Simultaneous regression of Philadelphia chromosome and multiple nonrecurrent clonal chromosomal abnormalities with imatinib mesylate in a patient autografted 22 years before for chronic myelogenous leukemia.

A 31-year-old patient developed chronic myelogenous leukemia (CML) in November, 1983. In November 1984, following a diagnosis of acceleration, he received an autologous hemopoietic transplant after conditioning with cyclophosphamide and total body irradiation. The autologous marrow was purged with mafosfamide. Over 20 years, the patient remained in chronic phase of CML. Multiple nonrecurrent clonal chromosomal abnormalities appeared leading to a very complex karyotype, including among others involvement of chromosomes 1, 7, 9, 13, 19, and X. Fluorescent in situ hybridization showed that the two chromosomes 9 were involved. Acute myeloid crisis was diagnosed in February, 2004. Treatment with imatinib mesylate resulted within 6 months in a total disappearance of all chromosomal abnormalities with a complete cytogenetic and molecular response, which persists 3 years later. We question whether the ex vivo purging procedure with mafosfamide has favored the occurrence of these particular cytogenetic abnormalities (with no independent oncogenic potential) within the original leukemic stem cell pool. It remains unclear whether the autologous transplantation has indeed resulted into some prolongation of the duration of the chronic phase, which lasted for 20 years. At time of acute crisis, the dramatic response to imatinib mesylate leading to a complete cytogenetic and molecular response is noteworthy.

Human GATA-3 trans-activation, DNA-binding, and nuclear localization activities are organized into distinct structural domains.

GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors.

Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: a retrospective analysis from the French intergroup of CML (Fi(phi)-LMC GROUP).

The emergence of ABL point mutations is the most frequent cause for imatinib resistance in chronic myelogenous leukemia (CML) patients and can occur during any phase of the disease; however, their clinical impact remains controversial. In this study, we retrospectively analyzed the predictive impact of 94 BCR-ABL kinase domain mutations (18 T315I, 26 P-loop, 50 in other sites) found in 89 imatinib-resistant CML patients. At imatinib onset, 64% of patients (57/89) were in chronic phase (CP), 24% (21/89) in accelerated phase (AP) and 12% (11/89) in blastic phase (BP). T315I and P-loop mutations were preferentially discovered in accelerated phase of BP CML, and other types of mutations in CP (P=0.003). With a median follow-up of 39.2 months (6.3-67.2), since imatinib initiation, overall survival (OS) was significantly worse for P-loop (28.3 months) and for T315I (12.6 months), and not reached for other mutations (P=0.0004). For CP only, multivariate analysis demonstrated a worse OS for P-loop mutations (P=0.014), and a worse progression-free survival (PFS) for T315I mutations (P=0.014). Therefore, P-loop and T315I mutations selectively impair the outcome of imatinib-resistant CML patients, in contrast to other mutations, which may benefit from dose escalation of imatinib, able to improve or stabilize disease response.

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.

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells.

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.

Chimerism analysis in peripheral blood using indel quantitative real-time PCR is a useful tool to predict post-transplant relapse in acute leukemia.

Detection of increasing mixed chimerism (IMC) using standard PCR correlates with relapse after allo-SCT for acute leukemias (ALs). Quantitative real-time PCR of insertion/deletion polymorphism (indel qrtPCR) is a much more sensitive method, which can be performed on peripheral blood. We studied the significance of low increases of recipient cells (0.1%) detected by indel qrtPCR in a cohort of 89 transplants. We did not observe relapse among the 32 patients with no IMC. Fifty-seven patients presented a first IMC, which was followed by four different scenarios: a decreasing MC (26 cases, no relapse), a stable MC (1 case, 1 relapse), a second IMC (24 cases, 15 relapse) or no control of chimerism (6 cases, 5 relapses). In multivariate analysis, detection of two successive IMCs was strongly associated with relapse (hazard ratio (HR): 9.4, 95% confidence interval (CI): 3.8-23; P<0.0001). Among the 57 patients who presented at least one IMC, 27 underwent immunomodulation (tapering of immunosuppression or donor lymphocyte injection), leading to a 1-year relapse rate of 15.7% vs 57.6% in the 30 other patients (P=0.0007). Altogether, these results indicate that chimerism analysis using indel qrtPCR in peripheral blood is a useful tool for detection of relapse in patients transplanted for AL.

Chimerism analysis by lineage-specific fluorescent polymerase chain reaction in secondary graft failure after allogeneic stem cell transplantation.

BACKGROUND: Chimerism analysis is essential in understanding the etiology of graft failure occurring after allogeneic stem cell transplantation. The detection of marrow and/or blood host cells suggests graft rejection, relapse of the underlying disease, or a state of stable mixed chimerism. However, complete donor chimerism may be observed in some cases. Our objective was to characterize, by a sensitive process of chimerism analysis, six cases of graft failure occurring after transplant. METHODS: Six cases of secondary graft failure, in which previous analysis had shown complete donor chimerism by standard polymerase chain reaction amplification of variable number of tandem repeats, were studied. In order to detect a minority population of recipient cells, we increased the sensitivity of the process by using fluorescent polymerase chain reaction and analyzing the origin of T, B, and natural killer lymphocytes at the time of graft failure. RESULTS: The complete donor origin of mononuclear cells and lymphocytic populations was confirmed with this method in five of six patients. In the remaining patient, diagnosis of graft failure was clarified by the detection of a previously undetected mixed chimerism, compatible with graft rejection. In the other five patients, graft rejection was thereby excluded and graft failure could be related to viral infection or to graft-versus-host disease. CONCLUSION: Our sensitive process of fluorescent lineage-specific chimerism analysis may help in distinguishing between graft rejection and other mechanisms of graft failure, which is essential for deciding appropriate therapy.

Methylenetetrahydrofolate reductase deficiency in four siblings: a clinical, biochemical, and molecular study of the family.

A diagnosis of methylenetetrahydrofolate reductase (MTHFR) deficiency was made in four sibs at different ages. The first three, including a pair of twins, had retarded psychomotor development, poor social contact, and seizures. Biologically, hyperhomocysteinemia and hypomethioninemia were found associated with low folate levels in serum and red cells, especially undetectable methyltetrahydrofolate in red cells. In the fourth child, prenatal diagnosis was not conclusive because of moderate decrease of enzymatic activity in chorionic villi and trophoblast. The girl was also affected, as shown by hyperhomocysteinemia and low folate levels found several days after birth. A 677C-->T (Ala-->Val) mutation was found in a homozygous state in the four children and in the father. Additionally, a second homozygous mutation, 1081C-->T, changing an arginine to cysteine also was identified in all of the children, whereas the distantly consanguineous parents were heterozygous. This amino acid substitution affecting an arginine residue in a sequence located at the end of catalytic domain seems critical for the function of the enzyme. The difficulty of prenatal diagnosis is discussed given the variability found in enzymatic activity and in the clinical phenotypes.

Autologous reconstitution combined with durable leukemic remission after allogeneic BMT for CML: absence of persistence of a donor-derived T cell effector population.

We report a case of autologous hematopoietic recovery with durable leukemic remission after BMT from a phenotypically HLA-identical donor in a 30-year-old male with CML. Graft loss was diagnosed from day 60 post-BMT by VNTR PCR. To assess for the presence of a minor donor-derived T cell population that could exert an anti-leukemic effect, we serially applied a sensitive process of chimerism analysis by fluorescent PCR on sorted T cells. No residual donor T cells could be detected. We also showed retrospectively that a very sensitive method of MRD analysis by real-time quantitative PCR could have permitted prediction of relapse in this case.

Plasma homocysteine levels related to interactions between folate status and methylenetetrahydrofolate reductase: a study in 52 healthy subjects.

Hyperhomocysteinemia, a risk factor for vascular disease, is related to vitamin B12, vitamin B6, and especially folate deficiency, or to genetic factors such as mutations in methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the remethylation pathway of homocysteine to methionine. Recently, a C677 --> T mutation identified in the MTHFR gene was found to be frequently associated with decreased MTHFR activity and an elevated plasma homocysteine concentration. Since hyperhomocysteinemia seems to be determined by both genetic and environmental factors, we studied the interactions between MTHFR (phenotype and genotype) and folate status, including methyltetrahydrofolate (methylTHF), the product of MTHFR, on the homocysteine concentration in 52 healthy subjects, (28 women and 24 men; mean age, 32.7 years). MTHFR activity seems to be dependent on folate status, as shown by a lower activity in folate-deficient subjects and a return to normal values after supplementation with folic acid, and also by a decreased enzymatic activity on phytohemagglutinin (PHA)-stimulated lymphocytes grown in a folic acid-deficient medium. Conversely, the C677 --> T mutation seems to influence folate metabolism. Subjects who were homozygous for this mutation (+/+) had significantly higher plasma homocysteine and lower plasma folate and total and methylfolate levels in red blood cells (RBCs) than heterozygous (+/-) and normal (-/-) subjects. The ratio of RBC methylfolate to RBC total folate was, respectively, 0.27 in +/+, 0.66 in +/-, and 0.71 in -/-. This mutation seems to have an impact on methylTHF generation. These data illustrate the interactions between nutritional and genetic factors.

[Chronic myeloid leukemia and imatinib: Experience at the Lome Campus teaching hospital (Togo)]. / Leucémie myéloïde chronique et imatinib, expérience du CHU Campus de Lomé au Togo.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal malignant myeloproliferative disorder characterized by the expansion of hematopoietic cells carrying the Philadelphia chromosome (t 9.22). Our main objective was to assess the efficacy of imatinib in CML patients, measured by their survival. METHODS: Over a six-year period (June 2003 through May 2009), 25 patients were seen regularly for CML at the Lomé Campus teaching hospital. Patients received imatinib after diagnosis and underwent regular laboratory monitoring (quantification of BCR-ABL ratio by RT-PCR). Patients' survival and treatment response were evaluated. RESULTS: Patients' mean age at diagnosis was 40 years (range: 9 to 72 years). Men predominated (17 compared with 7 women). Splenomegaly was found in 80% of cases. The mean leukocyte level was 188.71 g/L (24.4-350). Six patients (24%) had thrombocytosis with a mean platelet count of 491.15 g/L (108-2000). Six patients (24%) died after developing accelerated-phase CML or blast crisis. Estimated overall survival of patients at 6 years was 60%. Molecular biology monitoring detected a secondary G250E mutation with resistance to imatinib in one patient. Standard hematological side effects led to reduction in imatinib doses. The principal nonhematological side effects were weight gain and transient digestible disorders. CONCLUSIONS: At six years after diagnosis, imatinib was effective in treating patients with CML, even in sub-Saharan Africa. Mutation-induced resistance required regular molecular biological monitoring to determine the need to switch to later-generation tyrosine kinase inhibitors.

Fully functional NK cells after unrelated cord blood transplantation.

Promising results of umbilical cord blood transplantation (UCBT) from unrelated donors have been reported in patients with hematologic disorders. These transplants, having potential to trigger beneficial donor-versus-recipient natural killer (NK) cell-mediated alloreaction, we have conducted the first extensive analysis of the phenotypic and functional properties of NK cells after UCBT. NK cells from 25 patients with high-risk hematologic malignancies were compared with cells derived from both healthy adult and CB cells. We found that following UCBT, NK cells display not only some phenotypic features associated with maturity but also unique characteristics that make them fully functional against leukemic blasts. We propose that this full functionality of alloreactive donor-derived NK may drive graft-versus-leukemia reactions after UCBT.

Involvement of mature donor T cells in the NK cell reconstitution after haploidentical hematopoietic stem-cell transplantation.

We previously demonstrated that natural killer (NK) cells generated after haploidentical stem-cell transplantation (SCT) are blocked at an immature state characterized by phenotypic features and impaired functioning and that this may affect transplantation outcome. We hypothesize that the absence of mature donor T cells in the graft may affect NK cell differentiation. NK cells from 21 transplant recipients who underwent either partial (pTCD; n=11) or extensive (eTCD; n=10) T-cell depletion were compared with NK cells from their healthy donors. We report that despite the strong graft-versus-host disease (GvHD) reaction, pTCD patients, with T cells present during SCT, had a better clinical outcome than patients with eTCD transplants. In addition, the frequency of CD3- CD56(bright) and NKG2A+ NK cells was much lower in pTCD than in eTCD patients after transplantation, and the level of cytotoxicity against primary haplo-mismatched blasts was significantly more pronounced after pTCD than eTCD transplants. These finding strongly suggest that mature donor T cells in the graft may play a key role in NK cell differentiation in vivo, after haploidentical SCT.