ALBERT

Description: There is increasing evidence that the immune system can promote cytopenia in myelodysplastic syndromes (MDS) to a variable extent. The interaction between CD40 and its natural ligand CD40L (CD154) plays a critical role in the regulation of humoral and cellular immune responses. The aim of this study was to investigate the role of the CD40/CD40L interaction in the pathogenesis of MDS. For this purpose, we looked at the presence of the CD40 receptor and its ligand in MDS peripheral blood (PB) and bone marrow (BM), and we studied the in vitro effects of inhibition and stimulation of the CD40 receptor on MDS monocytes. 1) Expression of CD40/CD40L. We analysed PB samples of 40 patients with MDS (19 RCMD, 15 RCMD-RS, 3 RAEB-1, 3 5q- syndrome) and 29 age-matched control subjects by flow cytometry. We observed that MDS patients have significantly higher percentages of circulating CD40+/CD14+ cells (8.34 % +/− 1.28 vs. 2.78 % +/− 0.31, p=0.0004) and CD40L+ T helper cells (4.44 % +/− 0.52 vs. 2.16 % +/− 0.22, p=0.001). In addition, we found higher percentages of CD40+/CD14+ cells (9.47 % +/− 1.96 vs. 3.53 % +/− 0.63, p=0.03) and CD40L+ T helper cells (5.13 % +/− 0.86 vs. 2.60 % +/− 0.53, p= 0.008) in BM of 22 patients (1 RA, 11 RCMD, 4 RCMD-RS, 5 RAEB-1 and 1 5q- syndrome) compared to 11 donor samples. 2) Stimulation of CD40. Selected CD14+ cells from PB of 21 patients (1 RA, 12 RCMD, 6 RCMD-RS, 2 RAEB-1) and 25 control subjects were stimulated for 24h with LPS or an agonistic anti-human CD40 antibody (clone 64, PanGenetics NV, The Netherlands). We observed that anti-CD40 induced significantly higher TNF-alpha levels in patients than in controls (436 vs. 64 pg/mL, p=0.008), whereas LPS induced comparable TNF-alpha levels. 3) Blocking of CD40. We cultured 5x105 BM mononuclear cells of 22 patients (1 RA, 12 RCMD, 3 RCMD-RS, 4 RAEB-1 and 2 5q- syndrome) in methylcellulose supplemented with growth-factors in the presence or absence of 5D12 (antagonist chimeric monoclonal anti-human CD40 antibody, PanGenetics NV, The Netherlands). The presence of 5D12 significantly increased colony-formation at day 14 (140 vs. 112, p=0.002). 5D12 did not affect colony formation in CD14-depleted cultures, nor in controls. In conclusion, we have demonstrated elevated levels of CD40 receptor on PB and BM monocytes of patients with MDS compared to age-matched control subjects. In addition, we have demonstrated increased levels of its ligand CD40L on T helper cells in PB and BM. Stimulation of CD40 on monocytes from patients induces significantly higher TNF-alpha levels, a cytokine known to inhibit colony formation. Moreover, we demonstrated that inhibition of this CD40 receptor with 5D12 can increase colony formation and that its effect is specific monocyte-dependent. Our observation together with the proven safety of 5D12 in phase 1 trials for patients with inflammatory bowel disease, might justify a phase I trial of 5D12 to improve cytopenia in patients with MDS.

Description: Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph−) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph− progenitors are highly enriched in the CD34+HLA-DR− fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph− progenitor or stem cells, leaving the unanswered question whether Ph−CD34+HLA-DR- progenitors are normal. To show the clonal nature of Ph−CD34+HLA-DR− CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34+HLA-DR+ and CD34+HLA-DR− progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA−, CD34+HLA-DR−progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34+HLA-DR− derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph−, BCR/ABL mRNA−and polyclonal state in the CD34+HLA-DR−and CD34+HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34+ cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph−, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34+HLA-DR− cells that are Ph− and BCR/ABL mRNA− are polyclonal and therefore benign. This population is suitable for autografting in CML.

Description: Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph−) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph− progenitors are highly enriched in the CD34+HLA-DR− fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph− progenitor or stem cells, leaving the unanswered question whether Ph−CD34+HLA-DR- progenitors are normal. To show the clonal nature of Ph−CD34+HLA-DR− CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34+HLA-DR+ and CD34+HLA-DR− progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA−, CD34+HLA-DR−progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34+HLA-DR− derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph−, BCR/ABL mRNA−and polyclonal state in the CD34+HLA-DR−and CD34+HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34+ cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph−, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34+HLA-DR− cells that are Ph− and BCR/ABL mRNA− are polyclonal and therefore benign. This population is suitable for autografting in CML.

Description: BACKGROUND: Epoetin beta (NeoRecormon®) 30 000 IU/week raises hemoglobin levels, reduces transfusion need and improves quality of life in patients with cancer. Recent studies have also suggested that epoetin therapy may impact upon outcomes in these patients. A meta-analysis was performed to investigate the effects of epoetin beta on survival, tumor progression and thromboembolic events in patients with hematological malignancies receiving chemotherapy. METHODS: Data were pooled from all five randomized, controlled (placebo or standard care) clinical trials of epoetin beta that included anemic patients with hematological malignancies receiving chemotherapy. The study that showed epoetin beta once weekly to be as effective with the same safety profile as a three times weekly regimen was excluded from this analysis because of the lack of a non-epoetin-treated group. These studies were not designed to assess duration of survival and in all except one there was no follow-up beyond the duration of study treatment plus an additional 4-week period. Deaths reported during the study plus 4 weeks were therefore recorded in this analysis. All adverse event reports were assessed for evidence of disease progression or thromboembolism. Data were analyzed by standard Kaplan-Meier methods, Cox regression and log-rank tests. RESULTS: A total of 791 patients with hematological malignancies were included (epoetin beta, n=461; control, n=330) (the difference in number was due to two of the five studies containing multiple epoetin beta treatment groups versus one control group). The majority was diagnosed with lymphoma (56%) or multiple myeloma (42%); the remaining patients (2%) were diagnosed with acute myeloid leukemia. Treatment groups were well balanced with regard to baseline demographic characteristics. There were no obvious differences in baseline tumor stage between treatment groups, although these data were not consistently collected across studies. The median weekly dose of epoetin beta was 30 000 IU. Death rates were similar in both the epoetin beta and control groups (0.39 vs 0.37 deaths/patient year). There was no indication of a difference in survival with epoetin beta compared with control (relative risk 1.04, 95% CIs 0.69, 1.55, p=0.86). The rate of disease progression was lower in the epoetin beta group compared with the control group (0.69 vs 0.81 events/patient year). The results from Kaplan-Meier estimates and Cox regression did not indicate an increased risk of disease progression with epoetin beta compared with control (relative risk 0.84, 95% CIs 0.62, 1.13, p=0.25). In fact, the hazard rate of 0.84 indicated a 16% reduction in the risk of an event when treated with epoetin beta. Thromboembolic event rates were also similar in the epoetin beta and control groups (0.17 vs 0.14 events/patient year), corresponding to crude rates of 5.4% and 4.8% (observation times: 147 and 112 patient years). These event rates are well within the range of those reported in the literature. CONCLUSIONS: Epoetin beta has no effect on short-term survival, tumor progression or thromboembolic events when used to treat anemic patients with hematological malignancies, and the risk-to-benefit ratio of epoetin beta therapy remains favorable.

Description: T-cell Acute Lymphoblastic Leukemia (T-ALL) is a heterogeneous entity with several biologically distinct subtypes that differ in clinical outcome. Cytogenetics in T-ALL shows recurrent involvement of TCR a/d (14q11) and TCR b (7q34) rearrangements. The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-ALL. To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 rearrangements were detected, but 5 patients showed amplification of ABL1 and are described in a separate abstract and 2 patients showed a chromosomal rearrangement affecting ABL1. The first patient (male, age 16, 455x109 WBC/L, 99% blasts, cortical T immunophenotype) showed a cryptic t(9;14)(q34;q32). This was demonstrated in 7/12 metaphases and 60% of nuclei using FISH with the 5′/3′-ABL1 probes RACE-PCR detected a fusion between EML1 at 14q32 and ABL1 at 9q34. The fusion transcript joins exon 1–18 of EML1 to exon 2 of ABL1 and generates an ORF of 4926 nt. The functional characterization of the fusion protein is ongoing. The second patient (male, age 16, 25x109 WBC/L, 35% blasts, mature T-cell immunophenotype) showed a 47, XY, +10[5], idem, inv(9)(p21q34)[2}]/46, XY[9] karyotype. One third of the metaphases are tetraploid with the same distribution of abnormalities. The split signals obtained with the 5′-/3′ABL1 probes in the inv(9) subclone point to an ABL1 rearrangement that is being characterized at the present. These result suggest that ABL1 rearrangements are a rare but recurrent event in T-cell malignancies and support a role of ABL1 in T-cell biology. The therapeutic potential of ABL1 inhibitors for treating this group of T-ALL needs to be explored.

Description: Background: Fludarabine phosphate (F)-based nonmyeloablative conditioning regimens are associated with reduced transplant-related toxicity and allow transplantation in patients (pts) above 55 y.o. Aims: We prospectively studied the role of ATG to control the development of graft-versus-host-disease (GVHD), accelerate donor engraftment and maintain GVT effects in pts not eligible for classical allogeneic transplant. The subgroup of pts with 60 yo or more was analysed and compared with the younger population. Population: A total of 113 pts were enrolled in the phase II study. Pts with lymphoid malignancies were conditioned with F (30 mg/m²/day for 4 days) plus cyclophosphamide (1g/m²/day for 3 days) or cytarabine (Ara-C, 2g/m²/day) for myeloid malignancies. All pts received cyclosporine (3–5 mg/kg IV, daily) and ATG (10 mg/kg/day for 4 days - [ATG-4, n= 36] or 2 days - [ATG-2, n=67] ). Chimerism analyses were performed on d30, 45, 60 and 90. Results: 29 pts had 60 y.o. or more.Diagnoses were as follows: 9 MM, 5 NHL, 3 CLL, 5 AML, 4 MDS, 2 CML, 1 WM. The median age was 63 (range 60–74) years. The median follow-up was 24 months. Prior to transplantation, 35% of pts were in complete remission. 65% had poor prognostic disease(PR, RR or PD). Engraftment rate was 100% of the evaluable pts. Treatment-related mortality was 48%,much higher than in the younger population (15%) and due primarily to infectious complications. Grade II-IV acute GVHD (aGVHD) at d90 were similar in both populations (35%). Chronic GVHD rates were similar in both groups (40%). At d90, 23 pts were evaluable for T-cell chimerism analysis. Full (〉90%) donor chimerism was achieved in 20/23 pts. Overall survival was higher in patients with good prognostic diseases (60% vs. 48%). No pts developed veno-occlusive disease. Conclusion: These results confirm the feasibility of F-based nonmyeloablative conditioning in elderly population but infectious complications remain a concern compared to younger population.

Description: Background. For several decades, empirical antifungal therapy has been the standard of care for the early treatment of invasive aspergillosis (IA) in neutropenic cancer patients. However, this approach results in the undue exposure to potentially toxic drugs, may select for resistant strains, and jeopardizes the hospital budget. We conducted a prospective study to evaluate the feasibility and safety of a targeted pre-emptive approach (while avoiding empirical therapy), based on the incorporation of sensitive, non-invasive diagnostic tests. Methods. A total of 136 treatment episodes at high risk for IA (41 % ≥40 years of age; 26 % relapsed leukemia; 30 % high dose cytarabine; mean duration of neutropenia: 21.5 days; mean duration of broad-spectrum antibiotics: 15.9 days) were screened daily for circulating galactomannan by the Platelia (Bio-Rad) enzyme immunoassay (EIA) while receiving fluconazole prophylaxis. An index of ≥0.5 was considered positive if confirmed by the subsequent sample. In addition, well-defined clinical, radiological, and microbiological criteria triggered a diagnostics work-up for invasive fungal infection, including thoracic computed tomography scanning and bronchoscopy with bronchoalveolar lavage. Antifungal therapy (AmBisome 5 mg/kg/day) was withheld unless the patient became EIA-positive or had a positive culture or microscopy for molds with supportive radiological findings. Results A total of 4170 serum samples (mean 30.6/episode) was analyzed. The combined EIA-CT-scan approach identified 19 episodes (14 percent) of IA. Less than one quarter of the episode that qualified for empirical therapy (unexplained persisting or relapsing fever) received antifungal therapy (6.6 percent versus 30 percent). Conversely, early antifungal therapy was started in 10 episodes (7.3 percent) not otherwise suspected for IA due to treatment with corticosteroids or due to the presence of confounding co-factors/co-infections. Undetected cases of IA were not found at autopsy. Unexpectedly, autopsy identified one patient with disseminated zymogycosis. At 12 weeks, a 77 percent survival rate was noted in case patients with controlled underlying hematological disease (versus 30 percent for patients with refractory underlying disease) Conclusions. Replacing empirical therapy by a targeted, pre-emptive therapy offered effective antifungal control and spared patients from exposure to potentially toxic and expensive drugs without negative impact on mortality. This strategy should further be explored in a multicenter, randomized setting.

Description: High-dose therapy with allogeneic hematopoietic transplantation (allo-SCT) from matched sibling donors has been shown to induce durable remissions in some patients with relapsed / refractory mantle cell lymphoma (MCL). The aim of this retrospective analysis from the LWP of the EBMT was to investigate the outcome of patients with MCL treated with an unrelated donor allo-SCT (MUD-allo). From January 1994 to July 2005, 66 patients with MCL, 51 males and 15 females, with a median age of 50 years (range, 22 to 68) underwent a MUD-allo and were reported to the EBMT registry. The median time from diagnosis to MUD-allo was 34 months (range, 6–131). Thirty-five patients (53%) had previously failed an autologous procedure (ASCT). Forty-five patients (68%) had sensitive disease (including 22 patients in complete remission) at transplantation, whereas 21 patients (32%) were allografted with refractory disease. Reduced intensity conditioning regimens (RIC) were used in 44 patients (67%). Patients treated with RIC were older, more heavily pre-treated and had more frequently failed an ASCT (46% vs 30%, p = 0.01) than patients treated with a conventional conditioning protocol. Total body irradiation (TBI) was used in 68% of the patients receiving conventional protocols and low-dose TBI in 23% of the RIC patients. Grade II–IV acute graft versus host disease (GVHD) developed in 35% of the cases. The cumulative incidence (CI) of non-relapse mortality (NRM) was of 21% at 6 mo and 27% at 12 mo. The CI of relapse was 35% at 1 year and 45% at 2 years. After a median follow up of 15 months (1–73), 25 patients are alive without progression, with an estimated PFS and OS at 2 years of 28% and 42%, respectively. RIC protocols were not associated with a lower NRM or a better survival. Refractory disease at MUD-allo was an adverse prognostic factor for PFS (RR 1.7; p = 0.006). Patients allografted in sensitive disease presented a better 2-year PFS and OS (34% and 48%, respectively). In conclusion, although follow up is still short, MUD-allo is a feasible procedure in poor prognosis MCL patients, with almost one third of them being alive and progression-free in this series. RIC protocols do not seem to offer any advantage in terms of long-term outcome in relation to conventional conditioning regimens.