Amparo Sempere

A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling acute promyelocytic leukemia

Chromosomal translocations in hematological malignancies often result in novel fusion chimeric genes. We report a case of acute myeloid leukemia with a clonal translocation t(11;12)(p15;q13) displaying morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. The gene fused to NUP98 (nucleoporin 98) was detected by comparative genomic hybridization array as the retinoid acid receptor gamma gene (RARG). The involvement of RARG in a chimeric fusion transcript has not been reported previously in human leukemia.

Immunofluorescent analysis with the anti-PML monoclonal antibody PG-M3 for rapid and accurate genetic diagnosis of acute promyelocytic leukemia

Genetic diagnosis is currently considered the most reliable method to accurately identify patients with acute promyelocytic leukemia (APL) requiring tailored therapy including all-trans retinoic acid (ATRA). We investigated the clinical effectiveness of immunofluorescence techniques with the anti-PML monoclonal antibody PG-M3 for rapid and accurate diagnosis of APL. PML immunofluorescence staining was analyzed in 164 patients with acute myeloblastic leukemia (AML), including APL (110 patients) and non-APL subtypes (54 patients). All 54 patients with an AML phenotype, in whom tests for t(15;17) or its fusion gene PML/RARα were negative, showed a speckled (macrogranular) nuclear pattern. Of the 110 genetically diagnosed APL patients, 108 showed a microgranular pattern that confirmed PG-M3 positivity. The remaining two patients were not evaluable for PG-M3 reactivity because of scarcity of cells. No patient with APL showed a normal pattern. The high sensitivity and specificity of immunolabeling using PG-M3 monoclonal antibody show that it is a highly efficient and reliable tool to identify PML/RARα-positive patients with APL and that it should be standardized as a first-line diagnostic procedure. In addition, it is technically simple, fast, and cheap, only requiring small tissue samples and non-sophisticated equipment.

PROGNOSTIC VALUE OF CD34 EXPRESSION IN DE NOVO ACUTE MYELOBLASTIC LEUKAEMIA

Medical Research Council, the National Cancer Association, the Richardson and Kalisky bequests. As Dr Marsh and her colleagues point out, there is growing evidence of intrinsic abnormality in the haematopoietic progenitor cell function at least in some of the patients and this is reflected in our experience that included atypical blast colony formation over pre-formed stroma, decreased adhesiveness and impaired response to recombinant growth factors. It is therefore not surprising that the mature blood elements may be quantitatively abnormal in this disease (De Planque et a/. 1989). Although our own data is still inconclusive, a hypothesis could be proposed that in some cases of aplastic anaemia the haematopoietic defect may be present before the onset of the pancytopenia which is the result of an immunologically mediated surveillance mechanism mounted to suppress the abnormal haematopoiesis. Biological immune response modulation, with antilymphocyte globulin, leads to an improvement in the peripheral blood count values as well as in marrow cellularity but is unlikely to correct the underlying defect.

Recent improvements in outcome for elderly patients with de novo acute myeloblastic leukemia.

A retrospective analysis was performed on 263 consecutive patients aged over 60 with de novo acute myeloid leukemia (AML) diagnosed in a single institution between 1979 and 1998. Eighty-nine patients (33%) received only palliative treatment, while 174 patients (67%) were treated with different intensive chemotherapy regimens. The 5- and 10-year overall survival (OS) for the whole series was 7.7+/-1.2 and 4.3+/-1.6%, respectively. For patients receiving chemotherapy, OS was 10.5+/-2.5 and 7+/-2.6%, while for those patients receiving supportive treatment it was 1.1+/-1.1 and 0%, respectively (P=0.002). Within the group of patients receiving chemotherapy, the complete remission (CR) rate was 46%; treatment failure rate was 54% (36% due to treatment-related mortality and 18% due to resistant disease). Variables influencing CR rate were FAB subtype, CD7 positivity, chemotherapy regimen, creatinine level, hepatomegaly, and period of diagnosis. Median disease-free survival (DFS) duration was 8.4 months with a probability of being disease-free at 10 years of 10+/-5%. There were no significant differences in DFS according to age. According to the period of diagnosis (1979-1986 vs. 1987-1998), improvements in the CR rate (27 vs. 56%, P=0.0002), and OS (10.9 vs. 15.7 months, P=0.0007) were observed. This large single-center study of unselected de novo AML elderly patients substantiates the progressive improvement achieved in the management of elderly patients with AML, probably due to an improvement in supportive care and the administration of conventional induction chemotherapy.

Acute Myeloblasts Leukemia with Minimal Myeloid Differentiation (FAB AML-M0): A Study of Eleven Cases

The main clinical, morphological, cytochemical, immunological features and therapy results of eleven patients diagnosed as acute myeloblastic leukemia M0 (AML-M0) are reported here. There were no clinical characteristics, abnormalities on physical examination or initial laboratory parameters that distinguished these eleven patients. Bone marrow aspirates were hypocellular in four patients. The leukemic cells were undifferentiated by light microscopy and myeloperoxidase (MPO) and/or Sudan Black B (SBB) stains were negative in all cases. Myeloid differentiation antigens were present on the leukemic cells of all eleven patients, whereas B and T cell markers were clearly negative except for CD4 and CD7 antigens. Whatever the treatment employed survival was very short. Eight of the eleven patients were treated and two achieved complete remission (CR) but only one of them is alive in continuous CR. Our results like those previously reported, suggest that AML-M0 patients have a very poor prognosis with standard induction therapies and should perhaps be considered for experimental therapeutic approaches.

3 Immunophenotyping of AML and MDS and detection of residual disease

Immunophenotyping improves both accuracy and reproducibility of the FAB classification and is considered particularly useful for identifying poorly differentiated FAB subtypes of AML, such as AML with minimal differentiation (M0), microgranular promyelocytic leukaemia (M3V), and megakaryoblastic leukaemia (M7). Immunological studies of myeloid leukaemic blasts has become critical also in identifying biphenotypic leukaemias and AML expressing lymphoid-associated markers (Ly+ AML). At present, while the prognostic value of individual antigen expressions is still controversial, due to technical questions, the immunological detection of MRD seems to be important in monitoring AML patients in remission and, perhaps, in detecting leukaemic cell contamination into bone marrow or peripheral blood progenitor cells collected for autologous transplantation. In addition, the relationship established between genetic abnormalities and certain phenotypes within different FAB subtypes suggests that, in the future, immunophenotypical studies could be used for the screening of AML cases carrying specific genetic aberrations. Compared to acute leukaemias, little information is available concerning immunological patterns in MDS, and the role of the immunophenotype in diagnosis, subclassification, and prognosis of MDS is currently not well established.

A retrospective study of pregnancy-associated atypical hemolytic uremic syndrome

Pregnancy-associated atypical hemolytic uremic syndrome (aHUS) refers to the thrombotic microangiopathy resulting from uncontrolled complement activation during pregnancy or the postpartum period. Pregnancy-associated aHUS is a devastating disease for which there is a limited clinical understanding and treatment experience. Here we report a retrospective study to analyze the clinical and prognostic data of 22 cases of pregnancy-associated aHUS from the Spanish aHUS Registry under different treatments. Sixteen patients presented during the first pregnancy and as many as nine patients required hemodialysis at diagnosis. Identification of inherited complement abnormalities explained nine of the 22 cases, with CFH mutations and CFH to CFHR1 gene conversion events being the most prevalent genetic alterations associated with this disorder (66%). In thirteen of the cases, pregnancy complications were sufficient to trigger a thrombotic microangiopathy in the absence of genetic or acquired complement alterations. The postpartum period was the time with highest risk to develop the disease and the group shows an association of cesarean section with pregnancy-associated aHUS. Seventeen patients underwent plasma treatments with a positive renal response in only three cases. In contrast, ten patients received eculizumab with an excellent renal response in all, independent of carrying or not inherited complement abnormalities. Although the cohort is relatively small, the data suggest that pregnancy-associated aHUS is not different from other types of aHUS and suggest the efficacy of eculizumab treatment over plasma therapies. This study may be useful to improve prognosis in this group of aHUS patients.

In vitro all-trans retinoic acid sensitivity of acute myeloid leukemia blasts with NUP98/RARG fusion gene

Dear Editor, Retinoic acid receptors (RARs) are nuclear hormone receptors functioning as ligand-activated transcription factors which interact specifically in order to modulate transcription DNA elements. RARs include alpha (RARA), beta (RARB), and gamma (RARG) receptor types. They function as heterodimers with retinoid X receptors (RXRs). Heterodimers formed by RXRA-RARG are necessary for growth arrest and visceral and primitive endodermal differentiation [1, 2]. The RARA is known to be involved in the t(15;17) chromosome translocation uniquely associated with acute promyelocytic leukemia (APL) which generates the RARA/PML fusion [1]. In a few APL cases, RARA is rearranged with partner genes other than PML as a result of variant translocations [3, 4]. In a previous report, we described a novel fusion protein generated by a translocation t(11;12)(p15;q13) that involved the genes NUP98 and RARG in a patient with acute myeloid leukemia (AML) [5]. The involvement of RARG in a chimeric fusion transcript had not been reported previously in human leukemia. Interestingly, the patient blasts displayed morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. As reported, the patient achieved complete remission with standard AML chemotherapy followed by autologous peripheral blood transplantation [5]. Two years later, the patient relapsed with the same morphologic, immunophenotypic and molecular features displayed at diagnosis. The patient achieved second complete remission after the combination of all-trans retinoic acid (ATRA) with standard salvage treatment for relapse, but he died due to an infection complication after umbilical cord blood transplantation. Since it has been previously reported that fusion proteins generated with RARB and RARG also confer responsiveness to ATRA in vitro and in vivo [1, 2], we tested the in vitro sensitivity to ATRA of the patient leukemic blasts. Leukemic cells obtained from the bone marrow were resuspended in RPMI1640 medium, supplemented with 15% FCS and antibiotics at a concentration of 10cells/ml. ATRA (Sigma, St Louis,MO, USA) was added at a final concentration of 10 m. The differentiation effect was assessed by analyzing morphological characteristics (nucleus/ cytoplasm ratio, nuclear shape, granulations, and basophilia) on May-Grünwald-Giemsa stained cytospin slides and by multiparametric flow cytometry using the monoclonal antibodies CD10, CD11b, CD13, CD15, CD16, CD33, CD45, CD117, HLADR, and 7-AAD. Serial studies were performed after incubation at 3 and 5 days for all cell cultures. The experiment was performed in triplicate, and the ATRAsensitive NB4 cell line was used as a control. The analysis did not show changes either in morphological features or differentiation patterns measured by immunophenotype, whereas ATRA-treated NB4 cells showed both morphological differentiation and an increase in CD11b expression as expected (Fig. 1). Since ATRAwas given simultaneously with chemotherapy to our patient, we were unable to evaluate the effect of this agent in vivo. The in vitro studies, however, showed that AML with a NUP98/RARG rearrangement is resistant to Jose Cervera and Miguel A. Sanz senior authors contributed equally to this work. E. Such (*) : L. Cordón :A. Sempere : E. Villamón :M. Ibañez : I. Luna : I. Gómez-Seguí :M. López-Pavía :C. Alonso : J. Cervera :M. A. Sanz Hematology Department, Hospital Universitari i Politècnic La Fe, Valencia, Spain e-mail: such_esp@gva.es

Acute Promyelocytic Leukaemia. Relationship of Cytochemical Pattern with Clinical, Morphologic and Immunological Characteristics, and Outcome

In a series of 61 acute promyelocytic leukemia (APL) cases multiple clinical and biological characteristics were studied in order to assess their prognostic significance and to evaluate the relationship of cytochemical pattern with clinical, morphologic, and immunological characteristics and outcome. Alpha-naphthyl acetate esterase (ANAE) activi1:y was expressed in 23 out of 41 cases (56%). Sodium fluoride-sensitive naphthol AS-D acetate esterase was evident in only one out of 22 cases (5 %). ANAE reactivity was more frequently found in typical hypergranular APL (P = 0.003) and was associated with a shorter interval symptoms-diagnosis (P = 0.04). The expression of HLA-DK, CD33, CD13 and CD11b did not show any correlation with ANAE reactivity. Complete remission (CR) was achieved in 64 % of treated patients. The actuarial median remission duration was 22 ± 3 months. CR rate was significantly related to the presence of fever, age, absolute reticulocyte count, and albumin level, while the remission duration was associated with absolute reticulocyte, leucocyte and granulocyte counts, and albumin level. In our study ANAE activity did not reflect any important clinical and biological features of APL, apart from its obvious correlation with morphological subtype. In addition ANAE activity did not correlate with early response to therapy nor related with long-term prognosis.

Clonal genetic evolution at relapse of favorable-risk acute myeloid leukemia with NPM1 mutation is associated with phenotypic changes and worse outcomes

Acute myeloid leukemia (AML) is a dynamic disease caused by accumulating, somatically acquired driver mutations generating branching competing clones.[1][1] In favorable-risk AML, high resolution genomic profiling by single nucleotide polymorphism array analysis of paired diagnostic-relapse NPM1 mut