Molecular heterogeneity in chronic lymphocytic leukemia is dependent on BCR signaling: clinical correlation.

Chronic lymphocytic leukemia (CLL), the most frequent form of adult leukemia in Western countries, is characterized by a highly variable clinical course. Expression profiling of a series of 160 CLL patients allowed interrogating the genes presumably playing a role in pathogenesis, relating the expression of functionally relevant signatures with the time to treatment. First, we identified genes relevant to the biology and prognosis of CLL to build a CLL disease-specific oligonucleotide microarray. Second, we hybridized a training series on the CLL-specific chip, generating a biology-based predictive model. Finally, this model was validated in a new CLL series. Clinical variability in CLL is related with the expression of two gene clusters, associated with B-cell receptor (BCR) signaling and mitogen-activated protein kinase (MAPK) activation, including nuclear factor-kappaB1 (NF-kappaB1). The expression of these clusters identifies three risk-score groups with treatment-free survival probabilities at 5 years of 83, 50 and 17%. This molecular predictor can be applied to early clinical stages of CLL. This signature is related to immunoglobulin variable region somatic hypermutation and surrogate markers. There is a molecular heterogeneity in CLL, dependent on the expression of genes defining BCR and MAPK/NF-kappaB clusters, which can be used to predict time to treatment in early clinical stages.

Translocation (10;11)(p13;p15) in an infant acute myeloid leukemia with MLL gene rearrangement.

Molecular rearrangements of the MLL gene at the 11q23 region have been identified in most cases of infant leukemia, regardless of the phenotype. We present a case of acute myeloid leukemia which coexpressed myeloid and lymphoid markers in a 12-month-old girl. Karyotype analysis revealed the presence of a thus far unreported translocation t(10;11)(p13;p15). Although no 11q23 abnormalities were cytogenetically detectable, an MLL gene molecular rearrangement was found.

Apoptosis and mitotic arrest are two independent effects of the protein phosphatases inhibitor okadaic acid in K562 leukemia cells.

Treatment of human myeloid leukemia K562 cells with the serine/threonine protein phosphatases inhibitor okadaic acid induced mitotic arrest followed by apoptosis in a synchronized manner. The effect was observed at drug concentrations that inhibited the protein phosphatase type 2A but not type 1. We investigated whether apoptosis was a consequence of the preceding mitosis arrest or was induced independently by okadaic acid. We found that (1) apoptosis, but not mitotic arrest, was inhibited in cells with constitutive expression of Bcl-2; (2) pretreatment of cells with the DNA synthesis inhibitor hydroxyurea blocked the mitotic arrest but not the apoptosis mediated by okadaic acid; (3) down-regulation of c-myc gene was associated with apoptosis, but not with mitotic arrest; and (4) inhibition of protein synthesis abrogated mitotic arrest, but not apoptosis. The results suggest that inhibition of protein phosphatase 2A by okadaic acid provokes mitotic arrest and apoptosis of leukemia cells by independent mechanisms.

Spi-1/PU.1 proto-oncogene induces opposite effects on monocytic and erythroid differentiation of K562 cells.

Spi-1/PU.1 is a hematopoietic transcription factor of the Ets family. To analyze the effects of ectopic expression of spi-1 on the proliferation/differentiation of human myeloid leukemia cells, K562 cells were stably transfected with a spi-1 expression vector. The transfected cell lines expressed elevated levels of spi-1 mRNA and protein and high Spi-1-DNA binding activity. The spi-1 transfected cells showed reduced growth rates and reduced clonogenic cell growth. When the erythroid and monocytic differentiation markers were analyzed, spi-1 overexpression resulted in opposite effects: erythroid differentiation was significantly inhibited in spi-1 transfectants, while spi-1 overexpression increased the monocytic differentiation of cells. These results indicate a differential role of Spi-1 on the differentiation of human myeloid leukemia cells.

[Prognostic factors in low-grade lymphoma]. / Investigación de factores pronósticos en linfomas de bajo grado.

PURPOSE: Prognostic factors in low grade non-Hodgkin's lymphoma (LGL) are not well established. The aim of this study is to investigate prognostic factors on LGL treated in our institution during the last decade. PATIENTS AND METHODS: The study was carried out on 70 cases of newly diagnosed LGL, most treated with CVP or clorambucil and prednisone. The median follow-up was 37 months (1-132). Variables reported as prognostic factors in previous series were subjected to bivariate and multivariate analysis. RESULTS: Relevant clinical features were: Ann Arbor III-IV stage 74%, ECOG > or = 2-17%, bone marrow involvement 60% and large tumor burden according to MD Anderson criteria 21%. Complete response (CR) was achieved in 50% and partial response in 29%. In bivariate analysis factors related with poor CR were B symptoms, large tumor burden, high LDH and more than one extranodal site involvement. Logistic regression showed that large tumor burden (p = 0.02; OR = 0.07) and B symptoms (p = 0.07; OR = 0.14) were the best prognostic factors of poor CR. Five year global survival (GS) was 55%, with a median of 76 months. In univariate analysis factors related with GS were ECOG > or = 2, B symptoms, bulky, large tumor burden, retroperitoneo involvement and absence of CR. In multivariate analysis the only factor related with poor GS was large tumor burden (p < 0.00001; RR = 5.93). When therapeutic response was included in the model, absence of CR (p = 0.008; RR = 3.40) and large tumour burden (p = 0.005; RR = 3.86) were the factors selected. CONCLUSIONS: In LGL tumor burden was the most important prognostic variable. Tumor response showed less importance than in high grade lymphomas.

Down-regulation of c-Myc and Max genes is associated to inhibition of protein phosphatase 2A in K562 human leukemia cells.

Treatment of the human myeloid leukemia K562 cells with the protein phosphatase inhibitors okadaic acid or calyculin A resulted in down-regulation of both c-myc and max genes at the mRNA and protein levels. The extent of the down-regulation was similar for both genes and was dependent on the dose and on the treatment time. Interestingly, c-myc and max down-regulation was concomitant with apoptosis induced by okadaic acid and calyculin A in K562 cells. The expression of c-myc and max returned to control levels after the removal of okadaic acid from the media, although apoptosis was irreversible. These effects were observed at okadaic acid concentrations (15 nM) that inhibited the activity of protein phosphatase type 2A but not of phosphatase type 1. We conclude that the inhibition of protein phosphatase 2A is associated to decreased levels of c-Myc/Max heterodimers in K562 cells.

Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) administration after autologous bone marrow transplantation for acute myeloblastic leukemia enhances activated killer cell function and may diminish leukemic relapse.

Leukemic relapse is the major complication following autologous bone marrow transplantation (BMT) in acute myeloblastic leukemia (AML). Previously, we have shown that recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) infusion after autologous BMT has the ability to augment endogenous activated killer (AK) cell function which may play a role in the eradication of minimal residual disease. However, the clinical application of rhGM-CSF in patients with AML has been limited by its potential stimulatory effect on the malignant clone. Here we report the effect of rhGM-CSF 5 micrograms/kg/day infusion on AK cell function in 20 patients with AML undergoing autologous BMT. AK cell function was investigated before autologous BMT, during rhGM-CSF therapy and after withdrawal. In addition, its influence on the actuarial risk of relapse is analyzed and compared with a historical control group of 20 patients transplanted immediately before initiation of this study. rhGM-CSF significantly enhanced AK cell function. During rhGM-CSF treatment, median AK cell function rose from 1.8% before autologous BMT (range 0-8%) to 35% (range 3-80%) and remained increased after cessation of rhGM-CSF (median 20%; range 0-36%; P < 0.001). After a median follow-up of 24 months, the actuarial risk of relapse is 37.4% in rhGM-CSF-treated patients compared with 49.5% in controls (P = 0.05). Interestingly, none of the 7 patients with an AK cell activity > or = 20% in the first 2-5 weeks after autologous BMT have relapsed compared with 6 of 9 patients with an AK cell activity < 20% (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

[Research on prognostic factors in large-cell lymphomas]. / Investigación de factores pronósticos en linfomas de células grandes.

BACKGROUND: To carry out a study on the prognostic factors in large cell lymphomas (LCL) treated during the last decade and validate the international prognostic index (IPI). METHODS: One hundred twenty-four cases of newly diagnosed LCL, treated from 1978 to 1990, with a mean follow up of 27 months (1-142) were included in the study. The chemotherapy used was: CHOP (65%), ProMACE-CytaBOM (17%) and others (C-MOPP, MACOP-B). RESULTS: Complete remission (CR) was achieved in 71% of the cases and partial in 11%. Logistic analysis allowed the identification of three adverse factors to CR: Ann Arbor stage III, IV (p = 0.004; odds ratio, OR = 0.19), elevated tumoral load (p = 0.006; OR = 0.22) and age > or = 60 years (p = 0.02; OR = 0.31). Recurrence free survival (RFS) at 3 years was 67% (CI 95%; 55-79) with the median not having been achieved. Cox analysis allowed the identification to the ECOG > or = 2 scale as the only independent adverse factor (p = 0.0006; RR = 4.85) while Ann Arbor staging demonstrated marginal influence (p = 0.08). Global survival (GS) at 5 years was 45% (CI 95%; 35-55) with a median of 38 months. Multivariant analysis of independent adverse factors of GS were ECOG scale > or = 2 (p < 0.00001; RR = 6.07), Ann Arbor stage (p = 0.004; RR = 2.64) and hypoalbuminemia (p = 0.01; RR = 2.28). On inclusion of therapeutic response (TR) in the analysis, the factors chosen were absence of CR (p < 0.00001; RR = 9.58) and ECOG > or = 2 (p = 0.0004; RR = 4.24). CONCLUSIONS: Three variables evaluated at diagnosis, general state (ECOG), Ann Arbor stage and albumin, determined the prognosis in this series of large cell lymphoma. A prognostic model was designed from the same with three risk groups. The application of the international prognostic index to this series separated the patients into 4 groups of differentiated prognosis.

[Chronic eosinophilic pneumonia in a patient treated with allogenic bone marrow transplantation]. / Neumonía eosinófila crónica en un paciente tratado con trasplante de médula ósea alogénico.

A 39 year old patient diagnosed of severe aplastic anemia and treated with allogenic bone marrow transplantation and who presented chronic eosinophilic pneumonia eight months after the transplant is presented. The patient had no previous history of asthma or atopy. Conditioning was performed with cyclophosphamide and total body irradiation. Prophylaxis of the graft versus host disease was carried out with cyclosporin and short course of methotrexate. At day 30 mild graft versus host disease appeared which spontaneously resolved. A progressive increase in the number of eosinophils was observed from day +40 reaching 1.05 x 10(9)/l at day +180 coinciding with suspension of the cyclosporine. The patient remained asymptomatic with no evidence of chronic graft versus host disease. At 8 months following allogenic transplantation the patient developed three episodes of fever, cough, moderate dyspnea and pulmonary infiltrates. Respiratory tests showed a restrictive pattern. Bronchoalveolar lavage contained 20% of eosinophils. Upon lung biopsy alveolar infiltration by eosinophils, lymphocytes and mononuclear cells was observed. Diagnosis of chronic eosinophilic pneumonia was made with initiation of steroid treatment. A drastic response was observed. The patient remained asymptomatic without recurrence and without evidence of chronic graft versus host disease. This picture may have been caused by the donor eosinophils given that retrospective evaluation demonstrated a persistent moderate eosinophilia in the donor.

[Initial central nervous system involvement in a case of non-secretory multiple myeloma]. / Afectación inicial del sistema nervioso central en un caso de mieloma múltiple no secretor.

A 56-year old woman with non-secretory multiple myeloma presented with involvement of the base of brain and meningeal infiltration. Initial involvement of central nervous system is very rare in multiple myeloma, no such pathology being reported in non-secretory myeloma thus far.

Induction of apolipoprotein E expression during erythroid differentiation of human K562 leukemia cells.

Apolipoprotein E (ApoE) is the only apolipoprotein that is expressed in extrahepatic tissues. ApoE expression was studied in leukemia K562 cells differentiated towards erythroid or myelomonocytic lineages. When K562 cells were differentiated into the erythroid lineage by addition either of 1-beta-D-arabinofuranosylcytosine or hydroxyurea, an increase in ApoE mRNA and protein was detected. A weaker ApoE induction was also observed during phorbol ester-induced myelomonocytic differentiation. Previous work has associated ApoE expression to monocytic differentiation. The findings reported here indicate that ApoE overexpression is not associated with a specific lineage in myeloid differentiation and that may play a role in erythroid differentiation.

Recombinant human GM-CSF enhances T cell-mediated cytotoxic function after ABMT for hematological malignancies.

The interactions of GM-CSF with cells of lymphoid lineage are not well understood and their clinical use has been focused on the acceleration of hematopoietic recovery. However, several reports have shown that human GM-CSF can affect certain T lymphocyte in vitro cytotoxic functions. To assess whether recombinant human GM-CSF (rhGM-CSF) has a more broadly based activity in the immune system, we studied its in vivo effects on endogenously-generated killer function in patients undergoing ABMT for hematologic malignancies. Eleven patients received rhGM-CSF after ABMT: eight received rhGM-CSF as a 2-h infusion daily from days +3 to +17 and three received rhGM-CSF until reaching > 500 x 10(6)/l granulocytes. Eight patients not enrolled in the rhGM-CSF therapy protocol served as controls. Natural killer (NK) cell activity and activated killer (AK) cell activity were studied before conditioning, during rhGM-CSF therapy and after withdrawal of GM-CSF. rhGM-CSF therapy does not affect NK activity. Circulating lymphocytes with the ability to kill AK-sensitive targets appear spontaneously in control ABMT patients. AK activity was 1.6 +/- 0.8% before ABMT increasing to 9 +/- 2.5% and 14 +/- 2.1% at 2 and 3 weeks after ABMT, respectively (p = 0.002). In rhGM-CSF-treated patients this phenomenon also occurs. AK activity increased from 2.4 +/- 1.5% before ABMT to 33.6 +/- 8.1% during rhGM-CSF administration (p = 0.001) and 17.5 +/- 3.4% after withdrawal (p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of ras protooncogenes during in vitro differentiation of human erythroleukemia cells.

We have compared the expression of the ras protooncogene family (H-, K-, and N-ras) in leukemia cell differentiation utilizing as a model K562 and HEL erythroleukemia cells treated either with 1-beta-arabinofuranosylcytosine or 12-O-tetradecanoylphorbol-13-acetate (TPA). 1-beta-D-Arabinofuranosylcytosine induced terminal erythroid differentiation of K562 cells, while TPA induced myeloid differentiation of K562 and HEL cells, resulting in myelomonocytic-like cells expressing macrophagic and megakaryocytic markers. H-ras mRNA levels showed a dramatic decrease in K562 cells subjected to erythroid and myelomonocytic differentiation. The same result was found at the protein level for p21H-ras. Expression of K-ras and N-ras in K562 cells also decreased with differentiation, although significant mRNA levels remained despite cessation of cell proliferation. The decrease in K-ras expression was greater for TPA-treated cells than for 1-beta-arabinofuranosylcytosine-treated cells. TPA-induced myelomonocytic differentiation in HEL cells also resulted in a dramatic down-regulation of H-ras mRNA levels. Thus, by using a leukemia cell line able to differentiate along two different lineages, our results reveal a lineage-specific modulation of ras gene family expression.

Acquired von Willebrand disease in multiple myeloma secondary to absorption of von Willebrand factor by plasma cells.

A case of acquired von Willebrand disease (AvWD) associated with an IgA lambda multiple myeloma is reported. No form of inhibitor could be detected. SDS-agarose gel electrophoresis patterns of von Willebrand factor (vWF) both in plasma and platelet lysates were normal but a decrease in all-sized multimers with a type IA pattern was seen. After 1-deamino-8-D arginine vasopressin (DDAVP) infusion, vWF multimers larger than those seen in the resting state appeared in patient plasma, which were progressively cleared. Indirect immunofluorescence studies with a monoclonal antibody to vWF showed that vWF was selectively absorbed into myelomatous cells. This is the first case of AvWD associated with multiple myeloma resulting from the selective absorption of vWF into abnormal plasma cells. This feature established a new pathophysiological mechanism of AvWD in multiple myeloma and probably in other lymphoproliferative diseases.

[Prevention of acute graft versus host disease using 3 prophylactic schemes]. / Prevención de la enfermedad injerto contra huésped aguda con tres esquemas profilácticos diferentes.

During 7 years 81 patients received an allogeneic bone marrow transplant (BMT) for several diseases. The prevention of graft-versus-host disease (GVHD) was undertaken with methotrexate (MTX), MTX plus antilymphocytic gammaglobulin plus prednisone (MTX + ALT + P), and elimination of the T-lymphocytes of the donor's bone marrow with the monoclonal antibody CAMPATH-1. The actuarial survival of the patients who did not develop GVHD was significantly better than that of those who developed grade II-IV GVHD: 56% [95% confidence interval (CI) 39-71%] versus 10% (95% CI 3-25%) (p less than 0.0001). However, actuarial survival was similar in each of the three groups: MTX 35%, MTX + ALT + P 38%, and CAMPATH-1 43%. The incidence of GVH disease, when the sex of the donor and the receptor were different, was significantly higher than in cases where the donor and the receptor had the same sex: 45% (95% CI 31-58%) vs 15% (95% CI 8-28%) (p less than 0.005). By contrast, significant differences were not found between the three groups in the incidence of GVHD: MTX 36%, MTX + ALT + P 34%, and CAMPATH-1 20%. In patients with leukemia, a higher number of relapses occurred in the MTX group, because a higher number of patients in second or third complete remission (CR) or with active disease underwent transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prognostic value of the changes in the DNA of blast cells from patients with acute lymphoblastic leukemia]. / Importancia pronóstica de las alteraciones en el ADN de las células blásticas en pacientes con leucemia aguda linfoblástica.

The cytogenetic characteristics of 37 patients diagnosed of acute lymphoblastic leukaemia are presented. The studies were performed by cytofluorometry (CFM) after DNA staining with propidium iodide (34 cases) and/or chromosome identification with trypsin G bands (13 patients). Hyperdiploid DNA index was present in 15% of the patients, whereas none of them had hypodiploid DNA index. Abnormal karyotype was found in 69% of the evaluated cases. Good correlation was observed between the ploidy attained by CFM and by karyotyping cells. The highest percentage of aneuploidy corresponded to the L2 morphological subtype (55%), followed by L1 (36%). Structural alterations were the commonest in L2 variant, while numerical ones were commonest in the L1 variant. The 4 L1 patients with aneuploidy had the common immunophenotype, whereas the 6 aneuploidic patients of the L2 variant had common, early pre-B and undifferentiated immunophenotype. The actuarial survival of patients with diploid DNA index was 48.5% (IC 95%, 25-73%), whereas pseudodiploid patients have relapsed and died before 16 months from diagnosis (p less than 0.005). None of the patients with hyperdiploid DNA index and lacking structural alterations has relapsed. Patients with structural abnormalities have the poorest prognosis, while patients with hyperdiploid DNA index showed several favourable risk factors and are in the first complete remission.

Therapy of advanced myelodysplastic syndrome with aggressive chemotherapy.

Nine patients with advanced stages of myelodysplastic syndrome (MDS) received aggressive chemotherapy with high-dose cytarabine or with a standard acute myeloid leukemic regimen. Six of them were in frank acute myeloid leukemic phase. The mean age was 57 years (range 32-71). Seven patients obtained remission, 6 complete remission (CR) and 1 partial remission. The induction remission rate was 77.7%. There were 2 deaths in the aplasia period because of infectious complications. The mean duration aplasia was 36 days (range 21-69). In spite of this all responders received further consolidation chemotherapy. The mean duration of CR was 10 months. We concluded that patients with MDS with excess of blasts and blastic transformation may be treated with aggressive chemotherapy with low toxicity and high remission rate, similarly to de novo acute myeloid leukemia.