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PubMed1984

On the maturation order of AML cells: a distinction on the basis of self-renewal properties and immunologic phenotypes.

To investigate the heterogeneous cellular structure of human acute myeloid leukemia (AML), subpopulations of cells were distinguished by two combined criteria: proliferation and differentiation. Purified blast cells were fractionated from blood or bone marrow of patients with newly diagnosed AML and colonies and clusters grown in phytohemagglutinin (PHA)-leukocyte feeder cultures. Large colonies, small colonies, macroclusters, and microclusters were recloned separately to assess the replicative capacities as a function of clone size. Large colonies showed higher proliferative capacities than did small ones, etc. Anti-Ia and an antigranulocyte (B4.3) monoclonal antibody (MoAb) were then employed to evaluate the stage of differentiation of AML cells in two patients before and following colony culture. Alterations of the immunologic phenotypes appeared during colony formation. This suggested differentiation of cells to more mature B4.3 granulocyte antigen-positive stages. MoAb-dependent cell lysis with the two antibodies was subsequently performed to assess the phenotypes of the precursors of the colonies and clusters. Leukemic colony-forming cells were Ia-positive and B4.3-negative and different from cluster-forming cells, which were largely Ia-negative and B4.3-negative. These data suggest that the cell organization of AML fits a maturation scheme containing immature cells with relatively high proliferative capacities, intermediate cells with low proliferative capacities, and end cells that are nonreplicative, and each with specific phenotypes.

Adult↗

PubMed1995

Bone marrow morphology during induction phase of therapy for acute myeloid leukemia (AML).

Sequential bone marrow aspirates and sections from patients with acute myeloid leukemia (AML) were examined to determine if a correlation exists between bone marrow morphology during induction phase of therapy and outcome. Of 95 patients of AML diagnosed between July 1987 and December 1991, 53 uniformly treated patients (induction therapy with cytosine arabinoside and daunorubicin) had sequential bone marrow examinations performed in the 2- to 5-week period following initiation of induction therapy. Four morphologic patterns were recognized in these 53 patients: Group I (22 patients)--hypocellularity or normal regeneration (> or = 15% cellularity and < 5% blasts) on the initial 2-week marrow followed by marrows showing normal regeneration; Group II (10 patients)--hypocellularity followed by "reactive myeloblastosis" (> or = 15% cellularity, 5% to 34% blasts, with promyelocytes = or > blasts); Group III (12 patients)--residual blasts (> or = 5% blasts with blasts >> promyelocytes) in the initial posttherapy marrow; Group IV (9 patients)--atypical patterns not fitting any of the other categories. Complete remission was achieved in all 32 patients in Groups I and II without additional induction therapy, but was achieved eventually in only 10 of 21 patients in Groups III and IV combined (p < 0.005), 15 of whom received additional induction therapy. Remission duration and actuarial survival for each group were as follows: Group I: 344/596 days; Group II: 443 days/> 660 days; Group III and IV combined: 351/311 days (p = 0.017 for actuarial survival). Seven of 21 patients in Groups III and IV had unfavorable initial morphology (MO, hypoplastic AML and AML preceded by myelodysplasia) compared to only 3 of 32 patients in Groups I and II (p = 0.039). It was thus observed that "reactive myeloblastosis" with up to 34% blasts on the third or fourth week bone marrow following an initial hypocellular marrow does not require additional induction therapy to achieve durable remissions or favorable survival. Also, residual blasts that outnumber promyelocytes, and atypical patterns of regeneration correlate with lower remission induction rates, shortened survival, and unfavorable morphology on the initial diagnostic bone marrow.

Adult↗

PubMed1995

Autologous PBSC transplant for late onset AML after mafosfamide-purged and TBI-containing autologous BMT.

The prognosis of AML patients who fail autologous BMT and lack a donor for a salvage allogeneic BMT is very poor. We administered an alternative drug treatment including autologous PBSC transplant to a child who relapsed with AML 4 years after a mafosfamide-purged and TBI-containing BMT for second remission AML. Treatment comprised induction-consolidation with 'short' ICE courses (idarubicin-Ara C-etoposide for 2 days), high-dose CY plus G-CSF with collection of CD34+ PBSC and myeloablative treatment with high-dose mitoxantrone/BEAM supported by autologous PBSC reinfusion. The outcome was complete response, lasting 24 months. The patient died in remission of drug-related cardiotoxicity. This report emphasizes the possibility of a secondary AML after mafosfamide-purge autologous BMT, and documents the feasibility of an autologous PBSC transplant in such a high risk setting.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1994

No evidence for GM-CSF receptor alpha chain gene mutation in AML-M2 leukemias which have lost a sex chromosome.

Sixty-five percent of acute myeloid leukemias subtype M2 (AML-M2), displaying a translocation between chromosomes 8 and 21 (t8;21) have also lost one or other copy of the sex chromosomes (XO). This finding has led to the hypothesis that a recessive oncogene may be present in the DNA common to both sex chromosomes, that is the pseudoautosomal region. The alpha chain of the receptor for the hemopoietic growth factor GM-CSF has recently been mapped to the human pseudoautosomal region and, given the role of this molecule in the control of normal hemopoiesis, it is thought to be a good candidate gene. This paper examines the structure, expression, and sequence of the GM-CSF receptor alpha chain gene (GMR alpha) in three primary AML-M2,XO samples and one AML-M2,XO cell line. Results of this work show no gross rearrangement or loss of the remaining allele of GMR alpha, detectable expression of its transcript and protein, and no changes at the nucleotide sequence level. Thus, we find no evidence to support a role for the GM-CSF receptor alpha chain molecule in the development of AML-M2,XO leukemias.

Base Sequence↗

PubMed1994

Prevalence and clinical correlations of MLL gene rearrangements in AML-M4/5.

Rearrangements of the human trithorax gene (MLL, HRX, Htrx-1, All-1) were studied by Southern blotting in blast cells stored at presentation from 65 adults with de novo acute myelomonocytic (AML-M4) and acute monocytic leukemia (AML-M5). MLL rearrangements were demonstrated in 15 (23%) cases, including eight patients in whom karyotype analysis had failed to detect abnormalities of chromosome band 11q23. The patients with MLL rearrangements did not differ significantly from those with germline configurations in terms of the sex and age of the patients, the presence of lymphadenopathy, hepatosplenomegaly, or central nervous system involvement, and the absolute blast count at diagnosis. Kaplan-Meier analysis of the treated patients demonstrated no difference in survival for patients with MLL rearrangements compared with those without rearrangements. Therefore, in contrast to infantile acute leukemia, in adults with AML-M4 and AML-M5, MLL rearrangements do not identify a subgroup of patients with different clinical features or prognosis.

Adolescent↗

PubMed1993

Autologous bone marrow transplantation with marrow purged by mafosfamide in seven patients with myelodysplastic syndromes in transformation (AML-MDS): a pilot study.

Seven patients with acute myeloblastic leukemia (AML) occurring on myelodysplastic syndromes (MDS) were consolidated while in complete remission (CR) by autologous bone marrow transplantation (ABMT) with a marrow purged in vitro by mafosfamide. The median age of population was 44 years (range 39-55). MDS FAB diagnosis was established before progression to AML in five patients: refractory anaemia with excess of blast (RAEB) in three patients, RAEB in transformation (RAEB-t) in one patient, and chronic myelomonocytic leukemia (CMML) in one patient. In the remaining two patients, the diagnosis of MDS (as a secondary malignancy in one) was made retrospectively at time of overt AML. Three out the seven patients had karyotypic abnormalities. The median interval between the obtention of CR and ABMT was 7 months (range 6-18). One patient died from transplant related toxicity. Engraftment occurred at a median of 41 days (range 27-60), for white blood cells (> 10(9)/l) and 120 days (range 60-180) for platelets (> 50 x 10(9)/l). Four patients relapsed at 2.5, 6.8, and 25 months post-ABMT. Two patients are alive and well at 10 and 28 months, respectively. ABMT with marrow purged by mafosfamide is feasible in patients with AML following MDS with a prospect of cure. However, further studies are needed to assess the real value of this approach.

Adult↗

PubMed1996

The beta subunit common to the GM-CSF, IL-3 and IL-5 receptors is highly polymorphic but pathogenic point mutations in patients with acute myeloid leukaemia (AML) are rare.

The intracytoplasmic tail of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) beta c chain is essential for the activation of ligand-mediated signal transduction pathways in myeloid cells. Alterations in this region could deregulate normal signalling processes. We have therefore used RT-PCR-SSCP analysis of the receptor tail to look for point mutations in RNA from 35 patients with acute myeloid leukaemia (AML) and 10 haematologically normal controls. Patterns differing from those of the haemopoietic cell line TF-1 were detected in 25/35 (71%) AML patients and 8/10 (80%) normal controls. A total of six base substitutions were identified by sequencing. Three were conservative for the amino acid involved, three led to amino acid differences, valine652-->methionine, glycine647-->valine and proline603-->threonine. One alteration was found only in a normal control, the other five were all found in both AML patients and normal controls suggesting that they were DNA polymorphisms. Two substitutions were particularly common with allele frequencies of 0.23 (G1972-->A, unchanged proline648) and 0.13 (C1306-->T, unchanged serine426). These results indicate that the GM-CSFR beta c chain is highly polymorphic but point mutations of the intracytoplasmic tail do not appear to contribute frequently to the pathogenesis of AML.

Base Sequence↗

PubMed1996

Myelodysplasia and acute myelogenous leukemia in Down's syndrome. A report of 40 children of the AML-BFM Study Group.

Forty children with Down's syndrome have been identified among 633 patients in the cooperative pediatric BFM studies for acute myelogenous leukemia (AML) since 1987. The following features were characteristic for these patients: (1) a 500-fold higher than expected incidence of megakaryoblastic leukemia (M7) with a peak incidence under 4 years of age; (2) a myelodysplastic prephase with thrombocytopenia lasting for several months up to a few years; (3) frequent involvement of the red cell lineage as suggested by dyserythropoiesis in the bone marrow; (4) coexpression of the lymphoid cell surface antigen CD7 on the leukemic blasts; (5) absence of the translocation t(1;22), instead presence of complete or partial trisomies involving chromosomes 8 and 1; and (6) a good response to chemotherapy. Nearly half of the patients did not receive any or only palliative chemotherapy and subsequently died. In 21 patients treatment according to the AML-BFM protocols was intended, however, with major dose or protocol reductions in six patients. Four died early, 15 (71%) achieved remission. Nine of 11 patients remaining alive for 0.6-6.5 years had received intensive treatment including high-dose cytosine arabinoside (HD-Ara-C). The 5-year survival estimate of 48% +/- 12% was similar to patients without Down's syndrome. In conclusion, these clinical and biological features suggest that M7 leukemia in children with Down's syndrome (M7-Down) is distinct from megakaryoblastic leukemia in other children. Children with Down's syndrome show a favorable outcome if treated adequately. However, overtreatment should be avoided and life-threatening infections pose a particular problem. Therefore, standard-risk AML therapy (including HD-Ara-C) should be considered in children with Down's syndrome and AML.

Adolescent↗

PubMed1996

Anatomy of the 5q- deletion: different sex ratios and deleted 5q bands in MDS and AML.

Deletion of part of the long arm of chromosome 5 (5q-) is the most common structural chromosome aberration in patients with myeloid disorders. Most patients are women. Although widely varying 5q segments are deleted, the vast majority of cases share a common deleted region (5q31). Whether or not other deleted 5q regions are of clinical importance is unknown. In an attempt to throw light on this question, 439 published 5q- cases were analyzed. The female preponderance was found to be limited to refractory anemia (RA), RA with excess of blasts (RAEB) and myeloproliferative disease, all with 5q- as the only chromosome aberration. When other chromosome aberrations had developed or the diagnosis was AML, only marginal female preponderance was observed. The 5q31 was deleted in all patients with MDS and 5q- as the only aberration. In contrast, in AML and following cytogenetic progression other bands were deleted more often than 5q31. This difference, together with a different female preponderance suggests diverse pathogenetic mechanisms in MDS and AML. Furthermore, the bands 5q13-5q21 were found to be of importance for clinical progression from RA to RAEB, and 5q22-5q33 of importance for development of AML and cytogenetic progression. In conclusion, the 5q bands deleted affect both clinical and cytogenetic progression.

Acute Disease↗

PubMed1998

Pre-clinical evaluation of probes to detect t(8;21) AML minimal residual disease by fluorescence in situ hybridization.

The 8;21 translocation in acute myeloid leukemia (AML) results in a consistent fusion transcript, AML1/ETO. Long-term clinical remission occurs in some patients despite incomplete eradication of AML1/ETO as demonstrated by RT-PCR, thus limiting the usefulness of this assay. An important future goal will be to determine if there is a level of minimal residual disease (MRD) in patients below which relapse is unlikely. For the detection of MRD, we have developed reagents for fluorescence in situ hybridization (FISH) that identify both derivative 8 and 21 chromosomes with a high analytical sensitivity. In t(8;21) AML cells, two fused signals were detected in addition to the normal 8 and 21 alleles. The sensitivity and specificity of this probe mixture were analyzed in cell lines and patient bone marrows. One and two randomly juxtaposed signals were observed in 2.4 and 0.04% of normal cells, respectively. However, these were easily differentiated from t(8;21) cells by the absence of signals from the normal alleles. Using as criteria the presence of two fused signals plus the normal alleles, we observed no false positives among 5,000 normal cells. The probe correctly identified 20/20 patients with t(8;21) AML and 10/10 non-t(8;21) patients. In cell dilution experiments, the analytical sensitivity of this reagent was equal to that of the X chromosome and Y chromosome alpha-satellite probes. These optimized probes should facilitate the quantitative assessment and study of MRD in t(8;21) AML.

Bone Marrow Cells↗

PubMed1998

Treatment of elderly patients with AML: results of an individualized approach.

BACKGROUND AND OBJECTIVE: AML treatment in elderly patients must be individualized according to their characteristics. We report the results of a tailored treatment approach in all consecutive AML patients older than 60 years diagnosed at our institution during the last 2 years. DESIGN AND METHODS: Between December 1994 and December 1996, 43 AML patients over 60 years of age (median, 72; range 61-89) were managed according to their performance status (PS) and associated diseases. Twenty patients (46%) were eligible for intensive chemotherapy and received combination chemotherapy including an anthracycline (Idarubicin or daunorubicin), ara-C and VP-16. After complete remission (CR), consolidation chemotherapy with mitoxantrone and intermediate-high-dose ara-C was given to 13 of the 15 patients in remission (65% of all patients candidates for intensive treatment). Twenty-three patients who were not eligible for intensive chemotherapy received palliative measures. RESULTS: Patients treated with one course of intensive chemotherapy had a CR rate of 70% (95% CI: 48-92%)(n = 14) with a mortality rate of 20% (n = 4) and a resistance of 10% (n = 2). An additional patient reached CR after rescue therapy. Median CR duration was 10.5 months. Median survival was 10.5 months. Patients above 70 years had a median survival of 5 months compared to the median not reached for those aged between 60 and 70 years (p = 0.03). This latter group had a probability of survival of 52 +/- 18% at 18 months. None of the patients treated with palliative measures achieved CR and the median survival in this group was only 1.5 months. INTERPRETATION AND CONCLUSIONS: Patients with AML aged 70 years or less with good PS and without severe associated diseases should be intensively treated due to the high probability of achieving CR and an acceptable median-term survival. By contrast, results in patients 70 years or older and in those suitable only for palliative treatment because of a poor PS or severe associated diseases are very poor. Alternative treatment approaches for these patients should be investigated.

Aged↗

PubMed1998

Chromosomal abnormalities in secondary MDS and AML. Relationship to drugs and radiation with specific emphasis on the balanced rearrangements.

BACKGROUND AND OBJECTIVE: In therapy-related myelodysplasia (t-MDS) or acute myeloid leukemia (t-AML) balanced chromosome aberrations to bands 11q23 and 21q22 have been significantly related to previous chemotherapy with DNA topoisomerase II inhibitors. The purpose of the present study was to evaluate to what extent other balanced chromosome aberrations show the same association, and to evaluate a possible relationship to patient age and subgroups of drugs. DESIGN AND METHODS: All previously published cases of t-MDS and t-AML with any type of balanced aberration identified from Felix Mitelman's Catalog of Chromosome Aberrations in Cancer were analyzed for age and type of previous therapy, and the results were evaluated in univariate and multivariate analyses. RESULTS: A total of 422 cases were recorded, 328 had previously received well specified types of chemotherapy; 254 presented one out of five characteristic balanced aberrations, whereas 168 cases presented different uncharacteristic balanced aberrations. In univariate analysis cases with translocations to 11q23 had most often received DNA topoisomerase II inhibitors, whereas patients with the uncharacteristic balanced rearrangements most often had received alkylating agents (p < 0.00000001). Inv(16), t(15;17), and t(9;22) were likewise significantly associated with previous therapy with DNA topoisomerase II inhibitors and to almost the same extent as translocations to 21q22. Patients with translocations to 11q23 were significantly younger as compared to all other groups of patients. Translocations to 11q23 predominated following therapy with epipodophyllotoxins, whereas patients with translocations to 21q22, inv(16), t(15;17), and t(9;22) most often had received anthracyclines. In a multivariate analysis taking age into consideration, however, these drug-specific associations were no longer significant. INTERPRETATION AND CONCLUSIONS: Specific balanced chromosome aberrations in t-MDS and t-AML involving chromosome bands 11q23 and 21q22, inv(16), t(15;17), and t(9;22) are all significantly associated with previous therapy with DNA topoisomerase II inhibitors, as compared to the uncharacteristic balanced aberrations most commonly observed after therapy with alkylating agents. Younger age and not a specific type of DNA topoisomerase II inhibitor seems to predispose specifically to development of t-MDS and t-AML with translocations to chromosome band 11q23.

Acute Disease↗

PubMed2002

Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm.

To assess the prognostic relevance of activating mutations of the FLT3 gene in homogeneously treated adults 16 to 60 years of age with acute myeloid leukemia (AML) and normal cytogenetics, pretreatment samples from 224 patients entered into 2 consecutive multicenter treatment trials were analyzed for FLT3 internal tandem duplications (ITDs) and Asp835 mutations. Treatment included intensive double-induction therapy and postremission therapy with high cumulative doses of high-dose cytarabine. ITDs were detected in 32% of the patients and were related to de novo AML and to high white blood cell (WBC) counts, percentages of peripheral blood (PB) and bone marrow (BM) blasts, and serum lactate dehydrogenase levels. Asp835 mutations were present in 14% of the patients and were associated with WBC counts and percentages of PB and BM blasts that were higher than those of patients without FLT3 mutations. With a median follow-up of 34 months, remission duration and overall survival (OS) were significantly shorter for patients with Asp835 mutations or an ITD than for those without FLT3 mutations (P =.03 and P =.0004, respectively). These results were attributable mainly to the negative prognostic effect of FLT3 ITDs. On multivariate analysis, mutant FLT3 was an independent marker affecting remission duration and OS (hazard ratio, 2.35 and 2.11, respectively). Fluorescence in situ hybridization did not detect monoallelic FLT3 deletions in ITD-positive patients. FLT3 mutations identify a subset of young AML patients with normal cytogenetics who do not benefit from intensive chemotherapy, including double-induction and postremission therapy with high-dose cytarabine.

Acute Disease↗

PubMed2026

Day + 30 detection of minimal residual FLT3-ITD by high-sensitivity PCR-NGS predicts relapse risk and guides post-transplant maintenance in AML.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has improved outcomes in patients with acute myeloid leukemia (AML) harboring FLT3-internal tandem duplication (FLT3-ITD) mutations. However, relapse still occurs in 15-35% of these patients after transplantation. Therefore, early and highly sensitive detection methods are required to identify patients at risk of relapse and enable timely post-transplant intervention. METHODS: In this NICHE cohort study, a total of 136 patients were included, then we evaluated whether high-sensitivity polymerase chain reaction (PCR)-next-generation sequencing (NGS) for FLT3-ITD (limit of detection: 5 × 10-6) on day + 30 post-HSCT could identify patients at a high risk of relapse and inform decisions regarding maintenance therapy. RESULTS: Among the 136 patients, 37 patients (27.2%) had detectable FLT3-ITD clones on day + 30. These patients exhibited a significantly higher cumulative incidence of post-HSCT multiparameter flow cytometry (MFC)-measurable residual disease (MRD) relapse (40.3% vs. 18.8%, p = 0.001). Notably, FLT3-ITD-positive patients who received FLT3 inhibitor maintenance therapy had no relapses, while 6 out of the 13 patients who did not receive maintenance therapy relapsed. Conversely, FLT3-ITD-negative patients without high-risk factors (2022 European LeukemiaNet adverse-risk group, relapsed/refractory AML, MFC-MRD positivity pre-HSCT) showed limited benefit from maintenance therapy (MFC-MRD-free survival: hazard ratio (HR) = 0.25 (0.03-2.11), p = 0.204; OS: HR = 0.20 (0.02-1.70), p = 0.142). CONCLUSIONS: This is the first study to demonstrate that detection of minimal FLT3-ITD clones at the fixed time point of day + 30 post-HSCT can reliably stratify relapse risk in AML patients and provide a rationale for individualized post-transplant maintenance therapy.

Humans↗

PubMed2007

Tyrosine kinase mutations of JAK2 are rare events in AML but influence prognosis of patients with CBF-leukemias.

We investigated a large number of acute myeloid leukemia (AML) samples (n=959) for the presence of the JAK2 V617F mutation. We found a low incidence of the mutation in these AML samples (1%). JAK2 V617F mutations clustered in AML samples with an aberrant karyotype (p<0.05). The incidence of JAK2 V617F in patients with a core binding factor (CBF) leukemia was 3.6% (p<0.01). Moreover, JAK2 V617F mutations in CBF leukemias were associated with an aggressive clinical course with 80% of the patients relapsing.

Aged↗

PubMed2004

Feasibility and results of autologous stem cell transplantation in de novo acute myeloid leukemia in patients over 60 years old. Results of the CETLAM AML-99 protocol.

BACKGROUND AND OBJECTIVES: The benefits of high-dose cytarabine, anthracyclines and hematopoietic stem cell transplantation in the treatment of acute myeloid leukemia (AML) are greater in younger rather than in older patients. We assessed the proportion of patients over 60 years with de novo AML who qualified for intensive therapy and determined the feasibility and results of autologous stem cell transplantation (ASCT) in first complete remission (CR). DESIGN AND METHODS: Induction therapy included idarubicin, cytarabine and etoposide. Patients who achieved CR received one cycle of mitoxantrone and cytarabine and ASCT as consolidation therapies. RESULTS: Over a 4-year period, 258 patients were registered of whom 135 (52%) were enrolled for intensive treatment. The CR rate was 61%, advanced age (p=0.033) and unfavorable cytogenetics (p=0.015) emerged as independent negative prognostic factors for CR. The 2-year overall survival (OS) was 23 % (CI 14%-30%) and was poorer in patients with unfavorable cytogenetics (p=0.035), age over 70 years (p=0.019) or leukocytosis (p=0.006). Only 27% of the potential candidates underwent ASCT. The probability of 2-year leukemia-free survival after consolidation was 39% (CI 6%-71%) for these patients and 22% (CI 6% - 39%) for candidate patients not undergoing ASCT (p=0.07). INTERPRETATION AND CONCLUSIONS: Over 25% of the patients 60 to 70 years with de novo AML benefit from standard intensive treatment. In these patients, ASCT has a tolerable toxicity and may have a positive impact on leukemia-free survival.

Acute Disease↗

PubMed1997

Chemoattractant receptors for interleukin-8 and C5a: expression on peripheral blood leukocytes and differential regulation on HL-60 and AML-193 cells by vitamin D3 and all-trans retinoic acid.

Two homologous high-affinity receptors for the chemoattractant interleukin-8, IL-8RA and IL-8RB, and one for the chemoattractant C5a (C5aR) have been cloned. These membrane proteins are members of the rhodopsin superfamily of G-protein coupled seven-transmembrane segment receptors. New monoclonal antibodies (mAb) directed against the deduced N-terminal sequences of the IL-8RA (mAb SE2) and IL-8RB (mAb HC2) were generated to determine the IL-8R expression on human blood leukocytes and two human myeloid cell lines. The C5aR expression was detected using the mAb W17/1. Approximately 107,000 C5aR, 55,000 IL-8RA, and 25,000 IL-8RB molecules per cell could be detected on human granulocytes by flow cytometric analysis. On peripheral blood monocytes, 42,000 C5aR molecules/cell and 3000 IL-8RB molecules/cell were expressed. However, we were unable to quantitate IL-8RA expression, which was detectable but below 2500 molecules per cell and thus outside the standard range for the quantitation of receptor molecules by flow cytometry. On AML-193 cells, only the IL-8RB was constitutively expressed, whereas on HL-60 cells, we could not detect expression of any of the three receptors. Vitamin D3 (250 ng/ml, 7 days), which has been shown to induce differentiation of AML-193 and HL-60 cells into the monocytic phenotype, led to an up-regulation of IL-8RB and C5aR in both cell lines in the absence of any expression of IL-8RA. In contrast, all-trans retinoic acid (0.1 microM, 7 days), which induces differentiation into the granulocytic phenotype, led to an up-regulation of IL-8RB in AML-193 cells and to an expression of IL-8RB and C5aR in HL-60 cells. Again, neither cell line expressed IL-8RA. These findings suggest that regulation of IL-8RA expression differs from that of its IL-8RB homolog and may be a late event in leukocyte maturation.

Antigens, CD↗

PubMed2004

MLL/GRAF fusion in an infant acute monocytic leukemia (AML M5b) with a cytogenetically cryptic ins(5;11)(q31;q23q23).

More than 30 fusions involving the MLL gene at 11q23 have been reported in acute myeloid leukemia (AML). Some of these chimeras are rather common, such as MLL/MLLT3(AF9), but many are quite rare, with some, for example, MLL/GRAF, described only in a single case. The MLL/GRAF fusion, in which the reciprocal hybrid was not expressed, suggesting that the former transcript was the leukemogenic one, was detected in a juvenile myelomonocytic leukemia with a t(5;11)(q31;q23). Here, we report a second case--an infant acute monocytic leukemia (AML M5b)--with an MLL/GRAF fusion. By conventional G-banding, the karyotype was normal. However, Southern blot and fluorescence in situ hybridization analyses revealed that MLL was rearranged and that the 5' part of the MLL gene was inserted into 5q in the vicinity of 5q31, which harbors GRAF. Reverse-transcriptase polymerase chain reaction (PCR) showed that exon 9 of MLL was fused in-frame with exon 19 of GRAF. Extralong genomic PCR with subsequent sequence analysis demonstrated that the breakpoints occurred in intron 9 of MLL, nine base pairs (bp) downstream from exon 9, and in intron 18 of GRAF, 117 bp downstream from exon 18. A 6-bp insertion (ACACTC) of unknown origin was present at the junction. The putative MLL/GRAF fusion protein would retain the AT-hook DNA-binding domain, the DNA methyl transferase motif, the transcription repression domain of MLL, and the SH3 domain of GRAF. As expected, the reciprocal GRAF/MLL was neither expressed nor generated at the genomic level as a consequence of the ins(5;11)(q31;q23q23). On the basis of the now-reported two cases with MLL/GRAF, we conclude that this transcript--but not the reciprocal one--characterizes a rare genetic subgroup of infant AML.

Blotting, Southern↗