reinforced courses of chemotherapy on the prognostic value of FLT3-ITDs in 159 AML patients prospectively enrolled in the ALFA-9000 trial, which randomly compared three reinforced induction regimens (standard 3+7 including high-dose daunorubicin, double induction, and timed-sequential therapy). FLT3-ITD was present in 40/159 (25%) blast samples and associated with high WBC (P = 0.002) and cytogenetics (P < 0.001) with a higher incidence (35%) in patients with a normal karyotype. There was no difference in CR rate between FLT3-wt and FLT3-ITD patients (80% vs 78%). Relapse-free survival (RFS) was similar in both groups (5-year RFS, 33% vs 32%; P = 0.41), even after adjustment for age, sex, WBC, cytogenetics, and treatment arm. A trend to a worse survival was observed in the FLT3-ITD group (estimated 5-year OS, 23% vs 37%; P = 0.09), mainly in patients with a normal karyotype. This was associated with a dramatic outcome in relapsing FLT3-ITD patients (estimated 3-year post-relapse survival, 0% vs 27%; P = 0.04). These results suggest that the bad prognosis associated with FLT3-ITDs in AML might be partly overcome using reinforced chemotherapy. Early detection of FLT3 mutations might thus be useful to intensify induction as well as post-remission therapy in FLT3-ITD patients.
Acute Disease↗
BCR/ABL fusion gene detected on 9q34 by fluorescence in situ hybridization in an acute leukemia with two BCR/ABL positive clones, one Ph-negative and one Ph-positive.
We report cytogenetic, fluorescence in situ hybridization (FISH), and molecular analyses in the first reported case of an acute leukemia with two BCR-positive clones: one cell Ph-positive and all others Ph-negative. A BCR/ABL fusion gene on 9q34 was detected only with a BCR/ABL dual color translocation probe. These FISH interphase signals must be confirmed on a metaphase to avoid an erroneous interpretation. This observation appears to indicate a 2-step mechanism for this aberrant fusion gene localization: first, a classical t(9;22), and then the transfer of the fusion gene formed on chromosome 22 to chromosome 9 by a second translocation between the long arms of the derivative chromosomes 9q+ and 22q-, masking the first chromosome exchange.
Acute Disease↗
The incidence of clonal T-cell receptor rearrangements in B-cell precursor acute lymphoblastic leukemia varies with age and genotype.
B-cell precursor acute lymphoblastic leukemias (BCP-ALLs) are increasingly treated on risk-adapted protocols based on presenting clinical and biological features. Residual molecular positivity of clonal immunoglobulin (IG) and T-cell receptor (TCR) rearrangements allows detection of patients at an increased risk of relapse. If these rearrangements are to be used for universal follow-up, it is important to determine the extent to which they are informative in different BCP-ALL subsets. We show that IGH V-D-J rearrangements occur in 89% of 163 BCP-ALL, with no significant variation according to age or genotype (BCR-ABL, TEL-AML1, MLL-AF4, and E2A-PBX1). In contrast, TCRG rearrangements, which occur in 60% of patients overall, are frequent in BCR-ABL and TEL-AML1, are less so in MLL-AF4, and are virtually absent in infants aged predominantly from 1 to 2 years and in E2A-PBX1 ALLs. Incidence of the predominant TCRD Vdelta2-Ddelta3 rearrangement decreases with age but is independent of genotype. These differences are not due to differential recombination activating gene activity, nor can they be explained adequately by stage of maturation arrest. Analysis of MLL-AF4 BCP-ALL is in keeping with transformation of a precursor at an early stage of ontogenic development, despite the adult onset of the cases analyzed. We postulate that the complete absence of TCRG rearrangement in E2A-PBX1 cases may result from deregulated E2A function. These data also have practical consequences for the use of TCR clonality for the molecular follow-up of BCP-ALL.
Adolescent↗
The spectrum of mutations, including four novel ones, in the thiamine-responsive megaloblastic anemia gene SLC19A2 of eight families.
Thiamine responsive megaloblastic anemia (TRMA) is an autosomal recessive disorder with a triad of symptoms: megaloblastic anemia, deafness, and non-type 1 diabetes mellitus. Occasionally, cardiac abnormalities and abnormalities of the optic nerve and retina occur as well. Patients with TRMA often respond to treatment with pharmacological doses of thiamine. Recently, mutations were found in patients with TRMA in a thiamine transporter gene (SLC19A2). We here describe the mutations found in eight additional families. We found four novel mutations and three that were previously described. Of the novel ones, one is a nonsense mutation in exon 1 (E65X), two are missense mutations in exon 2 (S142F, D93H), and another is a mutation in the splicing donor site at the 5' end of intron 4 (C1223+1G>A). We also summarize the state of knowledge on all mutations found to date in TRMA patients. SLC19A2 is the first thiamine transporter gene to be described in humans. Reviewing the location and effect of the disease causing mutations can shed light on the way the protein functions and suggest ways to continue its investigation.
Alleles↗
Evaluation of minimal residual disease using reverse-transcription polymerase chain reaction in t(8;21) acute myeloid leukemia: a multicenter study of 51 patients.
PURPOSE: Most studies using various reverse-transcription polymerase chain reaction (RT-PCR) techniques reported that the detection of the AML1-ETO fusion transcript was a common finding in long-term complete remission (CR) in acute myeloid leukemia (AML) with t(8;21) translocation. However, larger prospective studies with interlaboratory quality control may be important to investigate more precisely the clinical usefulness of studying minimal residual disease with RT-PCR in t(8;21) AML. PATIENTS AND METHODS: We collected 223 marrow samples from 51 patients with t(8;21) AML diagnosed in five centers and tested all samples by two different RT-PCR techniques (a nested technique and a one-step technique, with a sensitivity of 10(-6) and 10(-5), respectively) in two different laboratories. RESULTS: Samples from 14 patients in long persistent CR (median follow-up duration, 112 months) were taken at least twice, and all were PCR-negative by both techniques. Samples were prospectively taken from 37 patients after achievement of first CR and/or second CR, before intensive consolidation treatment, and every 3 to 6 months after completion of therapy. Patients who converted to PCR negativity with the one-step technique (60%) or both techniques (48%) after CR achievement had a longer CR duration than those with persistently positive PCR results (two-sided log-rank test, P =.0001). Patients who became PCR-negative with the one-step technique before intensive consolidation (23%) had a lower relapse rate (11% v 72%) and a longer CR duration than those who remained persistently PCR-positive at that point (two-sided log-rank test, P =.0015). CONCLUSION: Patients with AML with t(8;21) in long-term remission were all PCR-negative. In prospectively studied patients, a good correlation was found between negative PCR results and absence of relapse. Early negative results with the one-step RT-PCR technique, before consolidation treatment, seemed to carry an especially good prognosis, suggesting that RT-PCR analysis could help in choosing the type of consolidation therapy in patients with t(8;21) AML.
Acute Disease↗
A prospective coagulation study including resistance to activated protein C and mutations in factors V and II in venous leg ulcers.
Hypercoagulable states have been reported to be associated with venous leg ulcers. In an attempt to investigate the prevalence of hypercoagulable states in patients with venous leg ulcers, we performed a prospective case-control study for the presence of coagulation defects in such patients, including resistance to activated protein C (APC), factor V Leiden mutation and a newly described mutation in factor II. Results were compared with those obtained in a control group. APC resistance was found in four of 33 patients tested, but only one was found to be heterozygous for the factor V Leiden mutation. Factor II mutation was found in two of 30 patients tested. Our findings show that the prevalence of coagulation abnormalities is not different in patients with venous leg ulcers from controls in our study, suggesting that only selected patients with venous ulcers and a history of recurrent deep venous thrombosis should be investigated for the presence of coagulation defects.
Activated Protein C Resistance↗
Germ-line deletion involving the INK4 locus in familial proneness to melanoma and nervous system tumors.
Joint predisposition to malignant melanoma and nervous system tumors (NSTs) is a puzzle. Several melanoma susceptibility genes have been identified, including p16, a clustered tumor suppressor. However, the molecular bases of inherited proclivity to NSTs in the absence of a recognizable genetic syndrome are unknown. We analyzed two families with joint proneness to melanoma and NSTs in view of genetic linkage and identification of the causal molecular lesions. Highly informative linkage markers were used for segregation analyses of the predisposition alleles in the two pedigrees. Characterization of the molecular lesions required hemizygosity mapping based on microsatellite markers physically mapped to contigs of the 9p21 region and a Southern blot approach using several PCR-generated probes. Both families were found to be allelic and linked to p16 markers. In the family segregating the melanoma/NST syndrome, a large germ-line deletion ablated the whole p16, p19, and p15 gene cluster (or INK4 locus), whereas a more circumscribed molecular lesion disrupting p16 and p19 but leaving p15 unaltered segregated with the melanoma-astrocytoma syndrome (MIM 155755). Our results suggest that multiple cancer susceptibility in these two families ensues from contiguous tumor suppressor gene deletion. Indeed, known phenotypes associated with germ-line p16 mutations and an apparent correlation between the deletion span and tumor spectrum in the two families suggest a new model of cancer pathogenesis based on the inactivation of contiguous tumor suppressor genes, an alternative to the established pleiotropic effects of single-gene disruption.
Adult↗
Mitochondrial DNA sequence variation in human leukemic cells.
The Long PCR followed by the RFLP technique has been used to search for abnormally structured mitochondrial DNA (mtDNA) and specific sequence differences implicated in the pathogenesis of acute lymphoblastic leukaemia (ALL). We have studied 54 specific sites whose combinations define groups of mtDNA types, in 30 leukemic patients of French Caucasian origin. Results were compared with those in 100 French healthy individuals. Nucleotide substitutions have been defined in 11 patients. This polymorphism is expressed by single base substitution at 6 sites which corresponds to 5 morphs, 2 of which were not found in the reference group. Combining the 11 observed morphs, we have identified 7 different mtDNA types, defined in 30 patients with ALL. Two of the morphs (MspI-2 and AvaII-3) and 3 of the types (17-2, 55-2, NewFr150) were not found in the group of healthy individuals. We have observed significant statistical changes in type 28-2 in ALL patients compared with the controls.
Adult↗
Genomic alterations of the p19ARF encoding exons in T-cell acute lymphoblastic leukemia.
We have previously shown that the disruption/deletion of the MTS(MTS1-MTS2) locus due to illegitimate V(D)J recombinase activity is a genetic event characteristic of T-cell acute lymphoblastic leukemia (T-ALL). Inactivation of the p16INK4a tumor suppressor protein, encoded by MTS1, is thought to be the major functional consequence of these chromosomal rearrangements. The two other cell cycle inhibitors encoded by genes identified in the locus (p19ARF by MTS1 and p15INK4b by MTS2), also represent possible candidates for inactivating events. By analyzing p16INK4a expression in three cases in which an identical 36-kb deletion had deleted MTS2 and disrupted the p19ARF, but spared the p16INK4a MTS1 encoding exons, we have excluded p16INK4a and pinpointed p19ARF and/or p15INK4b as the functional target(s) of this rearrangement. Moreover, by the study of the MTS genomic configuration of 149 rearranged alleles from a large series of T-ALL cases, we have shown that p19ARF encoding exons were always disrupted or deleted, whereas p16INK4a and p15INK4b encoding exons were spared in four and 21 cases, respectively. These results suggest that p19ARF may be targeted by the genetic events that occur in the MTS locus in the majority of T-ALLs.
Adolescent↗
Influence of CD34 cell selection on the incidence of mixed chimaerism and minimal residual disease after allogeneic unrelated donor transplantation.
Marrow transplantation from unrelated donors has been linked with an increased risk of graft-versus-host disease (GVHD). In an attempt to lower the risk of acute GVHD we used CD34 marrow cell selection for T cell depletion. Since T cell depletion has been linked to an increased risk of relapse and an increased risk of marrow failure, we used PCR amplification of minisatellite sequences to investigate donor cell engraftment and RT-PCR amplification of recurrent chromosomal translocations to investigate the residual disease post-transplant. Twenty-three patients who underwent BMT after positive selection of the CD34-positive cell population were studied. Results were then compared with those of 37 patients who underwent transplantation with unmanipulated marrow graft. Among the 23 patients who received CD34+ selected cell grafts, seven (30%) had evidence of full donor engraftment, 14 had evidence of residual recipient cells (61%), one had a non-take, and one autologous bone marrow recovery. Analysis of the chimaerism status post-transplant in 36 patients who received unmanipulated marrow grafts showed that 31 patients (86%) had evidence of full donor engraftment. The difference in the incidence of mixed chimaerism profile between patients who received unmanipulated marrow graft and those receiving CD34+ selected cell grafts was statistically significant (P< 0.01). Nine patients who received CD34+ selected cell grafts could be analysed for the presence of minimal residual disease post-transplant (one with t(9;22) acute lymphoblastic leukaemia and eight with CML). In the patient transplanted for a Ph-positive acute leukaemia, and in two out of the eight patients with CML, the search fora fusion transcript was consistently negative after transplantation. Among the six patients with evidence of residual disease, three patients also had a mixed chimaerism profile and were given donor lymphocyte infusions. Minimal residual disease study was performed post-transplant in 16 patients who received unmanipulated marrow grafts. In 10 of 14 patients with CML, and in two patients with acute leukaemia the search for a fusion transcript was consistently negative after transplantation. The difference in the incidence of minimal residual disease between patients who received an unmanipulated marrow graft and those receiving CD34+ selected cell grafts was not statistically significantly significant, but numbers of patients included in this analysis are still few. In conclusion, our study highlights the strong influence of graft manipulation on the incidence of mixed chimaerism after transplantation from an unrelated donor.
Adolescent↗
TAL1 expression does not occur in the majority of T-ALL blasts.
The TAL1 gene is disrupted by translocation or deletion (tal(d)) in up to 30% of T-cell acute lymphoblastic leukaemia (T-ALL), leading to aberrant transcriptional activation, as a SIL-TAL1 fused transcript in tal(d). It has been suggested that TAL1 transcription occurs in approximately 50% of a T-ALLs without apparent rearrangement. SIL-TAL1 was positive in 15/60 (25%) of T-ALL, whereas wild-type TAL1 transcripts were detected in all 13 SIL-TAL1 and in 19/43 (44%) T-ALL without SIL-TAL1. To investigate the cellular origin of TAL1 we exploited the fact that GATA1 and TAL1 are co-ordinately expressed in non-lymphoid haemopoietic cells, whereas only the latter is found in T-ALL. GATA1 was detected in 10/23 (43%) TAL1-negative T-ALLs but in 17/19 (89%) 'unexplained' TAL1-positive cases, suggesting a common non-lymphoid cellular origin. Immunocytochemical analysis with a TAL1-specific monoclonal antibody showed nuclear expression in the blasts of 10/34 (29%) cases, including 8/10 SIL-TAL1+ and two RT-PCR TAL1+, SIL-TAL1- cases. In the remaining cases TAL1 expression was restricted to a minor population (< 5%) of larger, strongly TAL1-positive cells which comprised erythroid cells, CD34+ CD3- precursors and an unidentified TAL1+ CD45- population which morphologically resembled monocytes/macrophages. We therefore suggest that appropriate diagnostic evaluation of T-ALL should include molecular detection of SIL-TAL1 transcripts and in situ immunocytochemical detection of TAL1 protein expression by leukaemic blasts. This approach will enable accurate analysis of the prognostic significance of TAL1 deregulation in T-ALL.
Adolescent↗
Disruption of the multiple tumor suppressor gene MTS1/p16(INK4a)/CDKN2 by illegitimate V(D)J recombinase activity in T-cell acute lymphoblastic leukemias.
We have recently shown that the multiple tumor suppressor gene 1 (MTS1 ) encoding the p16(INK4a) and p19(ARF) cell-cycle inhibitors is inactivated by deletion or disruption in most human T-cell acute lymphoblastic leukemias (T-ALLs), representing the most frequent genetic event thus far described in this disease. To analyze the mechanism of these chromosomal events, we used cloning, sequencing, and/or polymerase chain reaction mapping to study 15 rearrangements occurring in the MTS1 locus. We found that these breakpoints occur in two clusters (MTS1(bcralpha) and MTS1(bcrbeta) ). The three rearrangements occurring in MTS1(bcralpha) correspond to a recurrent recombination juxtaposing 5' MTS2 exon 1 and 5' MTS1 exon 1alpha sequences. Breakpoints for 10 of 12 rearrangements within MTS1(bcrbeta) are located at a polymorphic (CA) repeat, suggesting that this sequence might play a role in the clustering. For both MTS1(bcralpha) and MTS1(bcrbeta), sequence analyses and PCR mapping experiments show that the tightly clustered breakpoints are located in the vicinity of heptamers whose sequence is similar to those involved in the V(D)J recombination. Moreover, short deletions, GC-rich random nucleotide additions, and clone-specific junctional sequences are present in all cases, further suggesting that the rearrangements are due to illegitimate V(D)J recombinase activity. These data are the first demonstration that a tumor suppressor gene can be inactivated by the V(D)J recombinational mechanism. Moreover, they reinforce the view that this process, normally required for T-cell differentiation, plays a crucial role in the pathogenesis of T-ALL.
Base Sequence↗
Lymphomas in patients with Sjogren's syndrome are marginal zone B-cell neoplasms, arise in diverse extranodal and nodal sites, and are not associated with viruses.
The occurrence of non-Hodgkin's lymphoma (NHL) is the most serious complication of Sjogren's syndrome (SS). We performed a study of 16 NHLs occurring in patients with an underlying SS. These lymphomas arose not only in salivary glands (7 cases) but also in other mucosal extranodal sites (the stomach [4 cases], the lung [3 cases], the skin [3 cases], the buccal mucosa [1 case], the thymus [1 case]) and in nodal sites (8 cases). Low-grade marginal zone lymphomas (MZL) were diagnosed in 12 of the 16 patients, 9 of mucosa-associated lymphoid tissues (MALT) type in mucosal sites and 3 exclusively nodal. The 4 other patients presented with a high-grade B-cell lymphoma that was probably a histological transformation of an underlying low-grade MZL at least in 3 of the cases involving skin, stomach, and parotid, respectively. A t(14;18) translocation was detected in 1 of 8 lymphomas tested. We detected serum anti-p53 antibodies in 2 of the 14 studied patients. p53 protein was detected in 1 of 11 lymphomas tested. LMP protein and Eber RNAs of Epstein-Barr virus (EBV) were not detected in the 16 NHL biopsies. Using polymerase chain reaction, EBV was never detected except in 1 of 4 parotid lymphomas. No human T-lymphotropic virus 1 or human herpes virus 8 DNAs were detected in NHL biopsies. None of the patients had hepatitis C virus infection found using serological methods. Chemotherapy was usually efficient. In conclusion, lymphomas occurring in patients with an underlying SS are in most cases MZL. These lymphomas are not associated with viruses known to be present in other types of lymphomas. Some of the translocations or mutations of oncogenes or antioncogenes described in other lymphomas are detected in SS-associated lymphomas.
Adolescent↗