D. Rondelli

CD56 antigenic expression in acute myeloid leukemia identifies patients with poor clinical prognosis.

CD56 antigen, a 200–220 kDa cell surface glycoprotein, identified as an isoform of the neural adhesion molecules (NCAM), has been found frequently expressed in several lympho–hematopoietic neoplasms including acute myeloid leukemias (AML). In fact, in these latter diseases it has been reported that the presence of CD56 antigen on the blasts of AML patients with t(8;21) (q22;q22), and in those with M3 subtype, identifies a subgroup of patients with a more unfavorable prognosis. On the basis of these findings, we evaluated in 152 newly diagnosed AML patients CD56 surface expression, and results were correlated with morphology, immunophenotype, cytogenetic pattern and clinical outcome. CD56 antigen was recorded in 37 out of 152 cases (24%) and particularly in those with M2 and M5 cytotypes. Moreover, CD56 expression was significantly associated with P-glycoprotein (PGP) hyperexpression (P = 0.007), unfavorable cytogenetic abnormalities (P = 0.008) and with a reduced probability of achieving complete remission (CR) (36% vs 68%) (P = 0.035) as well as with a shorter survival (6 vs 12 months) (P = 0.032). In conclusion, CD56 antigenic expression on AML cells represents an important adverse prognostic factor and therefore its presence should be regularly investigated for a better prognostic assessment of AML patients at diagnosis.

High bcl-2 expression in acute myeloid leukemia cells correlates with CD34 positivity and complete remission rate

Flow cytometric expression of bcl-2 protein was analyzed in 90 newly diagnosed acute myeloblastic leukemia (AML) patients using an anti-bcl-2 monoclonal antibody by direct immunofluorescence technique and results were correlated with FAB cytotype, CD34 expression and clinical outcome. Bcl-2 was expressed in all AML cases with different intensity. The mean fluorescence index (MFI), expressed as the ratio of sample mean channel:control mean channel, ranged from 3.0 to 39.5 with a median value of 14. The MFI was significantly higher (P = 0.01) in M0 (20.9) and M1 (18.3) than in M2 (11.7), M3 (12.4), M4 (11.8) and M5 (9.5) cytotypes. In addition, bcl-2 MFI significantly correlated both with CD34 positivity (P = 0.001) and with CD34 MFI (P = 0.01), being CD34 antigen expressed in 65% of patients with a bcl-2 MFI >14, and only in 35% of AML cases with a bcl-2 MFI >14. When bcl-2 intensity expression was correlated with complete remission (CR) rate, a higher MFI was associated with a low CR rate after standard intensive chemotherapy. In particular, CR was achieved in 86% of patients with a bcl-2 MFI <14, but only in 57% of patients with a mfi >14 (P = 0.008). A further decrease of CR rate to 41% was observed in patients in whom a higher bcl-2 MFI was coupled with the presence of CD34 antigen on their blasts. By statistical analysis we also demonstrated that both bcl-2 high MFI (>14) and CD34 expression are independent prognostic factors for achieving CR in AML. These data raise the hypothesis that high values of bcl-2 may confer on myeloid blasts a higher resistance to standard chemotherapy. However, identification of patients with high expression of bcl-2 may be important for a different therapeutic approach.

Myeloperoxidase Expression by Histiocytes in Kikuchi's and Kikuchi-Like Lymphadenopathy

Forty-five examples of Kikuchis lymphadenitis (KL), 5 Kikuchi-like lupus erythematosus lymphadenopathies, 25 nonnecrotizing lymphadenitidies (5 toxoplasmic, 5 sarcoid-like, 6 dermatopathic, 4 suppurative, 3 tubercular, 2 with sinus histiocytosis), 4 examples of hyaline-vascular Castleman disease (CD), 2 plasmacytoid monocyte tumors (PM-Ts), and 61 accessory cell neoplasms were studied by a panel of antibodies, including the PG-M1 (against a macrophage-restricted CD68 epitope) and a polyclonal anti-myeloperoxidase (MPO). In KL and Kikuchi-like lupus erythematosus lymphadenopathies, 25 to 75% of CD68(+) histiocytes co-expressed MPO. This did not occur in nonnecrotizing lymphadenitidies and accessory cell neoplasms. MPO(+)/CD68(+) elements corresponded to nonphagocytosing mononuclear cells and some crescentic macrophages and phagocytosing histiocytes. Typical PMs were MPO(-)/CD68(+) in all cases, including CD and PM-T. Our observations suggest that in KL and KL-like lymphadenopathies: 1) MPO(+)/CD68(+) blood monocytes might be attracted into tissues because of the lack or paucity of granulocytes and the need of MPO for oxidative processes; 2) PMs are more likely to be involved in the cytotoxic immune reaction than in phagocytic phenomena; 3) the peculiar phenotype of the histiocytic component can be usefully used for the differentiation from malignant lymphoma and PM-T.

Generation and functional characterization of human dendritic cells derived from CD34 ˛ cells mobilized into peripheral blood: comparison with bone marrow CD34 ˛ cells

Dendritic cells (DCs) are the most powerful professional antigen‐presenting cells (APC), specializing in capturing antigens and stimulating T‐cell‐dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady‐state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony‐stimulating factor (G‐CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony‐forming unit‐dendritic cells (CFU‐DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM‐CSF and TNF‐α with or without SCF and FLT‐3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early‐acting growth factors SCF and FLT‐3L with IL‐4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+CD14− cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL‐4‐stimulated DCs was significantly higher than those generated in the absence of IL‐4. Furthermore, autologous serum collected during G‐CSF treatment was more efficient than fetal calf serum (FCS) or two different serum‐free media for large‐scale production of DCs. Thus, our comparative studies indicate that G‐CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early‐acting and intermediate‐late‐acting colony‐stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.

Long-lasting complete remission in patients with hairy cell leukemia treated with 2-CdA: a 5-year survey

Between January 1991 and January 1994, 40 patients with hairy-cell leukemia (HCL), 30 males and 10 females, with a median age of 54 years, were treated with a single course of 2-chlorodeoxyadenosine (2-CdA) at a dose of 0.1 mg/kg/day continuous infusion for 7 days. Thirteen patients were untreated and 27 had previously received α interferon. Thirty out of 40 patients (75%) achieved complete remission (CR) and 10 (25%) partial remission (PR). The median follow-up duration for patients in CR has been 48 months (range 30–66). Five of the complete responders (17%) relapsed at 12, 24, 26, 30 and 36 months after treatment as documented by the increase of hairy cells (Hc) in the bone marrow and two of them, who were retreated with 2-CdA after showing an initial impairment of peripheral blood values, obtained a second CR. The remaining three relapsed patients were never retreated and still show normal peripheral counts after 30, 38 and 40 months. Twelve of the continuous complete responder patients are still in CR after more than 5 years. In contrast, 8 out of 10 partial responders progressed after 8–36 months and all of them were retreated with 2-CdA at a dose of 0.15 mg/kg/day for 5 days i.v. Four of them (50%) achieved a CR, three a better PR and one patient died 6 months after the second 2-CdA course because of infectious complications. Two additional patients, both in CR, died after 28 and 37 months because of a second neoplasm. Toxic side-effects consisted of febrile episodes recorded in 16 patients: in seven of them, fever lasted only 24–48 h after the end of treatment and was apparently not infection-related. In the remaining nine patients, showing in addition severe neutropenia (neutrophils less than 1.0 × 109/l), fever was related to bacterial infection requiring systemic antibiotics in all of them and G-CSF in three cases. In conclusion, 2-CdA induces a very high proportion of complete and long-lasting remissions in patients with HCL. In a number of cases relapse at bone marrow level may not affect peripheral blood values for prolonged time. However, in those patients with initial pancytopenia a retreatment with 2-CdA is still effective in inducing a durable second CR.

Plasmacytoid dendritic cells: Do they have a role in immune responses after hematopoietic cell transplantation?

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) and are able to modulate immune responses. Investigators are studying methods to exploit the immunogenic and tolerogenic properties of DC. In the context of hematopoietic cell transplantation, DC might be helpful to facilitate engraftment and prevent graft-versus-host disease (GVHD) reactions. In this paper, we review circumstantial evidence that immature plasmacytoid DC might affect immune responses after transplantation of hematopoietic cells from allogeneic donors.

Expression of CD86 in acute myelogenous leukemia is a marker of dendritic/monocytic lineage

OBJECTIVE The aim of this study was to determine whether expression of the CD86 costimulatory molecule in acute myeloid leukemia (AML) can identify blast cells committed to the monocytic/dendritic lineage. MATERIAL AND METHODS One hundred ten consecutive AML patients observed at diagnosis were studied by flow cytometry. In selected experiments, in vitro cultures with CD34(+)CD86(+) or CD34(-)CD86(+) blasts were performed in the presence of granulocyte-macrophage colony-stimulating actor (GM-CSF) with or without tumor necrosis factor-alpha (TNF-alpha) or GM-CSF + interleukin-4 (IL-4), respectively, to induce a dendritic differentiation, documented by morphologic and immunophenotypic assays. T-cell alloreactivity to CD86(+) AML cells and leukemic dendritic cells (AML-DC) was tested in mixed leukocyte cultures and anti-leukemic cytotoxic assays. RESULTS CD86 was expressed in 54% AML, whereas CD80 and CD1a were only occasionally positive. CD86(+) AML samples included M5 and M4, but also 47% M0-M1 FAB types, and were more frequently CD14(+) (p < 0.00001) and CD34(-) (p = 0.00005) than CD86(-)AML. Six different patterns of CD86(+) AML were identified, according to CD34 or CD14 total or partial coexpression. Four samples enriched in CD34(+)CD86(+) AML cells differentiated into AML-DC CD86(+), CD80(+), CD40(+), CD11c(+), HLA-DR(++), CD14(+/-) that also were CD1a(+) or CD83(+), after 6 days of in vitro culture with GM-CSF +/- TNF-alpha. CD34(-)CD86(+) AML cells differentiated into AML-DC after 3 to 5 days (n = 5 experiments), and trisomy 8 was found in two AML and AML-DC samples by fluorescence in situ hybridization analysis. Finally, AML-DC induced more potent allo-T-cell proliferation, cytokine release, and anti-leukemic cytotoxicity than CD86(+) blasts. CONCLUSIONS In AML, CD86 is a marker of monocytic/dendritic lineage. Because CD86(+) blasts may differentiate into DC rapidly, CD86(+)AML patients could be optimal candidates for immunotherapy studies, both in autologous and allogeneic settings.

Fludarabine in patients with advanced and/or resistant B-chronic lymphocytic leukemia

Abstract:  In this study, we evaluated the efficacy of fludarabine (FLU), an adenine nucleoside analogue, in 35 previously treated patients with advanced and progressed B‐cell chronic lymphocytic leukemia (B‐CLL) and in 6 at diagnosis. All patients were treated at a dose of 25 mg/m2 per day for 5 consecutive days (mean number of courses was 5, with a range from 4 to 6). The majority of patients experienced a beneficial effect on hematological parameters. In particular, a remarkable reduction of lymphocyte count together with an increase of neutrophils and platelets was observed. The overall response rate was 42% with 1 complete response and 16 partial responses. Ten patients achieved minor responses and the remaining 14 showed no benefit from treatment. An increased response rate was achieved in 6 untreated patients who showed an overall response rate of 67% (4/6). The major complications observed were neutropenia (66%) and febrile episodes (44%) that were generally infection‐related and were fatal in 3 cases. Because we were dealing with patients whose disease was advanced and/or resistant to treatment, the overall results may be considered encouraging with acceptable toxic reactions not superior to those frequently observed with polychemotherapy.

2‐Chlorodeoxyadenosine in the treatment of relapsed/refractory chronic lymphoproliferative disorders

Abstract:  2‐Chlorodeoxyadenosine (2‐CdA) is a purine analog with cytotoxic activity on both resting and cycling lymphocytes which has been used as salvage therapy in advanced/resistant chronic lymphoproliferative disorders. In our study 39 patients (19 B‐CLL, 5 B‐PLL, 9 low‐grade B‐NHL, 5 CTCL and 1 high‐grade T‐NHL) who relapsed or became resistant after 1–4 chemotherapy regimens were treated with 2‐CdA 6 mg/m2 per day by 2 h infusion for 5 d every 28 d. The overall clinical response rate, including complete remission (CR) and partial remission (PR), was 66%. Two of 19 (10%) B‐CLL patients achieved a CR lasting 9 months, while 11/19 B‐CLL (58%) and 4/5 B‐PLL (3 B‐PLL/B‐CLL and 1 B‐PLL) (80%) achieved a PR. Interestingly, 5 of 6 patients who had been previously treated with fludarabine obtained a clinical response (2 CR and 3 PR). One of 9 (11%) low‐grade B‐NHL patients achieved a CR and relapsed after 26 months, and 5/9 (55%) achieved a PR. One of 5 (20%) CTCL achieved a CR lasting 32 months, while 2/5 (40%) achieved a PR. The overall mean duration of PR was 7.4 months and no differences were observed among different groups of patients. Toxicity was acceptable, as only a transient severe hematological impairment was observed in 20% of the patients while nonhematological toxicity was not documented. Two patients died because of bacterial pneumonia, 1 of meningitis due to Listeria and 9 from progression of the disease. In conclusion, treatment with 2‐CdA in heavily pretreated patients with chronic lymphoproliferative disorders is well tolerated and obtains high response rates, even in patients relapsed after treatment with fludarabine.

Increased expression of myeloid antigen markers in adult acute lymphoblastic leukaemia patients: Diagnostic and prognostic implications

Summary. By applying direct immunofluorescence with a dual‐staining technique we were able to demonstrate that 20/37 (54%) patients with acute lymphoblastic leukaemia (ALL) expressed both lymphoid and myeloid antigens on the same leukaemic cells. CD13 and CD33 myeloid antigens were detected in 18/20 and in 15/20 cases respectively, and both in 13. Molecular studies confirmed that the four patients with T‐cell phenotype had molecular rearrangement of T‐cell receptor (TcR) β chain, and 26 ALL patients with ‘B‐cell’phenotype showed JH rearrangement. Two ALL patients without (ALL/My‐) and three with myeloid antigens (ALL/My+) also demonstrated bcr/abl rearrangement. Both groups of patients had similar presenting features such as age, sex, Hb level, white blood cells, platelet counts and cytogenetic features. Complete response was achieved in 16/17 (94%) ALL/My‐ patients and in 15/18 (83%) ALL/My+ patients (two deaths occurred during induction) with a mean duration of 17 and 16 months respectively and with similar survival and event‐free survival curves.