Effective in vivo induction of NKG2D ligands in acute myeloid leukaemias by all-trans-retinoic acid or sodium valproate.

Engagement of NKG2D by their ligands (NKG2D-L), as the human major histocompatibility complex class I-related molecules MIC-A and the UL16-binding proteins, on cytolytic lymphocytes leads to the enhancement of antitumour effector functions. These ligands are missing or expressed at very low levels on leukaemic cells; furthermore, they can be shed by tumour cells and inhibit cytolytic activity of lymphocytes. Herein, we show that in vivo administration of all-trans-retinoic acid (ATRA) or the histone deacetylase inhibitor sodium valproate (VPA) to patients affected with acute myeloid leukaemia (AML) M3 or M1 respectively, leads to the induction of transcription and expression of NKG2D-L at the surface of leukaemic cells. Apparently, no detectable shedding of the soluble form of these molecules was found in patients' sera. Conversely, AML blasts from patients treated with chemotherapy not including ATRA or VPA did not show any induction of NKG2D-L transcription. Furthermore, upon therapy with ATRA or VPA, leukaemic blasts become able to trigger lytic granule exocytosis by autologous CD8(+) T and natural killer lymphocytes, as shown by CD107a mobilization assay, followed by leukaemic cell lysis. These findings indicate that ATRA and VPA may contribute to the activation of cytolytic effector lymphocytes in vivo, possibly enhancing their anti-leukaemic effect.

In vivo apoptosis of CD8(+) lymphocytes in acute myeloid leukemia patients: involvement of soluble HLA-I and Fas ligand.

In this study, we show that high serum levels of soluble human leukocyte antigens (HLA) class I molecules (sHLA-I, range: 0.7-1.7 micro g/ml) and soluble Fas ligand (FasL, range: 0.4-1.9 ng/ml) are detected in patients with acute myeloid leukemia (AML) at diagnosis, compared with healthy donors (HD) (sHLA-I, range: 0.1-0.6 micro g/ml; sFasL, range: 0.1-0.4 ng/ml). Patients' sera were able to induce transcription and secretion of FasL in CD8(+) T cells, followed by apoptosis in vitro; this apoptosis was inhibited by anti-HLA-I-specific monoclonal antibodies, suggesting that sHLA-I is responsible for cell death. These findings closely relate to the in vivo upregulation of FasL transcription observed in peripheral blood (PB) lymphocytes from AML patients; in the same cells, mRNA for the antiapoptotic proteins Bcl-2 and Bcl-x(L) was downregulated. Interestingly, caspase-8 and caspase-3, both downstream mediators of death receptor-induced apoptosis, were activated in CD8(+) cells of AML patients; one-third of these cells were already apoptotic in vivo, at variance with lymphocytes of HD. These data strongly suggest that in AML, increased levels of sHLA-I molecules may contribute to the elimination of potentially anti-tumor effector cells through a FasL/Fas interaction.

Allogeneic bone marrow transplantation (BMT) for adults with acute lymphoblastic leukemia (ALL): predictive role of minimal residual disease monitoring on relapse.

We developed a PCR-based method to monitor clonogenic IgH VDJ rearrangement as a possible predictor of relapse in patients with acute B-ALL after allogeneic bone marrow transplantation (BMT). We studied 23 patients at diagnosis, before and after BMT. At the time of BMT, 13 patients were in first complete remission, eight in second complete remission and two in relapse. Four patients were PCR negative before BMT and remained PCR negative also after BMT (-/- pattern). They are still in remission after a median follow-up of 41 months. Nineteen patients were MRD-positive before BMT: three were PCR negative at first determination after BMT (+/- pattern) and maintain remission. Sixteen patients were PCR-positive at first determination after BMT (+/+ pattern): five became PCR negative (+/+/- pattern) (four with chronic graft-versus-host disease (GVHD) and two after donor lymphocyte infusions (DLI)). Nine patients remained PCR-positive (+/+/+ pattern) (four remain in remission, and six relapsed); two patients died before transplant. In conclusion, PCR negative patients before BMT remained negative post-BMT; many pre-BMT positive patients had initial MRD positivity after BMT: 37% of them achieved a molecular remission with cGVHD or DLI.

Liposomal daunorubicin (DaunoXome) for treatment of poor-risk acute leukemia.

Toxicity limits the use of anthracyclines in elderly sick patients and in heavily pretreated patients. Since the liposomal preparation of daunorubicin (DNR) (DaunoXome, or DNX) is expected to be less toxic than conventional DNR, we tested DNX combined with high-dose arabinosyl cytosine (HDAC) in 42 adult poor-risk acute leukemia patients. Thirty-one patients had acute non-lymphocytic leukemia (ANLL). Of these, 12 patients were newly diagnosed but were not eligible for standard induction treatment, 13 were in first relapse, and 6 were in second or subsequent relapse. Eleven patients had acute lymphocytic leukemia (ALL), in first (eight cases) or second (three cases) relapse. DNX was given i.v. in three doses of 80 or 100 mg/m(2) each (days 1-3) by a 60-min infusion in glucose 5%, followed by a 4-h infusion of HDAC 2 g/m(2) (days 1-5). Among 31 ANLL patients there were 16 (51%) complete remissions (CR), 5 deaths during induction, and 10 failures. Among 11 ALL patients there were 10 CRs and 1 failure. The response rate was not affected by the overexpression of MDR-related proteins (PgP, MRP-1, and LRP). Non-hemopoietic toxicity was negligible, with no intestinal toxicity and only one case of gram-negative bacteremia. We conclude that DNX, in combination with HDAC, is an effective treatment for poor-risk adult AL. Because of the low non-hematologic toxicity, it can be used to reinduce remission in poor-risk patients who are candidates for allogeneic bone marrow transplantation. The high CR rate observed in ALL requires confirmation.

Fludarabine, ARA-C, idarubicin and G-CSF (FLAG-Ida), high dose ARA-C and early stem cell transplant. A feasable and effective therapeutic strategy for de novo AML patients.

Forty-three consecutive patients with de novo and untreated non M3 AML aged 60 or less entered the study. The mean age of patients was 50 (range 15-60). The induction regimen (FLAG-Ida) included fludarabine (30 mg/sqm), Ara-C (2 g/sqm) on days 1-5, and idarubicin (10 mg/sqm) on days 1, 3, 5. G-CSF (300 mcg/day) was administered s.c. 12 hours before starting fludarabine and was continued for five days. HDT with stem cell rescue was planned for all patients in first CR after one course of high dose Ara-C (HDAC) consolidation and in good clinical conditions. Forty-two (98%) patients were evaluable for response. One patient died during induction (2%). CR was achieved in 35 patients (82%). Twenty-three patients, 66% of those achieving CR, underwent autologous (N = 17) or allogeneic (N = 6) transplantation. With a median follow up of 24 months, the average median duration of CR is 17 months (range 3-66) and the median survival is 20 months (range 1-83). Overall the 5 year projected disease free survival (DFS) and overall survival (OS) were 37% and 43%, respectively. Among patients who underwent stem cell transplantation DFS and OS were 53% and 69%, respectively. The median time to PMN recovery (> 0.5 x 10(9)/l) was 17 days (range 10-28) and 50 x 10(9)/l platelets were reached at a median of 17 days (12-38). In conclusion FLAG-Ida regimen is effective, low toxic and improves feasibility of stem cell transplant.

Leukocyte-associated Ig-like receptor-1 prevents granulocyte-monocyte colony stimulating factor-dependent proliferation and Akt1/PKB alpha activation in primary acute myeloid leukemia cells.

The leukocyte-associated Ig-like receptor-1 (LAIR-1), a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motif (ITIM) is expressed on acute myeloid leukemia (AML) blasts isolated from peripheral blood or bone marrow of 17 patients (2 M0, 3 M1, 5 M2, 2 M4 and 5 M5 according to French, American and British classification). Further, we provide evidence thatLAIR-1 engagement inhibits granulocyte-monocyte colony-stimulating factor (GM-CSF)-induced proliferation of AML blasts. Indeed, leukemia cells stimulated with GM-CSF were blocked in the G0/G1 phase of the cell cycle and underwent apoptosis within 4 days after the engagement of LAIR-1. Remarkably, LAIR-1 was functional also in AML blasts which do not express CD33, mainly M4 and M5. Importantly, the LAIR-1 ligation led to a strong inhibition of both GM-CSF receptor-mediated intracellular calcium increases, phosphorylation and activation of Akt1/protein kinase B alpha, a substrate of the phosphatidylinositol-3 kinase. This last inhibitory effect was prevented by a synthetic peptide spanning the ITIM portion of LAIR-1, suggesting the involvement of SHP-1 phosphatase in LAIR-1-mediated inhibitory signal. Altogether, these findings indicate that the engagement of LAIR-1 can down-regulate GM-CSF-mediated survival and proliferation of AML blasts, suggesting an additional therapeutic approach to the treatment of AML patients.

Lymphoplasmacytic lymphoma/immunocytoma: towards a disease-targeted treatment?

Lymphoplasmacytic-lymphoplasmacytoid lymphoma (LPL)/Waldenstrom's macroglobulinemia (WM) or immunocytoma (IMC) consists of diffuse proliferation of small mature B lymphocytes, plasmacytoid lymphocytes, and plasma-cells. The nosographic definition includes the lack of histological, immunophenotypic, cytogenetic, and molecular markers considered specific of other types of lymphoma. The cells show surface Ig (usually IgM), B-cell-associated antigens and display the CD5-, CD23- and CD10- phenotype, which allows for differential diagnosis from B-CLL and mantle cell lymphoma. t(9;14)(p13;q32) chromosomal translocation has been found in 50% of all LPL cases. The cytogenetic rearrangement juxtaposes the PAX-5 gene, which encodes for an essential transcription factor for B-cell proliferation and differention, to the Ig heavy chain gene. The combination of chlorambucil and prednisone holds as the standard treatment and seems to guarantee good control of the disease in most patients. Similar therapeutic results have been described with the combination of cyclophosphamide, vincristine, prednisone with (CHOP) or without doxorubicin (CVP), or with a combination of other alkylating agents and prednisone. Nucleoside analogues, alone or in combination with alkylating agents and anthracyclines, provide good salvage therapy for IMC and being increasingly employed as first line therapy. In a multicentric European trial Foran et al. administered the chimeric anti-CD20-monoclonal antibody (Rituximab) to 28 patients with previously treated IMC. Seven out of 25 evaluable patients (28%) achieved a partial response. Byrd et al. examined the outcome of 7 previously treated WM patients who received weekly infusions of rituximab (375 mg/m2). Therapy was well tolerated by all patients, and there was no decrease in cellular immune function, or significant infectious morbidity. Partial responses were noted in three of these patients, including two with fludarabine-refractory disease. These data suggest that rituximab exerts clinical activity on heavily pre-treated patients with WM. Furthermore, Weide et al. first reported that WM-associated polyneuropathy can be treated effectively with a combination of chemotherapy and the anti-CD20 monoclonal antibody rituximab. Most published trials exploring the efficacy of high dose treatment as salvage therapy for relapsed or refractory low grade non Hodgkin's lymphoma have included prevalently follicular or lymphocytic lymphomas. In selected high risk patients radioimmunotherapy with autologous stem-cell rescue, and myeloablative therapy followed either by autologous stem cell transplantation (SCT) or allogeneic SCT might represent an alternative strategy.

Thalidomide in agnogenic and secondary myelofibrosis.

Myelofibrosis with myeloid metaplasia (MMM) is a clonal disorder involving disregulation of angiogenesis and immunomodulatory mechanisms. Thalidomide (Thal) retains antiangiogenic, immunomodulatory and cytokine regulatory properties and recently it has been used successfully in multiple myeloma. Here, we report our experience in 10 MMM patients treated with Thal. Patients with agnogenic MMM treated in an early phase of the disease obtained significant benefits from the therapy and remain transfusion-free. In contrast, all secondary MMM failed to respond. These preliminary findings confirm that Thal plays a role in MMM therapy, although the efficacy in the different phases of the disease must be further evaluated.

Cost of de novo acute myeloid leukemia induction therapy in adults: analysis of EORTC-GIMEMA AML10 and FLANG regimens.

Since the social and financial impact of AML therapy is becoming more and more relevant we analyzed the cost of induction therapy of two different regimens. The first one is part of the widely employed EORTC-GIMEMA AML-10 and consists often days of therapy. The second (FLANG) is a short (three day), Fludarabine, Ara-C, mitoxantrone and G-CSF containing regimen. We first retrospectively analyzed the outcome of 77 consecutive AML patients with comparable clinical and haematological features receiving FLANG (25) or AML-10 (52), between June 1993 and October 1999, and observed equivalent CR rate, as well as DFS and overall survival duration. We then selected 9 non pretreated patients per group who reached CR after one course of therapy. Patients treated with FLANG had a statistically significant earlier platelet recovery compared to those treated with AML-10, fewer days of intravenous antibiotic therapy (14/22, respectively, p < 0.05), and a shorter hospitalization period (22/33 days, p < 0.01). FLANG was significantly more expensive than AML 10 as far as the cost of antiblastic drugs (p < 0.01) and G-CSF support (p < 0.05) are concerned. On the contrary, the expense for antiemetic drugs (p < 0.01) and the cost of personnel and other services ($5,906/$3,970, p < 0.05) were higher for AML-10 than for FLANG. Overall, the average costs of FLANG and AML10 were $9,269 and $12,424 respectively (p < 0.05; difference = -25%). Our study seems to indicate that, compared to AML-10, FLANG induction is as effective, less expensive and it allows for a decrease in the length of hospitalization and thus for better exploitation of the financial resources of Hematology-Oncology departments.

Molecular analysis of patients with relapsed or refractory intermediate-high grade non-Hodgkin's lymphoma with bone marrow infiltration undergoing peripheral blood progenitor cell transplantation.

BACKGROUND AND OBJECTIVES: IgH gene rearrangement studies with a polymerase chain reaction (PCR) technique can detect the persistence of clonal cells at molecular level during the remission phase. This persistence of clonal cells can be used to establish the relationship between minimal residual disease (MRD) and clinical outcome. We have developed a three-step single strand conformational polymorphism PCR strategy which is able to detect clonal B lymphoid cells at a frequency as low as 1 clonal cell in 10(6) normal cells. DESIGN AND METHODS: Twenty patients with intermediate or high-grade B non-Hodgkin's lymphoma (NHL) were evaluated. Patients were pre-treated with a median of two (range 1-4) conventional chemotherapy lines before high-dose cyclophosphamide (HDCY). All patients had their bone marrow (BM) involved by disease (median 10%; range 5-50%). Nineteen patients were offered high-dose therapy followed by peripheral blood progenitor cells (PBPC) autografting. RESULTS: MRD analysis was performed for each patient at the end of conventional chemotherapy and every three months after high dose therapy. All these patients achieved complete response (CR) after high dose therapy (HDT). Six patients relapsed after a median time of 24.5 months. All the studied apheresis samples were positive at the molecular analysis. All 6 patients still positive at the molecular analysis after PBPC autografting relapsed. The remaining 13 patients who were negative maintained CR. INTERPRETATION AND CONCLUSIONS: Whereas the detection of clonal cells in the apheresis samples did not predict an unfavorable outcome, the disappearance of the clonal rearranged band from the BM sample after HDT proved to be a favorable prognostic factor and was associated with long-lasting disease-free status

First line therapy with fludarabine combinations in 42 patients with either post myelodysplastic syndrome or therapy related acute myeloid leukaemia.

Acute myeloid leukaemias (AML) evolving from a myelodysplastic syndrome (MDS) or secondary to chemoradiotherapy frequently display unfavorable biologic characteristics. This may explain the lower remission rate obtained with conventional chemotherapy. Recently, the association of Fludarabine with intermediate dose Ara-C has produced interesting results particularly in high risk AML patients. Here, we report on 42 secondary AML patients treated with a combination of Fludarabine, intermediate dose Ara-C, G-CSF with or without an antracycline (FLANG, FLAG-IDA or FLAG). Overall, complete remissions (CR) were documented in 14 patients (33%) and partial responses (PR) in 12 (29%), while 10 patients proved resistant (24%). Six patients (14%) died early. The presence of a prognostically unfavorable karyotype had a negative impact on the CR rate (20% compared to 50% for patients with an intermediate prognosis karyotype, p 0.05). Patients treated with FLAG, FLANG and FLAG-IDA had similar CR rates. At the time of this analysis, after a mean follow-up of 12 months, the mean duration of CR is 16 months (range 3-66) and the mean survival is 11 months (range 1-67). The median time to granulocyte recovery (neutrophils > 0.5 x 10(9)/l) was 20 days (range 12-39) and 50 x 10(9)/l platelets were reached at a median of 26 days (range 9-56). Taken together, these Fludarabine containing regimens proved to be an effective and tolerable treatment for patients with secondary AML. Patients above 70 years of age may also benefit from this therapy, however the problem of treating patients with adverse chromosomal abnormalities still remains unresolved.

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.

Detection of minimal residual disease in B-lymphoproliferative disorders: a three step SSCP-PCR method.

The most recent therapeutic approaches can improve the outcome of B-cell neoplasia. By PCR analysis we amplify tumor specific DNA sequences of clonal IgH rearrangement from a limited number of malignant cells against a background of normal B cells. Recently described PCR based techniques for tracking minimal residual disease (MRD) in B lymphoproliferative disorders have given promising but discordant results, with significant variations in the sensitivity and specificity of the procedures. We have developed a three step single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) strategy which is able to detect clonal malignant cells in B lymphoproliferative disorders at a frequency as low as 1 in 10(6) cells. Since this method is simple, rapid, reliable and as specific as ASO-PCR, it could be especially useful in monitoring patients affected by B lymphoproliferative disorders in complete haematological and immunophenotypic remission.

GM-CSF, ARA-C, VP-16 and idarubicin (GM-IVA), a short, and effective induction treatment for de novo AML, suitable for the elderly.

GM-IVA is a short and effective induction therapy of non M3 de novo AML including GM-CSF (300 mcg 12 hrs before starting therapy), Ara-C (250 mg/sqm c.i. x 3 days), VP16 (100 mg/sqm x 3 days) and idarubicin (12 mg/sqm x 3 days); it was followed by a fludarabine containing salvage protocol (FLANG). Patients <60 years of age achieving CR received 2 courses of FLANG and autologous or allogeneic BMT when possible. Patients >60 years of age in CR received a second course of GM-IVA. Twenty-one consecutive patients (mean age 64, range 29-85) entered the study. Three patients (14%) died during induction therapy. After one course of GM-IVA, CR was achieved in 12 patients (57%). Two further patients were salvaged with FLANG therapy so that the final CR rate was 14/21 (67%). In elderly patients the final CR rate (62%) is noteworthy, considering that 6 patients were >70 years of age and 3 were >80. All three patients >80 achieved CR (lasting 5 to 7 months). The median time of granulocyte and platelet recovery was 15 days. Our scheme was well tolerated. In the group of elderly patients 3 out of 14 died during induction (21%) and 4 life-threatening infections were observed (28%). The short duration of cytotoxic therapy and perhaps the use of G-CSF contributed to a reduction of the hospitalization period (median of 22 days), thus providing major savings on induction costs and allowing for better utilization of beds as well as significantly improving patients' quality of life.

First line Fludarabine treatment of symptomatic chronic lymphoproliferative diseases: clinical results and molecular analysis of minimal residual disease.

Fludarabine (25 mg/m2 for 5 d, every 4 wk, for 6 courses) was administered as first line therapy in 32 symptomatic chronic lymphoproliferative diseases. All CLL patients achieved at least partial response (5 CR, 2 nPR, 9 PR) but 44% of patients relapsed. In LG-NHLs response and relapse rate were similar. Haematological toxicity was low. VDJ rearrangement PCR analysis was performed on marrow samples at diagnosis and at the time of response evaluation. In the 3 patients who underwent high dose therapy with peripheral blood progenitor cell rescue analysis was also performed on apheresis samples and on marrow samples at the end of the procedure. Clonal VDJ rearrangement was always evident after Fludarabine therapy even in those patients who achieved histological and immunophenotypic complete remission, whereas it disappeared in 2 of 3 patients who underwent HDT. Our data confirm that Fludarabine monotherapy can reduce the neoplastic mass to a subclinical level and suggest the possibility that high dose therapy might produce true complete remission.

High efficacy of fludarabine-containing therapy (FLAG-FLANG) in poor risk acute myeloid leukemia.

BACKGROUND: Elderly patients with acute myeloid leukemia (AML) those refractory to induction chemotherapy and those with so-called secondary leukemia have unfavorable prognoses and require innovative therapeutic approaches. Fludarabine allows an increased accumulation of Ara-CTP in leukemic cells and inhibits DNA repair mechanisms; therefore its association with Ara-C and mitoxantrone results in a synergistic effect. MATERIALS AND METHODS: From May 1993 to February 1996, fludarabine-containing regimens (FLAG and FLANG) were employed as induction therapy in 51 high-risk AML patients. Diagnosis of AML in 22 patients was preceded by a myelodysplastic syndrome lasting more than six months; 8 of the 29 de novo AML cases (28%) were refractory to previous chemotherapy, 9 (31%) were treated for early relapse, 12 (41%) presented poor prognostic factors at diagnosis. The median age was 64 (range 33-76) years and the FAB subtypes were the following: M0 3, M1 5, M2 28, M4 7, M5 8. Forty-eight per cent of patients showed poor prognosis chromosomal abnormalities. FLAG (24 patients) consisted of both fludarabine 30 mg/sqm over 30 minutes followed 4 hours later by Ara-C 2 g/sqm over 4 hours (for 5 days) and G-CSF 300 micrograms/day administered 12 hours before fludarabine, for a total of 5 doses. FLANG (27 patients) had a shorter duration (3 days), reduced Ara-C dosage (1 g/sqm) and administration of mitoxantrone (10 mg/sqm) at the end of Ara-C infusion. RESULTS: Recovery of both neutrophils (PMN > 0.5 x 10(9)/L) and platelets (Plt > 20 x 10(9)/L) required a median of 16 days from the end of therapy. Overall, 30 patients (59%) achieved CR, 6 (11%) PR and 10 (20%) were refractory; 5 (10%) experienced early death (cerebral hemorrhage or infection). The length of complete response ranged from 2 to 26 months with a median follow-up of 8 months. De novo and secondary AML registered 62 and 54% CR rates, respectively. Eight out of 10 patients refractory to conventional schemes achieved CR (80%) but only 3 out of 10 treated for relapse obtained CR (30%). CONCLUSIONS: FLAG and FLANG showed similar activity and toxicity while proving to be highly effective and relatively well-tolerated treatments for high-risk de novo AML. Secondary leukemias seemed to be responsive as well, but the presence of an unfavorable karyotype alteration lowered the response rate.

Anaplastic large cell lymphoma: a clinicopathologic study of 53 patients.

Fifty-three consecutive cases of adult CD30+ anaplastic large cell lymphoma (ALCL) have been analyzed. Thirty-six were classified as Hodgkin's disease like variety (HL) (67%) and seventeen as so-called common type (CT) (33%). All cases strongly expressed the CD30/Ki-1 antigen; the neoplastic cells expressed CD15, CD45 and EMA in 60%, 44% and 33% of cases, respectively; T. B and null phenotypes were found in 37%, 17% and 46% of cases. Bulky mediastinal, B symptoms, and extranodal disease at diagnosis were present in 36%, 49% and 25% of cases. EBV encoded latent membrane protein (LMP-1) was found in 10 cases. Of the 13 tested cases only 4 expressed a weak positivity of the CD40 molecule, in a fraction of the tumor cells; in the same cases CD21 was never found. Patients were treated with various protocols; of the 50 evaluable patients, 39 (78%) obtained a complete remission (CR), 3 (6%) a partial remission (PR) and 8 (16%) did not respond. The projected overall disease free survival (DFS) at 36 months is 70%. Only patients with advanced disease stage (III-IV) showed a statistically decreased DFS and survival. Only symptomatic and extranodal disease significantly appeared to influence survival. This study confirms the good outcome of this group of lymphomas and differs from other reports for some clinical (lower percentage of advanced stage, extranodal disease and skin infiltration) and pathological (HL/CT ratio and immunophenotype) features.

Detection of platelet-associated antibodies by flow cytometry in hematological autoimmune disorders.

We describe our experience in the evaluation of platelet-associated immunoglobulins (PAIg) by flow cytometry in comparison to solid-phase assay in patients affected by idiopathic thrombocytopenic purpura and by Evans syndrome. Results show that the analysis of PAIg by flow cytometry is easy and reliable and correlates well with data obtained by the solid-phase technique. In addition, flow cytometry allows the evaluation of samples containing small numbers of platelets (< 20,000/mm3); the analysis is objective, not influenced by personal experience. Moreover, flow cytometry appears simple enough to be performed in a routine laboratory, and data might be retrieved to perform batch analysis. Our results appear to indicate that PAIg flow cytometry might be a sensitive tool for the evaluation of patients with autoimmune thrombocytopenia.