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The t(9;22)(q34;q11), generating the Philadelphia chromosome, is found in more than 90% of patients with chronic myelocytic leukemia (CML). Deletions adjacent to the translocation breakpoint on the derivative chromosome 9 have been described by several groups. These studies revealed two primary points: (1) genomic microdeletions were concomitant with the t(9;22) rearrangement; and (2) the location of the deleted sequence was centromeric to ABL and telomeric to BCR genes. We report on a detailed molecular cytogenetic characterization of chromosomal rearrangements in two CML patients bearing a complex variant t(9;22) and insertions of chromosome 22 sequences in 9q34. Our study shows that the location of the deleted sequences was downstream of the ABL gene and that genomic microdeletions were concomitant with the ins(9;22)(q34;q11q11) rearrangement.

Forty patients with relapsed diffuse large B cell lymphoma (DLBCL) autografted in partial response (PR) (n = 23) or in refractory relapse (RR) (n = 17) achieved complete remission (CR) after autologous stem cell transplantation (ASCT). Salvage treatment consisted of ifosphamide, epirubicin and etoposide (IEV) in 33 patients and Cisplatinum, ARA-C and dexamethasone (DHAP) in 7 patients. All PR and 8 RR patients were conditioned with BEAM, while 9 RR cases received the BCV regimen. There were no significant differences between the two groups as age, serum LDH, duration of CR1 and IPI at relapse are concerned. Relapse rate after ASCT was 39% in PR group as opposed to 88% in RR group (p = 0.003). Median relapse free survival from ASCT was 6 months for RR patients as opposed to 34 months for PR patients (p = 0.003); median overall survival from ASCT was 10 months for RR subset as opposed to not reached for RR subgroup (p = 0.001). These data demonstrate that CR achieved after ASCT in DLBCL patients who are refractory to previous salvage therapy does not result in long-term disease control. Alternative preparative regimens, allogeneic SCT and/or monoclonal antibodies in the post-ASCT phase should be considered for RR patients despite CR achievement. Copyright © 2006 John Wiley & Sons, Ltd.

The prognosis of early relapsing or refractory aggressive non-Hodgkin's lymphoma (NHL) is still poor. Effective salvage therapy should be able to induce high response rate as well as to mobilize hematopoietic precursors. A combination of ifosfamide, epirubicin and etoposide (IEV) was given to 28 patients with refractory or relapsing high grade NHL (4 lymphoblastic lymphoma and 24 large cell lymphoma). All patients were evaluated for response. After 2 courses of IEV, the overall and complete response rate were 64% and 39%, respectively. All patients were controlled for mobilization of peripheral blood stem cells, which was successful in 26 out of 28 (93%). Overall, 25 out of 26 patients proceeded to autologous stem cell transplantation (ASCT). Toxicity was mild, with no occurrence of severe persisting extra-hematologic side-effects. Following the entire therapeutic program, including IEV and ASCT, median progression free survival has not yet been reached and 21 patients are alive (18 in continuous complete remission) after a median follow-up of 18 months. Our results demonstrate that treatment with IEV regimen is effective in refractory or relapsing aggressive NHL, resulting in a high percentage of successful stem cell mobilization and feasibility of ASCT.

Objectives: Therapeutic results in advanced chronic lymphocytic leukemia (CLL) are still unsatisfactory in terms of complete remission achievement and duration, in spite of the extensive use of purine analogs. The objective of this study was to describe the clinical characteristics and treatment results from a series of 32 patients managed with a therapeutic program based on the combination of fludarabine and cyclophosphamide (CTX). Methods: Thirty-two patients (median age 63 yr, range 42–75 yr) with newly diagnosed (47%) or refractory-relapsed (53%) CLL were programmed to receive six courses of a 3-d combination of fludarabine at 30 mg/m2/d plus CTX at 300 mg/m2/d. Refractory-relapsed patients had previously received different chemotherapy lines from 1 to 5. Results: Fourteen of 32 (44%) patients achieved a complete remission, 16 (50%) obtained partial remission and two (6%) failed to respond. The CR rate was higher in untreated patients; in particular, CR was achieved in nine of 15 (60%) newly diagnosed cases as opposed to five of 17 (29%) among pretreated patients. Toxicity was caused by myelosuppression and/or infections in most cases. After a median follow-up of 24 months (range 8–48 months), 20 of 32 patients (62%) are alive, and 14 of 32 (44%) are free from progression. Median overall survival and median time to progression were 35 and 25 months, respectively. Conclusion: The combination of fludarabine with CTX is effective in advanced CLL with acceptable toxicity, either as first-line therapy or in refractory-relapsed patients. In particular, a considerable rate of complete remission can be achieved in untreated patients. Myelosuppression represents the major side-effect.

We describe a case of acute myeloid leukemia (AML) with t(8;21) in which complete remission (CR) was obtained with G-CSF given at 10 microg/kg in the absence of concomitant cytotoxic chemotherapy. CR was achieved following 2 weeks of therapy and confirmed by investigating minimal residual disease by four-color flow cytometry analysis. During treatment with G-CSF, maturing cells with cytoplasmic Auer Rods were observed in the peripheral blood, suggesting a differentiation effect. This case adds further evidence for a specific role of G-CSF in the treatment of AML with t(8;21), namely in patients who are not eligible for aggressive chemotherapy.

Seventeen patients affected by acute myeloid leukemia (AML) with t(8;21) were prospectively programmed to receive three courses of high-dose cytarabine (HDARA-C) as post-remission therapy. The median age was 39 years and in all cases t(8;21) was the only karyotypic abnormality. Complete remission (CR) was achieved in 14 out of 17 cases (82%) and, after first consolidation with NOVIA regimen (intermediate dose ARA-C plus mitoxantrone), all patients received the three planned courses of HDARA-C (3g/m(2) q12h on days 1, 3, 5). There were two documented infections, while all patients experienced fever of unknown origin (FUO). Nonhematological toxicity was mild. Thirteen out of 14 patients are in continuous CR after a median follow-up of 44 months. One patient relapsed at 16 months and, following CR2 achievement, underwent allogeneic transplantation; he died 3 months later while in CR from acute graft versus host disease (GVHD). Survival at 5 years is projected at 79%. Our data confirm the efficacy of repeated courses of HDARAC for patients with t(8;21) AML.

Objectives : To evaluate therapeutic results and prognostic factors from a series of 44 patients affected by de novo acute myeloid leukemia with multilineage dysplasia (MD-AML), treated with the combination of fludarabine, cytarabine and G-CSF (FLAG). Methods : Forty-four patients with de novo MD-AML were treated with the FLAG regimen. The median age was 61 yr (range 31–75 yr). Induction therapy consisted of the FLAG regimen; consolidation included idarubicin plus cytarabine. Patients with a compatible donor and aged less than 55 yr were programmed to receive allogeneic bone marrow transplantation (BMT), while in those without a donor and aged less than 65 yr autologous transplantation with peripheral blood stem cells mobilized by a consolidation regimen plus G-CSF was planned. Bone marrow harvest was performed in poor mobilizers. Results : Complete remission (CR) was achieved in 28 out of 44 patients (64%). Death in induction occurred in four patients (9%), while 12 patients (27%) were resistant to FLAG. Toxicity of consolidation was negligible. Most patients aged less than 60 yr and achieving CR were eligible for transplantation procedures, the main reason of exclusion being early relapse. Median overall survival and disease free survival were 16 and 22 months, respectively. Unfavorable cytogenetics was the only parameter significantly related to inferior clinical outcome following multivariate analysis. Conclusion : Multilineage dysplasia per se is not an adverse prognostic factor in AML patients treated with the FLAG regimen. Favorable results are obtained in patients with intermediate karyotype, while in those with adverse cytogenetics new approaches are clearly needed. The toxicity of the regimen is also acceptable in the elderly, and following induction/consolidation, most patients may be submitted to transplantation procedures.

Curative properties of some medicinal plants such as the Feijoa sellowiana Bert. (Myrtaceae), have been often claimed, although the corresponding molecular mechanism(s) remain elusive. We report here that the Feijoa acetonic extract exerts anti-cancer activities on solid and hematological cancer cells. Feijoa extract did not show toxic effects on normal myeloid progenitors thus displaying a tumor-selective activity. In the Feijoa acetonic extract, fractionation and subsequent purification and analyses identified Flavone as the active component. Flavone induces apoptosis which is accompanied by caspase activation and p16, p21 and TRAIL over-expression in human myeloid leukemia cells. Use of ex vivo myeloid leukemia patients blasts confirms that both the full acetonic Feijoa extract and its derived Flavone are able to induce apoptosis. In both cell lines and myeloid leukemia patients blasts the apoptotic activity of Feijoa extract and Flavone is accompanied by increase of histone and non-histone acetylation levels and by HDAC inhibition. Our findings show for the first time that the Feijoa apoptotic active principle is the Flavone and that this activity correlates with the induction of HDAC inhibition, supporting the hypothesis of its epigenetic pro-apoptotic regulation in cancer systems.

Apart from PML–retinoic acid receptor-α (RARα) acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)–selective agonists (“rexinoids”) the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid–resistant and insensitive AML cells and patients' blasts. Protein kinase A activation leads to corepressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in “desubordination” of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARβ is blunted in mouse embryo fibroblasts lacking RARs, but reintroduction of exogenous RARα reestablishes responsiveness, thus confirming that the RARα-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, tumor necrosis factor–related apoptosis inducing ligand, both of which are known to induce apoptosis in a tumor cell–selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of tumor necrosis factor–related apoptosis inducing ligand and DR5 in AML patient blasts cultured ex vivo. AML patients' blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and French-American-British classification status. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Our results suggest that despite the genetic, morphologic, and clinical variability of this disease, the combination of rexinoids and cAMP-elevating drugs, such as phosphodiesterase inhibitors, might lead to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

International audience; Chromatin is a dynamic macromolecular structure epigenetically modified to regulate specific gene expression. Altered chromatin function can lead to aberrant expression of growth regulators and may, ultimately, cause cancer. That many human diseases have epigenetic etiology has stimulated the development of 'epigenetic' therapies. Inhibitors of histone deacetylases (HDACIs) induce proliferation arrest, maturation and apoptosis of cancer cells, but not normal cells, in vitro and in vivo, and are currently being tested in clinical trials. We investigated the mechanism(s) underlying this tumor selectivity. We report that HDACIs induce, in addition to p21, expression of TRAIL (Apo2L, TNFSF10) by directly activating the TNFSF10 promoter, thereby triggering tumor-selective death signaling in acute myeloid leukemia (AML) cells and the blasts of individuals with AML. RNA interference revealed that the induction of p21, TRAIL and differentiation are separable activities of HDACIs. HDACIs induced proliferation arrest, TRAIL-mediated apoptosis and suppression of AML blast clonogenicity irrespective of French-American-British (FAB) classification status, karyotype and immunophenotype. No apoptosis was seen in normal CD34(+) progenitor cells. Our results identify TRAIL as a mediator of the anticancer action of HDACIs.

Introduction: There is a growing demand for autologous stem cell transplantation (ASCT) in newly diagnosed patients with multiple myeloma (MM), resulting in an increasing pressure on available hospital beds. In addition, more rational utilization of health resources should induce physicians to attempt therapeutic strategies aiming at reduction of costs. The aim of this study was to explore the feasibility and safety of performing ASCT on an outpatient basis, according to an early discharge method. Materials and methods: A total of 28 patients affected by MM and in complete or partial remission were selected to receive ASCT on an outpatient basis. In particular, after conditioning with high-dose melphalan and stem cell infusion, patients were programmed to go home and to be rehospitalized in the case of febrile neutropenia or other severe toxicities. Results: All patients accepted the outpatient-based procedure. Out of 28 patients, 18 (64%) did spend the aplastic phase entirely at home following high-dose chemotherapy and stem cell infusion. A second hospital admission was required in 10 patients (36%). Febrile neutropenia and severe mucositis needing total parenteral nutrition were the most frequent causes of hospitalization. However, there were no documented infections and either fever or mucositis was easily resolved at the time of hematopoietic recovery in all patients. Conclusion: ASCT on an outpatient basis is feasible and safe in patients with MM. More than 60% of patients are manageable at home, provided that a caregiver is available. The Hematology Journal (2004) 5, 222–226. doi:10.1038/sj.thj.6200349

Therapeutic results in advanced chronic lymphocytic leukemia (CLL) are still unsatisfactory in terms of complete remission achievement and duration, in spite of the extensive use of purine analogs. The objective of this study was to describe the clinical characteristics and treatment results from a series of 32 patients managed with a therapeutic program based on the combination of fludarabine and cyclophosphamide (CTX).Thirty-two patients (median age 63 yr, range 42-75 yr) with newly diagnosed (47%) or refractory-relapsed (53%) CLL were programmed to receive six courses of a 3-d combination of fludarabine at 30 mg/m2/d plus CTX at 300 mg/m2/d. Refractory-relapsed patients had previously received different chemotherapy lines from 1 to 5.Fourteen of 32 (44%) patients achieved a complete remission, 16 (50%) obtained partial remission and two (6%) failed to respond. The CR rate was higher in untreated patients; in particular, CR was achieved in nine of 15 (60%) newly diagnosed cases as opposed to five of 17 (29%) among pretreated patients. Toxicity was caused by myelosuppression and/or infections in most cases. After a median follow-up of 24 months (range 8-48 months), 20 of 32 patients (62%) are alive, and 14 of 32 (44%) are free from progression. Median overall survival and median time to progression were 35 and 25 months, respectively.The combination of fludarabine with CTX is effective in advanced CLL with acceptable toxicity, either as first-line therapy or in refractory-relapsed patients. In particular, a considerable rate of complete remission can be achieved in untreated patients. Myelosuppression represents the major side-effect.

To evaluate therapeutic results and prognostic factors from a series of 44 patients affected by de novo acute myeloid leukemia with multilineage dysplasia (MD-AML), treated with the combination of fludarabine, cytarabine and G-CSF (FLAG).Forty-four patients with de novo MD-AML were treated with the FLAG regimen. The median age was 61 yr (range 31-75 yr). Induction therapy consisted of the FLAG regimen; consolidation included idarubicin plus cytarabine. Patients with a compatible donor and aged less than 55 yr were programmed to receive allogeneic bone marrow transplantation (BMT), while in those without a donor and aged less than 65 yr autologous transplantation with peripheral blood stem cells mobilized by a consolidation regimen plus G-CSF was planned. Bone marrow harvest was performed in poor mobilizers.Complete remission (CR) was achieved in 28 out of 44 patients (64%). Death in induction occurred in four patients (9%), while 12 patients (27%) were resistant to FLAG. Toxicity of consolidation was negligible. Most patients aged less than 60 yr and achieving CR were eligible for transplantation procedures, the main reason of exclusion being early relapse. Median overall survival and disease free survival were 16 and 22 months, respectively. Unfavorable cytogenetics was the only parameter significantly related to inferior clinical outcome following multivariate analysis.Multilineage dysplasia per se is not an adverse prognostic factor in AML patients treated with the FLAG regimen. Favorable results are obtained in patients with intermediate karyotype, while in those with adverse cytogenetics new approaches are clearly needed. The toxicity of the regimen is also acceptable in the elderly, and following induction/consolidation, most patients may be submitted to transplantation procedures.

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 6.2610 6 /l (2.6‐16.1). Results: No case of transplant-related mortality occurred. The median number of days to neutrophil (40.5610 9 /l) and platelet (420610 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 highdose IDA plus Busulphan in AML. Results concerning antileukemic eAcacy are promising, but need confirmation on larger series with longer follow-up. The Hematology Journal (2001) 2, 214‐219

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 × 109 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.

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.

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.

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.

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.

Summary. In the present study we investigated the membrance expression of selectin ligands (CD15/Lex, CDw65/VIM2, CD15s/sLex), β2 integrins (CD11a/LFA-1, CD11b/Mac-1) and CD45 phosphatase isoforms (CD45RA, CD45RO on leukaemic cells from 28 patients with acute myeloid malignancies cultured with and without all-trans retinioc acid (ATRA). Within each adhesion system, ATRA was bale 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 and 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 differentation, in terms of surface molecule expression, can be sufficient to obtain therapeutic results.

Apart from PML-RARα acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, in our study we show that elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists (“rexinoids”) the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cell lines and patients’ AML blasts. Protein kinase A activation leads to co-repressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in “de-subordination” of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARβ is blunted in mouse embryo fibroblasts lacking RARs, but re-introduction of exogenous RARα re-establishes responsiveness, thus confirming that the RARα-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, the tumor necrosis factor-related apoptosis inducing ligand (TRAIL), both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of TRAIL and DR5 in AML patient blasts cultured “ex vivo”. AML patients’ blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and FAB classification. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Indeed, it is known that cAMP levels can be elevated also by treating cells with 3′, 5′-cAMP phosphodiesterase inhibitors (PDEi’s). These observation and the clinical availability of the corresponding drugs provide a rationale for initiating clinical studies addressing the efficacy of combinatorial PDEi-rexinoid therapy in AML patients. Together with our recent finding that a very promising class of epigenetic anti-tumor drugs operates through activation of TRAIL expression (Nat Med2005, 11(1):7784), the possibility to target both the ligand (TRAIL) by HDAC inhibitors and the cognate receptors (DR4, DR5) by the above described rexinoid crosstalk may represent a promising therapeutic option which might lead, despite the genetic, morphologic, and clinical variability of AML, to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

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.

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.

1. When NaBr, a chaotropic salt, is added, in concentrations ranging from 0.5m to 2m, to low-salt mammary cytosol, (i) age-dependent aggregation of oestrogen receptor is inhibited, (ii) the receptor sediments as a sharp peak at 4.2S on sucrose-gradient centrifugation, with complete disappearance of heavier forms, and (iii) on gel filtration with Sephadex G-200, the receptor is included in the gel matrix. On a calibrated column, the receptor has a Stokes radius of 3.7nm (+/-6%). 2. Because NaBr inhibits interaction of receptor with other components of cytosol, the values of the sedimentation coefficient, measured by sucrose-gradient sedimentation, and of the Stokes radius, measured by gel filtration, can be accepted with confidence. From these values, it can be computed that the oestrogen-receptor form in NaBr has a mol.wt. of 64000, with a frictional ratio of 1.4. 3. Also, inhibition of aggregation by NaBr allows a 30-90-fold purification of oestrogen receptor. Analysis of this partially purified receptor by sucrose-gradient sedimentation and gel filtration in NaBr gives the same results as for receptor in crude cytosol. On electrofocusing on a pH5-8 gradient, the partially purified oestrogen receptor focuses at pH6.2. On removal of NaBr, receptor aggregates even in this partially purified state. It seems likely that at the protein and ionic concentrations of cytoplasm in vivo, the 64000-mol.wt. receptor form is part of higher states of self- and/or hetero-association with other cytoplasmic components. 4. NaBr up to a concentration of 2m does not inhibit binding of oestrogen by receptor, nor does it decrease the affinity of the interaction (K(D) approximately 8.9x10(-10)m). The total number of binding sites in cytosol, however, decreases by approx. 10%, but this decrease may actually be the result of elimination of lower-affinity binding by non-receptor components of cytosol. 5. NaSCN, another chaotropic salt, was also tested but gave less satisfactory results with the mammary cytosol than with uterine cytosol. EDTA was omitted from the buffers because it favours aggregation of mammary oestrogen receptor. KCl (0.4m), sucrose (15%) and ZnSO(4) (3mm) did not prevent aggregation of receptor.