Donatella Raspadori

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.

The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors

Monoclonal B‐cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5·0 × 109/l circulating CLL‐phenotype B‐cells. The relationship between MBL and Rai 0 CLL, as well as the impact of biological risk factors on MBL prognosis, are unknown. Out of 460 B‐cell expansions with CLL‐phenotype, 123 clinical MBL (cMBL) were compared to 154 Rai 0 CLL according to clinical and biological profile and outcome. cMBL had better humoral immune capacity and lower infection risk, lower prevalence of del11q22‐q23/del17p13 and TP53 mutations, slower lymphocyte doubling time, and longer treatment‐free survival. Also, cMBL diagnosis was a protective factor for treatment risk. Despite these favourable features, all cMBL were projected to progress, and lymphocytes <1·2 × 109/l and >3·7 × 109/l were the best thresholds predicting the lowest and highest risk of progression to CLL. Although IGHV status, CD38 and CD49d expression, and fluorescence in situ hybridization (FISH) karyotype individually predicted treatment‐free survival, multivariate analysis identified the presence of +12 or del17p13 as the sole independent predictor of treatment requirement in cMBL (Hazard ratio: 5·39, 95% confidence interval 1·98–14·44, P = 0·001). Overall, these data showed that cMBL has a more favourable clinical course than Rai 0 CLL. Given that the biological profile can predict treatment requirement, stratification based on biological prognosticators may be helpful for cMBL management.

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.

Alloantigen presenting capacity, T cell alloreactivity and NK function of G-CSF-mobilized peripheral blood cells

In this study we addressed whether the proportion and the function of antigen presenting cells (APC), T and NK lymphocytes are modified in the apheresis product of six healthy donors who received a stem cell mobilizing treatment with glycosylated G-CSF at 10 μg/kg/day × 5 days s.c. Flow cytometry analysis showed comparable percentages of HLA-DR+, CD19+, CD86+, CD80+ and CD1a+ cells in preG-CSF-peripheral blood mononuclear cells (preG-PBMC) and after mobilization in G-PBMC, whereas the proportion of CD14+ monocytes significantly increased in G-PBMC (3 ± 1% vs 17 ± 8%, P = 0.003). Analysis of lymphocyte subsets in preG-PBMC and G-PBMC showed similar proportions of CD3+, CD4+, CD8+ and CD28+ T cells, but a significantly lower percentage of CD16+ (11 ± 7% vs 4 ± 1%, P = 0.01), CD56+ (15 ± 6% vs 5 ± 2%, P = 0.008), CD57+ (16 ± 9% vs 5 ± 2%, P = 0.04), CD25+ (19 ± 2% vs 9 ± 6%, p = 0.009) and CD122+ (5 ± 2% vs 2 ± 1%, P = 0.05) cells in G-PBMC. Unfractionated preG-PBMC and G-PBMC were irradiated and tested in primary mixed leukocyte culture (MLC) with two HLA-incompatible responders and induced efficient alloresponses in four of six cases, whereas G-PBMC stimulated poorly in the remaining two cases. Also, in allo-MLC with irradiated G-PBMC we detected lower amounts of IFN-γ (P = 0.04) and of IL-2 (P = 0.06) than in allo-MLC with preG-PBMC. Furthermore, freshly isolated preG-PBMC and G-PBMC from each donor exerted comparable allogeneic responses to HLA-incompatible irradiated mononuclear cells in all cases. However, G-PBMC showed no NK activity against K562 target cells at any effector:target ratio tested. These data suggest that normal G-PBMC may prevent Th1 alloresponses, maintain efficient alloreactivity to HLA mismatched antigens and have impaired NK activity.

P-glycoprotein (PGP) and lung resistance-related protein (LRP) expression and function in leukaemic blast cells

P‐glycoprotein (PGP) lung resistance protein (LRP) and multidrug resistance associated protein (MRP) expressions and function were evaluated by flow cytometry in 65 leukaemic patients (38 acute non‐lymphocytic leukaemias, eight acute lymphocytic leukaemias, 19 Ph‐positive chronic myeloid leukaemias in blastic phase). By using the MRK‐16, the LRP‐56 and the MRPm6 MoAbs, 34% of the cases did not over‐express any proteins (−); 24.5% over‐expressed (+) only PGP, 11% only LRP, 1.5% only MRP, 24.5% both PGP and LRP, and 4.5% both PGP and MRP. The mean intracellular daunorubicin accumulation (IDA) and rhodamine 123 (Rh123) retention in the presence or absence of the reversal agent SDZ PSC 833 (PSC) of the PGP−/LRP−/MRP− cases were comparable to the ones observed in normal leucocytes. With respect to the non‐over‐expressing cases, the PGP−/LRP+/MRP− cases showed only an impaired IDA (mean 204 ± 29; P < 0.001). The PGP+/LRP+/MRP− cases had a defect both in IDA (mean 166 ± 47, P < 0.001) and Rh123 retention (mean 0.42 ±0.14; P < 0.001), which were both corrected by PSC. All the PGP+/LRP+/MRP− cases had a defect in IDA (mean daunorubicin (DNR) accumulation 192 ± 44; P < 0.001. However, only in 8/16 of them an evident defect in Rh123 retention was found. In conclusion, both PGP and LRP over‐expression were common in leukaemia. An impaired IDA was found in all cases over‐expressing PGP, LRP or both. The study of Rh123 retention could give incorrect information about the blast cells’ ability to accumulate cytotoxic drugs in patients over‐expressing both PGP and LRP.

Hairy cell leukemias with unmutated IGHV genes define the minor subset refractory to single agent cladribine and with more aggressive behavior

Hairy cell leukemia (HCL) is generally responsive to single-agent cladribine, and only a minority of patients are refractory and with poor prognosis. HCLs generally express mutated (M) and, in a minority, unmutated (UM) IGHV. In a multicenter clinical trial in newly diagnosed HCL, we prospectively investigated clinical and molecular parameters predicting response and event-free survival after single-agent cladribine. Of 58 HCLs, 6 expressed UM-IGHV (UM-HCL) and 52 M-IGHV (M-HCL). Beneficial responses were obtained in 53 of 58 patients (91%), whereas treatment failures were observed in 5 of 58 patients (9%). Failures were associated significantly with UM-IGHV (5 of 5 failures vs 1 of 53 beneficial responses had UM-IGHV, P < .001), leukocytosis (3 of 5 vs 3 of 53, P = .006), and bulky spleen (4 of 5 vs 4 of 53, P < .001). The UM-HCL not benefiting from cladribine characteristically had bulky spleen (4 of 5, 80%), leukocytosis (3 of 5, 60%), and TP53 defects (2 of 5, 40%), and progressed rapidly after first treatment (median event-free survival, 7.5 months). Our data suggest that UM-HCLs identify the minor subgroup failing cladribine treatment and with more aggressive disease. High incidence of TP53 dysfunction indicates a potential mechanism of resistance to cladribine in the UM-HCL group. Overall, our data provide new molecular elements relevant for treatment concerns in HCL.

Genome‐wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion

Deletion of 17p (TP53) identifies a rare subset of chronic lymphocytic leukaemia (17p‐ CLL) with aggressive behaviour. Genome‐wide DNA‐profiling was performed to investigate 18 patients with 17p‐ CLL. All cases had multiple copy‐number (CN) changes. Among the several recurrent CN changes identified, 8q24.13‐q24.1‐gain (MYC), 8p‐loss (TNFRSF10A/B, also known as TRAIL1/2) and 2p16.1‐p14‐gain (REL/BCL11A) appeared frequently represented. 8p‐loss and 2p16.1‐p14‐gain also appeared clinically relevant and predicted significant shorter time from diagnosis to treatment (8p‐loss) and overall survival (8p‐loss and 2p16.1‐p14‐gain, P < 0·05). These observations document a highly unstable genome in 17p‐ CLL and suggest that additional genes outside the TP53 locus may be important for tumour behaviour.

Discrepancy between phenotypic and functional features of natural killer T-lymphocytes in B-cell chronic lymphocytic leukaemia

Summary. The phenotypic expression and functional capacity of natural killer (NK) T‐lymphocytes (E+, OKT3 +) were analysed in a series of untreated patients with B‐cell chronic lymphocytic leukaemia (B‐CLL).

“Fludarabine Containing-Regimens May Adversely Affect Peripheral Blood Stem Cell Collection in Low-Grade Non Hodgkin Lymphoma Patients”

Fludarabine (FLUDA) based chemotherapy has shown promise in both initial and salvage treatment of low-grade non Hodgkins lymphomas (LG-NHL). Recently, more aggressive therapies followed by autologous hemopoietic progenitor cell rescue, have also been successfully employed in these patients. However, this procedure, due to several factors including previous therapeutic regimens, is often limited by an inadequate collection of peripheral blood stem cell (PBSC). At present, very little data is available on the effect of FLUDA containing regimens in PBSC collection. We report our preliminary experience showing a possible correlation between FLUDA based chemotherapy regimens employed before mobilization and inability to collect an adequate number of blood derived hematopoietic progenitors for autologous PBSC transplantation in LG-NHL patients.

A comparative analysis of the sensitivity of multidrug resistant (MDR) and non MDR cells to different anthracycline derivatives

Because of the fact that tumor cell sensitivity to cytotoxic agents may play a major role in cancer treatment, and several anthracyclines are widely used for first-line treatment of leukemia, lymphoma and other tumors, and since the overexpression of the mdr-1 gene-coded 170 Kd glycoprotein (P170) decreases cell sensitivity to anthracyclines, we investigated the relationship between P170 overexpression and the cytotoxicity of two classic anthracyclines (Daunorubicin or DNR and Doxorubicin or DX) and two lipophilic anthracycline derivatives (Idarubicin or IDA and Iododoxorubicin or IDX). For these purposes, we used multidrug resistant (MDR) and non-MDR tumor and leukemia cell lines and the MTT-microcultured tetrazolium colorimetric assay. We showed that mdr-1 gene overexpression was strongly associated with the development of a high level of resistance to DNR and DX, but not to the derivatives IDA and IDX. These data suggest that more lipophilic anthracycline derivatives may also be active in MDR cell systems.