Vincristine as treatment for recurrent episodes of thrombotic thrombocytopenic purpura.

The clinical course of thrombotic thrombocytopenic purpura has dramatically improved after the introduction of plasma-based therapy, including plasma exchange and plasma infusion. However, a considerable number of patients still experience relapse after initial successful treatment. In this study, vincristine (VCR) was given as salvage treatment in 12 episodes of recurrent thrombotic thrombocytopenic purpura in seven patients, concomitantly with short-term plasma infusion. Complete remission (CR) was defined by normal platelet, hemoglobin, and serum lactic dehydrogenase (LDH) values as well as by absence of clinical signs. Of 12 patients, 12 achieved CR following therapy with VCR. The median duration of CR was 15 months (range: 2-16). Toxicity was mild consisting of paresthesias in three cases, leukopenia in one case, and autonomic neuropathy leading to paralytic ileus in one case. We conclude that VCR is remarkably effective for recurrent thrombotic thrombocytopenic purpura with acceptable toxicity.

Differentiation of myeloid cell lines correlates with a selective expression of RIZ protein.

BACKGROUND: The retinoblastoma-interacting zinc-finger gene RIZ is expressed in two forms (RIZ1 and RIZ2) that differ for the presence near the N-terminus of RIZ1 of a conserved domain, defined PR (PRDI-BF1-RIZ homology), homologous to a similar domain present in other proteins recognized as tumor suppressor gene products. The RIZ1 form is usually absent or expressed at low levels in tumor cells, whereas RIZ2 is frequently expressed. We investigated a possible involvement of RIZ1 in differentiation control using a myeloid cell maturation model that is easily modulated by retinoids and other agents. MATERIALS AND METHODS: HL60 or NB4 cell lines or patients' leukemic promyelocytes were treated with all- trans -retinoic acid or other agents to induce differentiation. RIZ gene expression was determined with reverse transcriptase polymerase chain reaction (RT-PCR) and RNase protection assay. Immunocytochemistry was performed to assess variation of the intracellular distribution of RIZ protein on all- trans-retinoic acid treatment. Forced expression of RIZ1 protein was obtained with a recombinant adenovirus containing RIZ1 cDNA. RESULTS: Treatment with retinoic acid induced a selective expression of RIZ1 in HL60 cell line. Retinoic acid effect was maximal at 7 days and correlated to the granulocytic differentiation of cells. A similar effect was obtained in retinoic acid-sensitive NB4 cell line or in patients' leukemic promyelocytes, but not in the retinoic acid-resistant cell line NB4.007/6 or in the U937 cell line. Selective expression of RIZ1 was also induced by 12-O-tetradecanoyl-phorbol-13-acetate in the U937 and HL60 cell lines and by 1,25-dihydroxyvitamin D(3) only in HL60 cells. In HL60 cells, RIZ1 was also induced by activation of a retinoid alpha receptor-independent maturation pathway based on retinoid X receptor agonist and protein kinase A synergism. In addition, retinoic acid produced a redistribution of the antigen within the nucleus in these cells. Forced expression of RIZ1 protein induced growth arrest and death of HL60 cells. CONCLUSIONS: The correlation between the selective expression of RIZ1 induced by retinoic acid, 12-O-tetradecanoyl-phorbol-13-acetate, or 1,25-dihydroxyvitamin D(3) and differentiation suggested that RIZ protein was involved in myeloid cell differentiation induced by these agents.

Aggressive salvage treatment is not appropriate for the majority of elderly patients with acute myeloid leukemia relapsed from first complete remission.

BACKGROUND AND OBJECTIVES: The prognosis of acute myeloid leukemia (AML) in the elderly is still poor because of different reasons, including a high incidence of relapse. The aim of this study was to investigate whether aggressive salvage chemotherapy (CHT) results in an actual survival advantage in elderly patients with relapsed AML, as well as to compare hospitalization and load of supportive treatment between patients receiving aggressive management or only palliation. DESIGN AND METHODS: One hundred and fifty consecutive patients with relapsed AML (median age 66 years) were analyzed. At relapse, 99 (66%) were treated with CHT, and 51 had palliative management. RESULTS: Second complete remission (CR2) was achieved in 36/99 patients (36%) receiving CHT, while no CR was observed in the other group (p<0.001). Induction death rate was 22%, while 41% were resistant to CHT. The median survival from relapse was 4 months for the whole patient population; according to management, it was 5 months and 3 months for CHT and palliation, respectively (p=0.01). As to patients given CHT, a CR1 duration of more than 12 months was the only parameter significantly related to a better clinical outcome (survival from relapse: 8 vs. 4 months, p=0.002; CR2 duration: 11 vs. 5 months, p=0.001, respectively). Finally, patients managed with palliation required less hospitalization and less supportive therapy as compared to the CHT group. INTERPRETATION AND CONCLUSIONS: Aggressive chemotherapy results in an actual survival advantage only for a minority of elderly patients with relapsed AML, i.e. those with CR1 lasting for more than 12 months.

High-dose idarubicin and busulphan as conditioning for autologous stem cell transplantation in acute myeloid leukemia: a feasibility study.

INTRODUCTION: Between 30 and 50% of patients with acute myeloid leukemia still relapse after autologous stem cell transplantation. We investigated the feasibility of a new conditioning regimen consisting of high dose IDA plus oral busulphan in patients undergoing autologous transplantation. MATERIALS AND METHODS: Patients (n = 13) were given three days continuous infusion IDA, followed by four days conventional dose oral busulphan as conditioning. Peripheral blood stem cells were used in all cases. Eleven patients were in CR1. Patients with t(8;21) and inv(16) as well as those with acute promyelocytic leukemia were excluded from the study. The median of CD34+ cells infused was 6.2 x 10(6)/l (2.6-16.1). RESULTS: No case of transplant-related mortality occurred. The median number of days to neutrophil (>0.5 x 10(9)/l) and platelet (>20 x 10(9)/l) recovery was 10 (7-21) and 20 (9-26), respectively. Patients needed a median of 3 platelet units (1-6) and 3 blood units (0-12), respectively. Left ventricular ejection fraction remained unmodified after ASCT. Twelve out of 13 patients (92%) had variable grade of mucositis (two grade 2, five grade 3 and five grade 4). Total parenteral nutrition was needed in nine patients (69%). After a median follow-up of 14 months from ASCT, 11 patients out of 13 (85%) are alive in continuous CR; the other two patients experienced relapse at 12 and 14 months. CONCLUSION: Our data demonstrate the feasibility of a conditioning regimen based on high-dose IDA plus Busulphan in AML. Results concerning antileukemic efficacy are promising, but need confirmation on larger series with longer follow-up.

Estradiol induces functional inactivation of p53 by intracellular redistribution.

Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm.

CD56 expression is an indicator of poor clinical outcome in patients with acute promyelocytic leukemia treated with simultaneous all-trans-retinoic acid and chemotherapy.

PURPOSE: Preliminary reports suggest that leukemic cell expression of CD56, a neural cell adhesion molecule, is associated with adverse clinical outcome in either acute myeloid leukemia with t(8;21) or acute promyelocytic leukemia (APL). We investigated the prognostic relevance of CD56 in a series of patients with APL who were treated homogeneously with all-trans-retinoic acid (ATRA) and chemotherapy. PATIENTS AND METHODS: Clinicobiologic presenting features and therapeutic results were analyzed in a series of 100 patients with genetically proven APL who were treated, according to the example of the Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto multicenter trial, with ATRA plus idarubicin (AIDA) and for whom data on CD56 expression were available at diagnosis. RESULTS: Fifteen patients (15%) showed expression of CD56 in greater than or equal to 20% blasts at diagnosis and were considered as CD56(+). No differences were found regarding age, sex, WBC and platelet counts, incidence of coagulopathy, hemoglobin and fibrinogen levels, promyelocytic leukemia/retinoic acid receptor (PML/RAR) alpha fusion type, or complete remission (CR) rate in the comparison of the CD56(+) and CD56(-) populations. Conversely, compared with patients who were CD56(-), patients with CD56(+) APL had shorter CR duration (P =.04) and overall survival (P =.002). In the multivariate analysis, CD56 positivity and initial WBC count greater than 10 x 10(9) cells/L retained statistical significance in overall survival (P =.04 and P =.02, respectively). CONCLUSION: The expression of CD56 is significantly associated with inferior CR duration and survival in patients with APL who were treated with modern frontline treatment that included ATRA and simultaneous chemotherapy. Combined with other well-established prognostic factors such as WBC count, CD56 expression at diagnosis might be used to build prognostic scores for risk-adapted therapy in APL.

Interstitial 9q deletion is associated with CD7+ acute leukemia of myeloid and T lymphoid lineage.

We report the clinical, hematological and immunophenotypic characteristics from four cases of acute leukemia with interstitial deletion of chromosome 9, ie del(9)(q12-q22), as a single chromosomal abnormality. Three patients had acute myeloblastic leukemia (AML) and one T origin acute lymphoblastic leukemia (ALL). According to FAB classification, blasts were classified as M1 (two patients), M2 (one patient), and L2 (one patient). In two out of three AML cases a myelodysplastic syndrome, one AREB-t and one AREB diagnosed 6 and 11 months before respectively, preceded the onset of AML. Morphological examination showed dysgranulopoiesis, dyserythropoiesis and cytoplasmic vacuoles in two AML patients, while a strong positivity to myeloperoxidases was observed in all AML cases. As concerns immunophenotypic findings, blast cells from two of three AML patients expressed CD7 and CD34, while those from the T-ALL case displayed CD33 and CD34 along with CD7. These observations suggest that del (9q) is associated with CD7+ acute leukemia of myeloid or lymphoid lineage.

Differential regulation of GPI-linked molecules on leukaemic promyelocytes treated in vitro with all-trans retinoic acid.

It has been demonstrated that certain cell-surface proteins are anchored to the cell membrane by a unique structure known as the glycosylphosphatidylinositol (GPI) anchor whose absence has been reported on blood cells from patients with paroxysmal nocturnal haemoglobinuria. We have investigated the expression of CD16/Fc(tau)R-III and CD66b GPI linked molecules at the surface of blast cells from five acute promyelocytic leukaemia (APL) patients before and after in vitro stimulation with all-trans retinoic acid (ATRA). We observed that whereas CD66b antigen exhibited a strong ATRA-driven up-regulation in all cases studied, CD16 expression was unaffected by the treatment with the drug.

All-trans retinoic acid (ATRA) and the regulation of adhesion molecules in acute myeloid leukemia.

A review of recent information on the expression and the ATRA-driven modulation of cell surface adhesion molecules of acute myelogenous leukemia blast cells is presented. Cytofluorometric studies on fresh blast cells have demonstrated that CD11a, CD11b CD11c, CD15, CD45RO and CD54 expression is significantly lower in acute promyelocytic leukemia (APL) than is acute myeloid leukemia of other subtypes (AML). In vitro treatment with ATRA dramatically modifies the adhesion phenotype of APL blast cells, promoting a consistently striking up-regulation of CD11b, CD11c, CD15, CD65, CD54, and CD38. Which is in general, poorly demonstrable in AML. The behaviour of CD15s is variable and fully independent from CD15 and CD65 in induction experiments, suggesting a differential enzyme regulation within the selectin ligand system. ATRA is capable, in both APL and AML, of producing a switch from the high- (RA) to the low- (RO) molecular weight isoform of CD54, Moreover, treatment with this retinoid exerts a negative regulation of the membrane expression of CD49e, CD58 and CD11a in APL as well as in AML. Of particular interest is the fact that the negative effect on CD1 1a expression generates an asynchronous phenotype in APL (CD11a-, CD11b+, CD15+), undetectable on normal maturing myeloid cells. In the last part of this review the possible implications of adhesion molecule modulation in the pathogenesis of ATRA syndrome are discussed.

Stem cell factor receptor (c-kit, CD117) is expressed on blast cells from most immature types of acute myeloid mallignancies but is also a characteristic of a subset of acute promyelocytic leukaemia.

Investigating 208 patients with acute haematological malignancies, we found that stem cell factor receptor (SCFR) was expressed on high numbers of blast cells from the vast majority of patients (93%) with refractory anaemia with excess of blasts in transformation. SCFR was also detected in 62% of AMLs, in which it was directly associated to the expression of CD7, interleukin 6 receptor and CD34, and inversely to that of CD11b and CD14. SCFR-positive cases were preferentially represented in AML-M1 (70%) and in AML-M2 (83%) subsets, whereas only 45% of the remaining samples (M3-M4-M5) exhibited SCFR positively. Interestingly, 50% of cases with acute promyelocytic leukaemia expressed SCFR and this molecule was heterogenously regulated by in vitro treatment with all-trans retinoic acid.

Expression of the leucocyte common antigen (LCA, CD45) isoforms RA and RO in acute haematological malignancies: possible relevance in the definition of new overlap points between normal and leukaemic haemopoiesis.

The membrane expression of CD45RA and CD45RO on fresh leukaemic cells taken from 529 cases of acute haemopoietic malignancies, including 117 B-origin acute lymphoblastic leukaemia (B-origin ALL), 37 T-origin acute lymphoblastic leukaemia (T-origin ALL0, 297 de novo acute myeloid leukaemia (AML), 42 refractory anaemia with excess of blasts in transformation (RAEB-T) and 36 myeloid blastic phase of chronic myelogenous leukaemia (CML-BP-my), was analysed. B-origin ALLs were characterized by the lack of the RO isoform along with the consistent presence of RA. Conversely, a differential expression of the two isoforms was detected in different subsets of T-origin ALL, in that T-stem cell leukaemias (T-SCL: CD7+, CD4-, CD8-, CD1-) preferentially expressed CD45RA whereas conventional T-acute lymphoblastic leukaemias (T-ALL: CD7+, CD4+ and/or CD8+ and/or CD1+) were consistently marked by CD45RO. Within myeloid malignancies, most of AMLs displayed CD45RA, while a substantial group of CML-BP-my preferentially exhibited CD45RO. As a general rule, a reciprocal exclusion of the two isoforms was observed in AML as well as in ALL. Nevertheless, a frequent coexpression of CD45RA and CD45RO was observed in CD14+ AML. In vitro treatment with all-trans retinoic acid (ATRA) was able to promote a switch from CD45RA to CD45RO expression in 27 de novo AML, independently from morphological subtyping. To our knowledge, this is the first report on CD45 isoform expression in a large series of patients with acute leukaemia. The knowledge of the differential expression of CD45RA and CD45RO can ameliorate our classificative approach to haematological malignancies, as well as disclose new multiple overlap points between normal and leukaemic cell differentiation.

Expression and ATRA-driven modulation of adhesion molecules in acute promyelocytic leukemia.

On fresh leukemic cells taken from 30 patients with acute promyelocytic leukemia (APL) the membrane expression of a series of adhesion molecules including beta 2 integrins (CD11a/LFA-1, CD11b/Mac-1), selectin ligands (CD15/Le(x), CD15s/Le(x)) and tyrosine-phosphatase isoforms (CD45RA, CD45R0) was analyzed. The expression of these molecules was also studied in nine of these patients following the APL cells' culture with and without all-trans retinoic acid (ATRA). The fresh APL promyelocytes expressed CD45RA and CD15s on their surface, while CD11a, CD11b, CD15, and CD45R0 were constantly absent. In vitro treatment with ATRA consistently increased the expression of CD15, CD11b, and CD45R0 on leukemic promyelocytes; these changes were paralleled by a decrease of CD45RA display. The expression of sialylated antigen CD15s was fully independent from CD15 suggesting a differential enzymatic regulation within this selectin ligand system. ATRA was, however, incapable of promoting the up-regulation of CD11a in APL. As a result, asynchronous phenotype (CD11a-, CD11b+, CD15+, CD15s+/-, CD45RA-, CD45R0+) was generated that is normally undetectable on maturing myeloid cells. In order to provide a further control a case of acute agranulocytosis was also investigated, in which > 75% bone marrow cells were arrested at the promyelocyte stage; these bone marrow cells showed a surface phenotype identical to non-leukemic promyelocytes (CD11a+, CD11b+, CD15+, CD45R0+, CD45RA-) with a spontaneous ability to differentiate in vivo towards the more mature stages of myeloid differentiation. We therefore suggest that in fresh and ATRA-induced APL cells distinct, regular phenotypic changes are identifiable that are probably associated with t(15;17) and not seen in normal and activated bone marrow.

All-trans retinoic acid promotes a differential regulation of adhesion molecules on acute myeloid leukaemia blast cells.

In the present study we investigated the membrane expression of selectin ligands (CD15/Le(x), CDw65/VIM2, CD15s/sLe(x), beta 2 integrins (CD11a/LFA-1, CD11b/Mac-1) and CD45 phosphatase isoforms (CD45RA, CD45O) on leukaemic cells from 28 patients with acute myeloid malignancies cultured with and without all-trans retinoic acid (ATRA). Within each adhesion system. ATRA was able to differentially regulate distinct molecules. Furthermore, it was able to exert effects specific for acute promyelocytic leukaemia (APL) blast cells, as well as to induce a series of non-cytotype-restricted phenotypic changes. An impressive feature of ATRA induction was a simultaneous increase in the expression of CD15, CDw65 and CD11b on leukaemic promyelocytes. The sialylated antigen CD15s, however, showed results independent from the other two carbohydrates (CD15 and CDw65), suggesting a differential enzymatic regulation within the selectin ligands system. In spite of the well-recognized expression of CD11a throughout all stages of normal myeloid differentiation, APL blast cells were found to virtually lack LFA-1 expression. Moreover, ATRA was unable to promote an up-regulation of this antigen in APL, while inducing a frequent down-modulation in non-APL cases constitutively expressing this antigen. In APL cases ATRA generated an asynchronous phenotype (CD15+, CDw65+, CD11b+, CD11a-), undetectable on normally maturing myeloid cells, but consistent with the concept that incomplete differentiation, in terms of surface molecule expression, can be sufficient to obtain therapeutic results.

Expression and ATRA-driven modulation of adhesion molecules in acute promyelocytic leukemia.

On fresh leukemic cells taken from 30 patients with acute promyelocytic leukemia (APL) the membrane expression of a series of adhesion molecules including beta 2 integrins (CD11a/LFA-1, CD11b/Mac-1), selectin ligands (CD15/Le(x), CD15s/sLex) and tyrosine-phosphatase isoforms (CD45RA, CD45R0) was analyzed. The expression of these molecules was also studied in nine of these patients following the APL cells' culture with and without all-trans retinoic acid (ATRA). The fresh APL promyelocytes expressed CD45RA and CD15s on their surface, while CD11a, CD11b, CD15, and CD45R0 were constantly absent. In vitro treatment with ATRA consistently increased the expression of CD15, CD11b, and CD45R0 on leukemic promyelocytes; these changes were paralleled by a decrease of CD45RA display. The expression of sialylated antigen CD15s was fully independent from CD15 suggesting a differential enzymatic regulation within this selectin ligand system. ATRA was, however, incapable of promoting the up-regulation of CD11a in APL. As a result, asynchronous phenotype (CD11a-, CD11b+, CD15+, CD15s+/-, CD45RA-, CD45R0+) was generated that is normally undetectable on maturing myeloid cells. In order to provide a further control a case of acute agranulocytosis was also investigated, in which > 75% bone marrow cells were arrested at the promyelocyte stage; these bone marrow cells showed a surface phenotype identical to non-leukemic promyelocytes (CD11a+, CD11b+, CD15+, CD45R0+, CD45RA-) with a spontaneous ability to differentiate in vivo towards the more mature stages of myeloid differentiation. We therefore suggest that in fresh and ATRA-induced APL cells distinct, regular phenotypic changes are identifiable that are probably associated with t(15;17) and not seen in normal and activated bone marrow.

Myeloid antigen expression in adult acute lymphoblastic leukemia: clinicohematological correlations and prognostic relevance.

Fifty adult patients with acute lymphoblastic leukemia (ALL) were prospectively studied to determine the clinical and hematological relevance of surface immunophenotypes. Before treatment, blast cells were assayed for reactivity to monoclonal antibodies to B-cell, T-cell, and myeloid (My) antigens. My antigens (CD13, CD33, and VIM2, singly or in combination) were demonstrated in 16 cases (32%) along with lymphoid specificities. Bone marrow and peripheral blood stains were classified according to French-American-British (FAB) Cooperative Group criteria and evaluated for myelodysplastic changes and azurophilic granules. Mean age of My+ patients was significantly higher. Furthermore, a greater number of My+ cases showed azurophilic cytoplasmic granules and acid ANAE positivity. FAB subtypes and myelodysplastic features did not significantly differ in the two groups analyzed, but patients with myelodysplastic abnormalities represented a significantly older age group. Response to treatment was comparable in My+ and My- cases, in terms of either complete remission rate or median survival duration.

Immunodiagnosis of acute leukemia displaying ectopic antigens: proposal for a classification of promiscuous phenotypes.

The nature of the blast cells in 163 cases of acute leukemia was investigated by immunophenotyping, with particular emphasis on the expression of "ectopic" surface membrane structures. Although no antigen included in our panel except CD3 revealed absolute lineage restriction, immunological typing allowed a definite characterization of blast cells in more than 90% of cases. Four groups of patients were identified (A, B, C, D) with different degrees of antigen ectopic expression. We classified as group A leukemias (74%) those expressing conventional antigenic patterns, in absence of cross-lineage markers. Samples classified as group B (18%) showed a single ectopic membrane specificity, apparently discordant with the overall composite phenotype; such a "low-grade deviation" did not prevent a definite immunodiagnosis. Pattern C specimens (5%) revealed a promiscuous coexpression of markers related to different lineages (biphenotypic leukemias), whereas group D included unclassifiable phenotypes, characterized by no antigen or DR-only expression. Our findings suggest the possibility of interpreting complex phenotypic constellations of membrane markers in a consistent and logical manner.