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The present study reports the Spanish PETHEMA group experience in 31 heavily pretreated relapsed/refractory acute lymphoblastic leukemia (ALL) and lymphoma (LL) patients treated with clofarabine-based regimens. The complete remission (CR) rate was 31% (median CR duration of 3 months [range 2–28]) and the overall survival probability at 1 year was 10% (95%CI 4–16%). Responses were seen in B and T lineage diseases and in patients with adverse cytogenetics. Hematological and infectious grade >3 toxicities were found in 100 and 67% of the patients, respectively, with 7 (23%) treatment-related deaths. Other organ toxicities were infrequent. Clofarabine-based chemotherapy regimens might induce CRs in ALL and LL patients, but hematological toxicity and infections may limit their use in heavily pretreated patients.

BACKGROUND: Dendritic cell (DC)-based immunotherapeutic protocols are being developed to treat acute myeloid leukemia (AML). So far, DCs for clinical use are obtained from leukemic blasts or from monocytes, after 6 to 10 days of ex vivo culture. However, DC precursors are easily driven to DCs in short-term culture. We tested if DC precursors contained in peripheral blood stem cell (PBSC) products obtained from AML patients can be used to induce antileukemia responses. STUDY DESIGN AND METHODS: PBSCs obtained from 30 consecutive AML patients were tested. Myeloid DCs (MDCs) were purified by immunomagnetic selection and screened for cytogenetic and/or molecular abnormalities by fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) assays. MDCs were matured and pulsed with autologous blast lysates and tested for stimulatory capability against AML cells. RESULTS: A median of 0.62 × 106 MDCs (range, 0.04-3.25)/mL were quantified in PBSC products. Isolated MDC expressed Class I and II HLA but CD86, CD54, and CCR5 partially. By FISH or PCR assay, these MDCs lacked cytogenetic or molecular abnormalities detected in leukemia cells at diagnosis. MDCs achieved a maturated stage (mature-MDCs) after 24-hour ex vivo culture with tumor necrosis factor-α and autologous blast lysates. These mature-MDCs were capable of stimulating autologous peripheral blood effectors to exert cytotoxicity against autologous leukemia cells and HL-60 cell line. CONCLUSION: We conclude that PBSCs obtained for autologous stem cell transplantation can constitute a novel source of MDCs to design feasible vaccination trials.

Background and Aim: In human blood, two main subsets of antigen-presenting-cells (APCs) have been described: plasmocytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) which are further subdivided in CD11c-mDC and CD16-mDC DC. In ulcerative colitis patients (UC) peripheral blood APCs express significant levels of the activation and lack immature-tolerogeneic APCs. Adacolumn selective granulocytapheresis (GCAP) has been associated with clinical efficacy in patients with UC. In the present study we sought the effect of sequential GCAP procedures in peripheral blood APCs in patients with UC and the effect on soluble cytokines. Methods: We used multiparametric flow cytometry to quantify peripheral blood APCs and serum cytokines in 210 samples obtained from seven patients with steroid-dependent or steroid resistant UC undergoing GCAP treatment. Samples were drawn before, after 30 and 60 min of each session. Results: Each GCAP session resulted in a dramatic tenfold reduction of peripheral blood CD16-mDC (P

Graft-versus-host disease (GVHD) is still a major complication after allogeneic stem cell transplantation. In murine models, freshly isolated or ex vivo expanded CD4(+)CD25(high) regulatory T cells (Treg) are able to ameliorate GVHD while maintaining graft-versus-leukaemia reactions. However, in the human setting, prospective studies of this population and its interaction with activated non-regulatory CD134(+) (OX40) lymphocytes during post-transplant follow-up are lacking. In this study, we prospectively quantified CD4(+)CD25(high) and activated CD134(+) lymphocytes in 119 peripheral blood samples from 35 consecutive patients who underwent allogeneic bone marrow transplantation (BMT). Fifty-five samples obtained less than 100 d after allogeneic BMT, were not statistically different regarding CD4(+)CD25(high) Treg or CD134(+) lymphocytes compared with those obtained from patients with (n = 35) or without (n = 20) acute GVHD. Chronic GVHD was associated with a small, but not statistically significant, increase in the number of Treg (9.9 vs. 6.7 x 10(6)/L). However, the CD134/CD25(high) ratio was significantly higher during chronic GVHD (cGHVD) when compared with either patients without cGVHD (67.7 +/- 40.3 vs. 4.0 +/- 0.9, P < 0.01) or cGVHD after treatment (67.7 +/- 40.3 vs. 3.7 +/- 0.8, P < 0.01). Our findings suggest that the suppressive activity of CD4(+)CD25(high) Treg could be abrogated in vivo during cGVHD by CD134 expression in a much higher number of activated donor T lymphocytes. In addition to CD4(+)CD25(high)ex vivo expansion protocols, OX40 blocking might be crucial to optimize the use of Treg to prevent GVHD.

Summary. In this study, we used multiparameter flow cytometry to quantify minimal residual disease (MRD) in 165 serial bone marrow samples from 40 patients diagnosed with acute lymphoblastic leukaemia (ALL) who underwent allogeneic stem cell transplantation (allo-SCT) from siblings (n = 34) or unrelated donors (n = 6). Samples were prospectively taken from 24 patients before starting the conditioning regimen, at days +30, +60 and +90 and subsequently every 2–3 months. Samples from 16 patients in complete remission (CR) after allo-SCT were taken at least twice. Six of 24 patients harboured MRD (0·2–10% of mononuclear cells) at transplant and 18 were negative. Estimated disease-free survival for the MRD+ and MRD– groups at transplant was 33·3% and 73·5% respectively (P = 0·03). During follow-up, increasing MRD levels were detected in nine patients, a finding that preceded marrow relapse by 1–6 months. Two patients with stable low MRD levels remained in CR. When we used flow cytometry to test the effect of donor leucocyte infusions (DLI) in six patients, we observed that the only sustained remission was achieved when DLI was applied prior to overt relapse. We conclude that MRD by flow cytometry can rapidly assess tumoral burden before transplant to predict outcome, and can be clinically useful for the timing of DLI for increasing levels of leukaemia after transplant.

Summary The impact of lenalidomide treatment on long-term outcomes of patients with lower risk myelodysplastic syndromes (MDS) and chromosome 5q deletion (del(5q)) is unclear. This study used time-dependent multivariate methodology to analyse the influence of lenalidomide therapy on overall survival (OS) and acute myeloblastic leukaemia (AML) progression in 215 patients with International Prognostic Scoring System (IPSS) low or intermediate-1 risk and del(5q). There were significant differences in several relevant characteristics at presentation between patients receiving (n = 86) or not receiving lenalidomide (n = 129). The 5-year time-dependent probabilities of OS and progression to AML were 62% and 31% for patients receiving lenalidomide and 42% and 25% for patients not receiving lenalidomide; differences were not statistically significant in multivariate analysis that included all variables independently associated with those outcomes (OS, P = 0·45; risk of AML, P = 0·31, respectively). Achievement of RBC transfusion independency (P = 0·069) or cytogenetic response (P = 0·021) after lenalidomide was associated with longer OS in multivariate analysis. These data clearly show that response to lenalidomide results in a substantial clinical benefit in lower risk MDS patients with del(5q). Lenalidomide treatment does not appear to increase AML risk in this population of patients.

In this study, we examined myeloperoxidase (MPO) gene expression in a series of 31 non-infant pro-B acute lymphoblastic leukaemia (ALL) patients that included 16 cases with the t(4;11) translocation and/or the resultant ALL1/AF4 chimaeric gene. Sixteen out of 31 cases (51%) were MPO mRNA positive/enzyme negative. MPO mRNA was detected in nine out of 16 (56%) and seven out of 15 (47%) patients with and without the ALL1/AF4 fusion transcript respectively. The comparative study between MPO mRNA positive and negative cases showed statistically significant differences with regard to age and white blood cell (WBC) count, and was 39.5 years vs. 26.3 years (P = 0.016) and 71.4 x 10(9)/l vs. 157.8 x 10(9)/l (P = 0.046) in the MPO mRNA positive and negative groups respectively. The correlation analysis between MPO mRNA expression, age, WBC count and leukaemic relapse according to the presence/absence of the ALL1/AF4 fusion showed that the statistically significant differences observed in the whole group were related mostly to the ALL1/AF4-positive ALL patients. In fact, in this latter group, the mean WBC count and patients' age were 85 +/- 79 x 10(9)/l vs. 289.8 +/- 102 x 10(9)/l (P = 0.0005) and 44.8 +/- 15.3 years vs. 26.7 +/- 13.7 years (P = 0.01) in patients with and without MPO mRNA expression respectively. It appears, therefore, that the assessment of MPO mRNA expression enables a further dissection of leukaemia heterogeneity in apparently homogeneous genetic/immunophenotypic ALL subsets.