Purine metabolism in B-cell lymphocytic leukemia: a microarray approach.

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.

Effect of a p210 multipeptide vaccine associated with imatinib or interferon in patients with chronic myeloid leukaemia and persistent residual disease: a multicentre observational trial.

BACKGROUND: Although imatinib is the standard treatment for chronic myeloid leukaemia, not all patients reach complete cytogenetic remission (CCR) and most maintain detectable disease at the molecular level. We investigated whether a vaccine targeting the BCR-ABL-derived p210 fusion protein was an active and specific immunotherapy. METHODS: We recruited 16 patients who had chronic myeloid leukaemia (with the b3a2 fusion point of p210), stable residual disease, a minimum treatment of 12 months of imatinib or 24 months of interferon alfa, and no further reduction of residual disease for at least 6 months preceding enrollment. They were given six vaccinations with a peptide vaccine derived from the sequence p210-b3a2 plus molgramostim and QS-21 as adjuvants (CMLVAX100) before assessment of immunological and disease response, which included detecting amounts of b3a2 transcripts by standardised quantitative real-time reverse-transcriptase PCR. RESULTS: Of ten patients on imatinib, nine started CMLVAX100 having had a median of 10 months' stable cytogenetic disease (median 10% Philadelphia-chromosome-positive metaphases), whereas one started in stable CCR. All patients' cytogenetic responses improved after six vaccinations, with five reaching CCR. Notably, three of these five patients also had undetectable amounts of b3a2 transcript (BCR-ABL:beta2 microglobulin ratio <0.00001). Six patients on interferon alfa treatment with a median of 17 months' stable residual disease (median 13% Philadelphia-chromosome-positive cells) were also vaccinated. All but one had improved cytogenetic responses, and two reached CCR. Overall, we recorded peptide-specific delayed-type hypersensitivity (in 11 of 16 patients), CD4 cell proliferation (13 of 14 assessed), and interferon gamma production (five of five assessed). INTERPRETATION: Addition of CMLVAX100 to conventional treatment in patients with chronic myeloid leukaemia might favour further reduction of residual disease and increase the number of patients reaching a molecular response.

Efficacy of anti-CD20 monoclonal antibodies (Mabthera) in patients with progressed hairy cell leukemia.

BACKGROUND AND OBJECTIVES: Recently, a chimeric monoclonal antibody (MoAb) directed against the CD20 antigen (rituximab) has been successfully introduced in the treatment of several CD20-positive B-cell neoplasias and particularly of follicular lymphomas. Based on these premises we evaluated the efficacy and the toxicity of chimeric anti-CD20 monoclonal antibody (MoAb) in relapsed/progressed hairy cell leukemia (HCL). DESIGN AND METHODS: Ten patients with relapsed/progressed HCL entered the study. Eight patients were males and two females with a median age of 55 years (range 41-78) and all of them had been previously treated with 2-chlorodeoxyadenosine and/or deoxycoformycin and a-interferon. Two out of 10 patients were anemic (Hb < 10 g/dL), 4 thrombocytopenic (Plt < 100 x 10(9)/L), 3 had fewer than 1.0 x 10(9)/L neutrophils and 3 had circulating hairy cells (HC). All patients received 375 mg/m2 i.v. of anti-CD20 MoAb once a week for 4 doses. RESULTS: All patients were evaluable for response, one patient showing a complete remission and 4 a partial response. Adverse reactions, such as fever, chills, bone pain, hypotension and thrombocytopenia, were transient and mild (grade 1-2) and occurred only during the first course of treatment. One month after the last infusion, patients who had had anemia, neutropenia or thrombocytopenia, recovered normal peripheral blood values. Circulating HC also disappeared within one month. Immunostained bone marrow biopsies were checked 1, 3 and 6 months after the end of therapy and in 5 out of 10 patients a >50% reduction of bone marrow HC infiltration was recorded. INTERPRETATION AND CONCLUSIONS: On the basis of these preliminary results observed in 10 patients with progressed HCL, it appears that treatment with anti-CD20 MoAb is safe and effective in at least 50% of patients, particularly in those with a less evident bone marrow infiltration (50%) and in those previously splenectomized.

Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing.

Hairy cell leukemia (HCL) derives from a mature B cell and expresses markers associated with activation. Analysis of immunoglobulin variable region genes has revealed somatic mutation in most cases, consistent with an origin from a cell that has encountered the germinal center. One unusual feature of hairy cells (HCs) is the frequent expression of multiple immunoglobulin heavy chain isotypes, with dominance of immunoglobulin (Ig)--G3, but only a single light chain type. The origin and clonal relationship of these isotype variants have been unclear. In order to probe the isotype switching status of HCL, RNA transcripts of V(H)DJ(H)--constant region sequences from 5 cases of typical HCL, all expressing multiple surface immunoglobulin isotypes, were analyzed. Tumor V(H)DJ(H)--C(mu) sequences were identified and found to be somatically mutated (range, 1.4% to 6.5%), with a low level of intraclonal heterogeneity. Additional immunoglobulin isotypes of identical V(H)DJ(H) sequence were also identified, including IgD (5 of 5), IgG3 (5 of 5), IgG1 (3 of 5), IgG2 (2 of 5), IgA1 (4 of 5), and IgA2 (1 of 5). Derivation of multiple isotypes from individual cells was demonstrated by analyzing transcripts in single sorted cells from one patient, with evidence for coexistence of isotype variants in 10 of 10 cells. These findings indicate that clonally related multiple isotypes coexist in single HCs, with individual isotypes presumably generated via RNA splicing. Production of IgG3 appears common, but IgG1, IgG2, IgA1, and IgA2 also arise, indicating a continuing influence of a directed process on the tumor clone. These HCs appear to be arrested at the point of isotype switch, where RNA processing may precede deletional recombination. (Blood. 2001;98:1174-1181)

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.

Fludarabine, arabinosyl cytosine and idarubicin (FLAI) for remission induction in poor-risk acute myeloid leukemia.

Progress in treatment of acute myeloid leukemia (AML) is slow and treatment intensification alone has limited effects, particularly in poor-risk cases. Poor-risk cases, that are identified mainly by prior history, leukemic cell mass and cytogenetic abnormalities, share multiple mechanisms of drug resistance that are responsible for treatment failure. Since Pgp-mediated resistance to anthracycline can be reduced with Idarubicin (IDA) and resistance to arabinosyl cytosine (AC) can be reduced with Fludarabine (FLUDA), we tested a combination of high dose AC (2000 mg/sqm, 5 doses), FLUDA (30 mg/sqm, 5 doses) and IDA (12 mg/sqm, 3 doses) for remission induction and consolidation in 45 consecutive cases of poor-risk AML. The complete remission (CR) rate was 71% after the first course and 82% overall, with a projected 2-year survival and relapse-free survival of 44% and 50% respectively. Non-hematologic toxicity was very mild, that is very important in elderly patients, but hemopoietic toxicity was substantial, with a time to hematologic recovery of 3 to 4 weeks and two cases of death in CR. Peripheral blood stem cells (PBSC) could be mobilized and collected successfully only in 11 cases. This three-drug combination is effective and has a limited non-hematologic toxicity, but FLUDA may increase the difficulty of obtaining PBSC early after remission induction.

Expression of the CD11c antigen in B-cell chronic lymphoproliferative disorders.

This study analyzed the expression of the beta2 integrin CD11c in 155 patients with well-characterized B-cell chronic lymphoproliferative disorders: 106 B-cell chronic lymphocytic leukemias (B-CLL), 21 hairy cell leukemias (HCL), 9 B-cell prolymphocytic leukemias (PLL) and 19 low grade non-Hodgkin's lymphomas (NHL) in leukemic phase. CD11c was expressed in 100% of patients with HCL and B-PLL, while in B-CLL and NHL it was expressed in only 49 and 57%, respectively. Furthermore, in B-CLL the expression of CD11c was found mainly in patients with early stage of disease. In addition, when the fluorescence intensity of CD11c, calculated by MFI, was evaluated, it proved significantly higher in HCL and B-PLL compared to the values recorded in B-CLL and NHL (325 and 387 vs 34 and 56, respectively) (p < 0.05). Our results demonstrate that the evaluation of CD11c, both in terms of overall positivity and of fluorescence intensity, represents an additional useful parameter for a more precise differential diagnosis within the spectrum of B-cell chronic lymphoproliferative disorders.

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 Hodgkin's 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.

Different immune reconstitution in multiple myeloma, chronic myeloid leukemia and acute myeloid leukemia patients after allogeneic transplantation of peripheral blood stem cells.

In this study we compared the lymphocyte reconstitution in 13 multiple myeloma (MM), nine acute myeloid leukemia (AML) and 10 chronic myeloid leukemia (CML) patients after allogeneic G-CSF-mobilized PBSC transplantation from HLA-identical siblings. Conditioning regimens included standard total body irradiation + cyclophosphamide (CY), or busulphan + CY, whereas VP-16 was added in patients with advanced disease. Overall comparable numbers of mononuclear cells, CD34+ cells and CD3+ T cells were infused in each group. A significantly higher CD3+ T cell number was observed in MM and AML than in CML patients 1 month after transplant. However, MM patients showed a faster and better recovery of CD4+ T cells than both AML and CML patients at 3 months (P = 0.01 and P = 0.01, respectively) and 12 months (P = 0.01 vs AML, while P = NS vs CML) after transplant, and had a CD4:CD8 ratio > 1 with a median CD4+ T cell value > 400/microl 1 year after transplant. Development of acute graft-versus-host disease (GVHD) did not affect CD4:CD8 ratios but patients who experienced acute GVHD > grade I had lower CD4+ and CD8+ T cell numbers at all time points. However, after excluding patients with GVHD > grade I, MM patients still showed a significantly higher CD4+ T cell value than patients with myeloproliferative diseases 1 year after transplant. These findings suggest that although allogeneic PBSC transplantation induces rapid immune reconstitution, different kinetics may occur among patients with hematological malignancies. In particular, the rapid reconstitution of CD4+ T cells in MM patients may contribute to the low transplant-related mortality achieved in this disease.

Long-lasting decrease of CD4+/CD45RA+ T cells in HCL patients after 2-chlorodeoxyadenosine (2-CdA) treatment.

The CD45RA and CD45RO isoforms of the leukocyte common antigen identify functionally distinct CD4+ T cell subsets: CD4+/CD45RA+ cells which represent a more 'naive' stage of T cell compartment and CD4+/CD45RO+ 'memory' cells. Phenotypic and functional abnormalities in T cell compartment have been frequently reported in patients with hairy cell leukemia (HCL) and, in more recent studies, a significant reduction in the absolute number of CD4+ lymphocytes bearing the CD45RO antigen has also been recorded. In our study we evaluated the CD45RA and CD45RO expression on CD4+ T cells by three-color staining in flow cytometry in 38 HCL patients, 19 untreated and 19 previously treated with 2-chlorodeoxyadenosine (2-CdA), administered at a daily dose of 0.1 mg/kg c.i. for 7 days. In HCL untreated patients, the proportion and the absolute number of CD4+/CD45RA+ and of CD4+/CD45RO+ T cell subsets were similar to normal controls. In contrast, HCL patients at 3-5 years by the end of treatment with 2-CdA, together with a reduction in the absolute number of CD4+ T cells, showed a persistent and significant decrease in the proportion and absolute number of CD4+/CD45RA+ cells as compared with both untreated HCL patients and normal controls (41 +/- 16% vs 57 +/- 14% and vs 65 +/- 7%) (P = 0.01 and 0.0001) and (0.201 +/- 0.137 x 10(9)/l vs 0.549 +/- 0.238 x 10(9)/l and vs 0.696 +/- 0.078 x 10(9)/l) (P = 0.00009 and P = 0.00001). In addition, together with the reduction of CD4+/CD45RA+ cells, we recorded a concomitant increase in the proportion of the CD4+/CD45RO+ cells as compared to untreated HCL patients and normal controls (62 +/- 16% vs 47 +/- 15% and vs 42 +/- 12%) (P = 0.08 and 0.02). These findings may suggest that CD4+/CD45RA+ cells are more sensitive than CD4+/CD45RO+ to the toxic effect of 2-CdA.

Weekly administration of 2-chlorodeoxyadenosine in patients with hairy-cell leukemia is effective and reduces infectious complications.

BACKGROUND AND OBJECTIVE: It has been widely demonstrated that one single 7-day course continuous infusion (c.i.) 2-chlorodeoxyadenosine (2-CdA) at a dose of 0.1 mg/kg daily is dramatically effective in inducing high and prolonged complete remission (CR) rates in patients with hairy-cell leukemia (HCL). However, 2-CdA administration often results in severe neutropenia and lymphocytopenia both responsible for the infectious complications observed in these patients. We previously reported preliminary data regarding the effectiveness and toxicity of a modified protocol of 2-CdA administration (0.15 mg/kg 2 hours infusion once a week for 6 courses) in 25 HCL patients. This treatment schedule produced a similar overall response rate compared to standard 2-CdA regimen and appeared to be followed by a lower incidence of infectious episodes. In the present study we report response rate and toxicity of weekly 2CdA administration in a larger cohort of patients and with a longer follow-up. DESIGN AND METHODS: In a group of HCL patients with a pronounced decrease in neutrophils count (< 1 x 10(9)/L), we modified the standard protocol (0.1 mg/kg daily x 7 days c.i.) by administering 2-CdA at a dose of 0.15 mg/kg 2 hours infusion once a week for 6 courses. Thirty HCL patients, 24 males and 6 females with a median age of 56 years (range 37-76), entered into this protocol. Seventeen out of 30 patients were at diagnosis while the remaining 13 had been previously treated with alpha-interferon (alpha-IFN) (7), or 2-CdA (4) or deoxycoformycin (DCF) (2). RESULTS: Overall, 22/30 (73%) patients achieved CR and 8 (27%) partial remission (PR) with a median duration of response at the time of writing of 35 months, ranging from 6 to 58 months. Five patients (1 CR and 4 PR) have so far progressed. The treatment was very well tolerated. Five out of 30 patients (16%) developed severe neutropenia (neutrophils < 0.5 x 10(9)/L) and only in two of them we did register an infectious complication which required treatment with systemic antibiotics and granulocyte colony-stimulating factor (G-CSF). INTERPRETATION AND CONCLUSIONS: In conclusion, we confirm that weekly administration of 2-CdA at a dose of 0.15 mg/kg for 6 courses appears to be very effective in HCL inducing a high CR rate, similar to that observed with daily c.i. administration. CR durability and relapse/progression rates are also comparable to standard 2-CdA schedule. Moreover this new regimen seems to be safer in pancytopenic patients, markedly reducing life-threatening infectious complications.

P-glycoprotein, lung resistance-related protein and multidrug resistance associated protein in de novo acute non-lymphocytic leukaemias: biological and clinical implications.

P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance associated protein (MRP) expression and the blast cells' intracellular daunorubicin accumulation (IDA) were evaluated in 96 previously untreated cases of de novo acute non-lymphocytic leukaemia (ANLL). 47/96 patients (49%) were classified as PGP+ 44/ 96 (46%) as LRP+, and 8/96 (8%) as MRP+. The more frequent MDR clusters were PGP-/LRP-/MRP- (32/96 cases, 33%) and the PGP+/LRP+/MRP- (27/96 cases, 28%) followed by PGP+/LRP-/MRP- (15/96 cases, 16%) and PGP-/LRP+/MRP- (14/96 cases, 14%). A favourable karyotype was observed more frequently in PGP- and LRP-cases. A highly significant correlation was found between either PGP or LRP overexpression and leukaemic blast cell IDA. All the patients received standard induction and consolidation treatments containing MDR-related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs. Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival. Other independent prognostic factors were age and cytogenetics. LRP overexpression did not reach statistical significance, although for LRP+ cases the trend was unfavourable. Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission. This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare. It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value. It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine.

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 microg/kg/day x 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-gamma (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 Thl alloresponses, maintain efficient alloreactivity to HLA mismatched antigens and have impaired NK activity.

P-glycoprotein (PGP), and not lung resistance-related protein (LRP), is a negative prognostic factor in secondary leukemias.

BACKGROUND AND OBJECTIVE: In cell lines, there is an ongoing debate about the role of the lung resistance-related protein (LRP) whereas the role played by P-glycoprotein (PGP) in determining a multidrug resistance is well known. The aim of this study was to evaluate the frequency and the role of a PGP and an LRP overexpression in affecting the intracellular daunorubicin accumulation (IDA) and in predicting the therapy outcome on a subset of overt secondary acute non lymphocytic leukemias (ANLL). An adjunctive point was to evaluate the efficacy of the reversal agent SDZ PSC 833 (PSC) in counteracting impaired IDA. DESIGN AND METHODS: By flow cytometry, PGP and LRP expression and the IDA were evaluated on 54 overt secondary ANLL PGP and LRP overexpressions were respectively defined by an MRK-16 mean fluorescence index (MFI) > or = 6 (PGP+) and by an LRP-56 MFI > or = 5 i.e. by MRK-16 and LRP-56 MFIs higher than the one observed in normal leukocytes. The blasts' IDA was studied after a two-hour incubation in 1000 ng/mL daunorubicin in the presence or in the absence of the MDR reversal agent SDZ PSC 833 (PSC) 1.6 mumol. RESULTS: A PGP overexpression was detected in 40/54 (74%) cases while an LRP overexpression was observed on 33/54 (61%) cases. No differences were found in terms of PGP and LRP expressions between ANLL developing after chemo/radiotherapy (therapy-related ANLL) or evolving from a myelodysplastic syndrome (MDS-related ANLL). Compared to the PGP-, the PGP+ cases showed a significantly lower mean IDA (DNR NMFI 196 +/- 46 vs. 267 +/- 53, p < 0.001). The co-incubation of DNR with the PSC significantly increased only the mean IDA of the PGP+ cases, that grew from a DNR NMFI of 196 +/- 46 to a DNR NMFI of 284 +/- 67 (p < 0.0001). With respect to normal leukocytes, even the PGP- cases had an impaired IDA suggesting that other mechanisms, including an LRP overexpression, could affect the IDA. A strongly negative correlation was observed between PGP overexpression and therapy outcome, in fact, 8/10 (80%) PGP- but only 2/27 (7%) PGP+ patients obtained complete remission (p = 0.0002). Moreover, 7/33 (21%) cases showing an impaired IDA (NMFI < 280) but 4/4 (100%) with NMFI > 280 had complete remission (p = 0.006). No correlation was found between therapy response and LRP or CD34 expression. INTERPRETATION AND CONCLUSIONS: This data suggests that an important role in determining therapy outcome is played by PGP in secondary leukemias. Even if the LRP is frequently overexpressed in secondary leukemias and is likely to contribute to the reduction of the intracellular drug accumulation, the role played by LRP in determining the therapy-outcome has still to be cleared.

Incidence and prognostic relevance of CD34 expression in acute myeloblastic leukemia: analysis of 141 cases.

In 141 adult patients with diagnosis of acute myeloid leukemia the overall expression and intensity of expression of CD34 antigen on leukemic cells was investigated. Myeloid blasts were tested by applying direct immunofluorescence staining using anti-CD34 fluorescein monoclonal antibody in flow cytometry. CD34 antigen was found in 73 out of 141 (51%) cases and in particular in M0, M1 and M4 French-American-British (FAB) cytotypes, while M3 and M5 cases were rarely positive. In patients whose blasts expressed CD34 antigen a significantly lower rate of complete remission (CR) was observed as opposed to CD34 negative cases (61% vs 88%) (P = 0.001). Furthermore, a negative correlation between high intensity of CD34 expression, measured as a mean fluorescence index (MFI), and CR rate was observed. In particular, patients with a higher CD34 fluorescence intensity (MFI > 23), showed a further reduction in CR rate (48%). Also, these patients had a significantly lower overall survival (P = 0.03) as compared to patients with no expression of CD34 and patients with CD34 MFI < 23. In conclusion, these findings confirm that CD34 expression is frequently associated with "immature" FAB cytotypes (M0, M1 and M4) and with a reduced probability to achieve CR. Furthermore, a high CD34 intensity of expression should be considered as a reliable poor prognostic factor.

Amplification of third-complementary-determining-region (CDR-III) of heavy chain immunoglobulin gene (IgH) in one hundred adult acute leukemias.

We applied a simple polymerase chain reaction (PCR) based method for detecting immunoglobulin heavy-chain (IgH) gene rearrangement, using its CDR-III region to assess B-cell clonality in a series of 100 acute lymphoblastic leukemias (ALL) (84 B-cell lineage, 4 null-ALL and 12 T-ALL). The amplified CDR-III regions can be generated in all the B-lineage ALL and separated by size by polyacrylamide gel electrophoresis (PAGE), thereby providing a specific diagnostic marker for each B cell clone. Size heterogeneity resulting from independent IgH rearrangement events and the high resolution power after electrophoresis and silver staining of the PAGE gels can be used to generate a "fingerprint" of the PCR fragments representing either the spectrum of B-cell clonality in complex populations of B lymphocytes or the partially genomic configuration of the VH-N-DH-N-JH region. At diagnosis, we found the presence of one clonal IgH heavy-chain gene rearrangement in 80 B-cell lineage and null ALL and a biclonal rearrangement in 8 cases. The CDR-III bands were of sizes ranging from 80 to 130 bp. The PCR analysis of the IgH gene enabled us to obtain a CDR-III leukemia specific product in all cases, thereby providing a specific and diagnostic marker for each B-cell clone.

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 alpha-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 x 10(9)/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.

Hairy-cell leukemia and alpha-interferon treatment: long-term responders.

BACKGROUND AND OBJECTIVE: In the 1980s alpha-interferon (alpha-IFN) dramatically improved the management of hairy cell leukemia (HCL), producing normalization of hematologic parameters including the disappearance of circulating hairy cells in the majority of treated patients, within 6 months. The quality and durability of the response depended on the duration of alpha-IFN treatment; progression of the disease consistently followed discontinuation of alpha-IFN. In this report, we examine the characteristics of long-term responders from our series of 44 HCL patients treated with alpha-IFN. METHODS: We report follow-up data on 44 HCL patients who underwent alpha-IFN as first-line treatment between 1985 and 1990. The alpha-IFN dose was 3 x 10(6) U daily for 12-15 months, with 20 patients continuing to receive the same dose three times a week as maintenance treatment for an additional 6-12 months. Of the 44 patients, 8 achieved a CR, 28 a PR and 8 a MR, with an overall response rate of 82%. Thirty-eight (86%) of these patients showed disease progression and were retreated with alpha-IFN (2 pts), 2-chlorodeoxyadenosine (35 pts), or pentostatin (1 pt). So far, all 38 patients are alive and in good unmaintained second response, except for two patients who developed a second neoplasm. RESULTS: Six of the 8 first complete responders are alive and have not required further treatment after completing alpha-IFN. These long responders most often (5/6) presented a hairy cell index (HCI) < 0.50 at diagnosis; all 6 registered a significant reduction in bone marrow infiltration (HCI < 0.10) after induction therapy and underwent alpha-IFN maintenance treatment. These three parameters turned out to be statistically significant when the long-term responders were compared with the failure patients subset (p = 0.003 for HCI at diagnosis; p = 0.001 for HCI at the end of the induction phase; p = 0.003 for the maintenance phase). The median progression-free survival of these 6 long-term responders was 75 months (range, 62 to 78). INTERPRETATION AND CONCLUSIONS: Overall, alpha-IFN represents an excellent palliative treatment for most HCL patients. A small subset of these patients could become long-term responders following first-line alpha-IFN therapy alone.