Pedro Gómez

Clinical value of immunological monitoring of minimal residual disease in acute lymphoblastic leukaemia after allogeneic transplantation

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.

Kinetic of regulatory CD25high and activated CD134+ (OX40) T lymphocytes during acute and chronic graft-versus-host disease after allogeneic bone marrow transplantation

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+CD25high 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+CD25high 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+CD25high 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 × 106/L). However, the CD134/CD25high 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+CD25high 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+CD25highex vivo expansion protocols, OX40 blocking might be crucial to optimize the use of Treg to prevent GVHD.

Population Pharmacokinetics of High-Dose Methotrexate in Children with Acute Lymphoblastic Leukaemia

AbstractObjective: To develop and a priori validate a methotrexate population pharmacokinetic model in children with acute lymphoblastic leukaemia (ALL), receiving high-dose methotrexate followed by folinic acid rescue, identifying the covariates that could explain part of the pharmacokinetic variability of methotrexate. Methods: The study was carried out in 49 children (aged 6 months to 17 years) who received high-dose methotrexate (3 g/m2 per course) in long-term treatment. In an index group (37 individuals; 1236 methotrexate plasma concentrations), a population pharmacokinetic model was developed using a nonlinear mixed-effects model. The remaining patients’ data (12 individuals; 278 methotrexate plasma concentrations) were used for model validation. Age, sex, total bodyweight (TBW), height, body surface area, lowest urine pH during infusion, serum Creatinine, ALT, AST, folinic acid dose and length of rescue were analysed as possible covariates. The final predictive performance of the pharmacokinetic model was tested using standardised mean prediction errors. Results: The final population pharmacokinetic model (two-compartmental) included only age and total bodyweight as influencing clearance (CL) and volume of distribution of central compartment (V1). For children aged ≤10 years: CL (L/h) = 0.287 · TBWO-876; V1 (L) = 0.465 · TBW, and for children aged >10 years: CL (L/h) = 0.149 · TBW; V1 (L) = 0.437 · TBW. From the base to the final model, the inter-individual variabilities for CL and V1 were significantly reduced in both age groups (30–50%). The coefficients of variation of the pharmacokinetic parameters were <30%, while residual and inter-occasional coefficients maintained values close to 40%. Validation of the proposed model revealed the suitability of the model. Conclusion: A methotrexate population pharmacokinetic model has been developed for ALL children. The proposed model could be used in Bayesian algorithms with a limited sampling strategy to estimate the systemic exposure of individual patients to methotrexate and adapt both folinic acid rescue and methotrexate dosing accordingly.

Intermediate dose of imatinib in combination with chemotherapy followed by allogeneic stem cell transplantation improves early outcome in paediatric Philadelphia chromosome-positive acute lymphoblastic leukaemia (ALL): results of the Spanish Cooperative Group SHOP studies ALL-94, ALL-99 and ALL-2005

Philadelphia‐chromosome acute lymphoblastic leukaemia (Ph+ ALL) is a subgroup of ALL with very high risk of treatment failure. We report here the results of the Sociedad Española de Hematología y Oncología Pediátricas (SEHOP/SHOP) in paediatric Ph+ ALL treated with intermediate‐dose imatinib concurrent with intensive chemotherapy. The toxicities and outcome of these patients were compared with historical controls not receiving imatinib. Patients with Ph+ ALL aged 1–18 years were enrolled in three consecutive ALL/SHOP trials (SHOP‐94/SHOP‐99/SHOP‐2005). In the SHOP‐2005 trial, imatinib (260 mg/m2 per day) was given on day‐15 of induction. Allogeneic haematopoietic stem‐cell transplantation (HSCT) from a matched related or unrelated donor was scheduled in first complete remission (CR1). Forty‐three patients were evaluable (22 boys, median age 6·8 years, range, 1·2–15). Sixteen received imatinib whereas 27 received similar chemotherapy without imatinib. Seventeen of 27 and 15 of 16 patients in the non‐imatinib and imatinib cohort, respectively, underwent HSCT in CR1. With a median follow‐up of 109 and 39 months for the non‐imatinib and imatinib cohorts, the 3‐year event‐free survival (EFS) was 29·6% and 78·7%, respectively (P = 0·01). These results show that, compared to historical controls, intermediate dose of imatinib given concomitantly with chemotherapy and followed by allogeneic HSCT markedly improved early EFS in paediatric Ph+ ALL.

Chromosome fragility in patients with Fanconi anaemia: diagnostic implications and clinical impact

Background Fanconi anaemia (FA) is a rare syndrome characterized by bone marrow failure, malformations and cancer predisposition. Chromosome fragility induced by DNA interstrand crosslink (ICL)-inducing agents such as diepoxybutane (DEB) or mitomycin C (MMC) is the ‘gold standard’ test for the diagnosis of FA. Objective To study the variability, the diagnostic implications and the clinical impact of chromosome fragility in FA. Methods Data are presented from 198 DEB-induced chromosome fragility tests in patients with and without FA where information on genetic subtype, cell sensitivity to MMC and clinical data were available. Results This large series allowed quantification of the variability and the level of overlap in ICL sensitivity among patients with FA and the normal population. A new chromosome fragility index is proposed that provides a cut-off diagnostic level to unambiguously distinguish patients with FA, including mosaics, from non-FA individuals. Spontaneous chromosome fragility and its correlation with DEB-induced fragility was also analysed, indicating that although both variables are correlated, 54% of patients with FA do not have spontaneous fragility. The data reveal a correlation between malformations and sensitivity to ICL-inducing agents. This correlation was also statistically significant when the analysis was restricted to patients from the FA-A complementation group. Finally, chromosome fragility does not correlate with the age of onset of haematological disease. Conclusions This study proposes a new chromosome fragility index and suggests that genome instability during embryo development may be related to malformations in FA, while DEB-induced chromosome breaks in T cells have no prognostic value for the haematological disease.

EFFECT OF CYCLOSPORIN ON PLASMA LIPOPROTEINS IN BONE MARROW TRANSPLANTATION PATIENTS

The effects of cyclosporin and prednisone on plasma lipid and lipoprotein levels were studied in 20 allogeneic bone-marrow transplantation patients receiving cyclosporin plus prednisone therapy, and in 14 allogeneic patients treated only with cyclosporin during 100 days. Eighteen autologous bone-marrow patients not requiring cyclosporin were used as a control group. Patients were studied 5 days prior to transplantation, and on days 30, 60, and 100 after transplantation. To determine the reversibility of the changes, lipid parameters were analyzed 30 days after completion of the treatment. Nutritional supplementation, conditioning regimens, and concomitant medications were not significantly different between groups. Furthermore, no significant differences in age, weight, lipid, or lipoprotein levels were found at baseline. Our results indicate that cyclosporin therapy induces a reversible increase of plasma cholesterol, LDL-cholesterol, triglycerides, VLDL-triglycerides, and apolipoprotein B and a decrease of HDL-cholesterol, HDL2-cholesterol, and apolipoprotein A-I. The addition of prednisone to cyclosporin therapy induces a higher increase in plasma cholesterol mainly due to an increase in HDL-cholesterol. Total cholesterol/HDL-cholesterol ratio increased significantly in patients treated only with cyclosporin. No differences were found in this ratio in patients treated with prednisone compared to those submitted to autologous bone-marrow transplantation. Lipid changes observed in this study were reversible 30 days after cessation of cyclosporin treatment.

Voriconazole as primary antifungal prophylaxis in patients with neutropenia after hematopoietic stem cell transplantation or chemotherapy for acute myeloid leukemia

To the Editor: Invasive fungal infections (IFI) remain a major cause of illness and death in patients with prolonged and severe neutropenia (PSN) following treatment for hematological malignancies and allogeneic hematopoietic stem cell transplantation (HSCT). Treatment success is higher in early stages of the disease making some clinicians to consider antifungal prophylaxis as an adequate strategy to diminishing the IFI morbidity–mortality in patients under PSN (1, 2). Early evidence-based guidelines recommended fluconazole for the prevention of IFI after HSCT (3). By contrast, initial trials did not show a marked advantage of antifungal prophylaxis in acute myeloid leukemia (AML) patients undergoing chemotherapy, although there was a tendency toward a reduced morbidity and mortality with the use of fluconazole or itraconazole (4). More recently, oral posaconazole is recommended in HSCT recipients with graft-versus-host disease as well as in patients with AML or myelodysplastic syndrome at high risk for invasive aspergillosis (5). Voriconazole, a new generation azole with both oral and intravenous formulations available, has been successfully employed as treatment of IFI. For these reasons, voriconazole is also used as antifungal prophylaxis in patients with PSN (6). We conducted a prospective study to determine the efficacy, and safety of voriconazole for the primary prevention of IFI in 127 high risk patients with 181 evaluate episodes of PSN after chemotherapy for AML or HSCT between June 2004 and July 2007. PSN was defined as an absolute neutrophil count of <500 neutrophils ⁄mm with an expected duration of at least 7 d. Antifungal prophylaxis consisted of voriconazole 400 mg in oral or i.v. formulation (200 mg tablet, b.i.d.; 8 mg ⁄kg for patients weighing <50 kg). Diagnostic procedures included twice weekly serum galactomannan levels. When IFI was suspected, a lung or sinus high-resolution CT was performed. Cultures of bronchoalveolar lavage fluid were performed if available. Neutropenic fever was managed according to Infectious Diseases Society of America (IDSA) guidelines. IFI was defined as stated by the Mycoses Study Group ⁄European Organization for Research and Treatment of Cancer (MSG ⁄EORTC). Baseline demographic and clinical characteristics are presented in Table 1. Median duration of prophylaxis with voriconazole was 24 d (range 11–70 d). Antifungal prophylaxis was considered successful when patients completed prophylaxis showing no evidence of proven, probable, or suspected IFI, and empirical or pre-emptive therapy was not used. The overall treatment success rate was 72.9%. Thirtyone of 181 episodes (17.1%) failed voriconazole prophylaxis because of the use of empirical antifungal therapy [with caspofungin or Liposomal amphotericin B (L-AmB)]. Seven proven or probable IFIs were documented (3.9%). Five corresponded to invasive aspergillus pneumonia and four resolved after combined therapy with caspofungin and voriconazole (n = 2) or caspofungin and L-AmB (n = 2). The remaining patient died because of graft failure. Additionally, we documented two cases of breakthrough opportunistic fungal infections: a surgically controlled nasal zygomycosis and another case of systemic fusariosis unsuccessfully treated with L-AmB. Ten (7.8%) of 127 voriconazole-treated patients died during the study period but only one patient died because of IFI (0.7%). Overall survival at 28 d from the end of antifungal prophylaxis was high, 92.8 ± 2.1%. Fourteen patients discontinued voriconazole prophylaxis because of possible–probable drug-related adverse events. Interruptions were mainly because of mild to moderate liver disturbances resolving within a range of 1–7 d after drug discontinuation. Furthermore, treatment interruptions occurred in most instances (all but one) in patients undergoing HSCT and only in one pediatric patient. Two patients needed definitive withdrawal because of visual hallucinations. This study showed that voriconazole effectively prevented IFI during PSN after chemotherapy for AML and HSCT. Because of this heterogeneous population, favorable results would need to be confirmed in a specific group of allogeneic HSCT patients. A drawback of this prospective study was non-randomized design but results are in agreement with those reported by two prior nonrandomized retrospective studies (7, 8), which showed low rates of IFI and, most impressively, no cases of aspergillosis among allogeneic HSCT recipients. In contrast, we still documented some cases of invasive doi:10.1111/j.1600-0609.2009.01367.x European Journal of Haematology 84 (271–273)

Simultaneous Hodgkin's disease in three siblings with identical HLA‐genotype

We report 3 cases of Hodgkins disease (HD) within a family of seven children. All three children had mixed cellularity type in different stages of the disease (IV‐B, III‐A, and II‐A). The diagnosis was made within a one‐month interval. HLA typing shows an identical genotype in the 3 HD patients, which was different from the rest of the family. One of the patients developed an idiopathic thrombocytopenic purpura (ITP) with temporary response to prednisone and MOPP‐C chemotherapy that subsided after splenectomy. The HLA identity, in addition to the simultaneous onset of the HD, suggests a combination of both genetic and environmental factors in the pathogenesis of this disease.

Control of Epstein-Barr Virus Load and Lymphoproliferative Disease by Maintenance of CD8+ T Lymphocytes in the T Lymphocyte- Depleted Graft after Bone Marrow Transplantation

Of 100 bone marrow transplant recipients, 30 (30%) received a CD4(+) lymphocyte-depleted graft (1x10(6) CD8(+) T lymphocytes/kg of body weight). Replication of Epstein-Barr virus (EBV) was observed in 40 patients (40%). The use of a CD4(+) lymphocyte-depleted graft was the only independent risk factor for replication of EBV (relative risk, 11.5; 95% confidence interval, 5.8-22.8; P<.0001). Nevertheless, EBV load in those patients was not higher than in the rest of patients, and the low EBV load prevented the development of lymphoproliferative disease. These results suggest that the presence of CD8(+) T lymphocytes in the bone marrow graft can control EBV load, thereby reducing the risk of developing lymphoproliferative disease.

High dose brachytherapy (real time) in patients with intermediate- or high-risk prostate cancer: technical description and preliminary experience

IntroductionIt has been well documented that the outcome of prostate cancer treatment depends on the dose administered. Hence, techniques have been developed that allow high-dose administration without increasing the complications, e.g. external radiotherapy combined with high-dose radiation (HDR) brachytherapy. In this article we analyse the technique and protocol of real-time HDR brachytherapy together with the preliminary results that support its use.Materials and methodsBetween June 1998 and December 2004, 100 patients with adenoma of the prostate were treated with 46 Gy of external irradiation to the pelvis and 2 HDR brachytherapy fractions (each of 1150 cGy) at the end of weeks 1 and 3 of a 5-week radiotherapy course. The 1997 American Joint Commission on Cancer (AJCC) system was used to establish disease stage. Patients with intermediaterisk (PSA 10–20 ng/ml or Gleason=7 or T2c) and high-risk (two intermediate risk factors or PSC >20 ng/ml or Gleason >7 or >T2c) without metastases were eligible for the brachytherapy. Biochemical failure was defined according to the American Society for Therapeutic Radiology and Oncology (ASTRO) consensus panel statement. SPSS statistical package was used to quantify survival (Kaplan-Meier method). Toxicity was scored according to RTOG guidelines.ResultsThe mean age of patients was 67 years (range 49–78). Clinical stage was T2a in 22% of the patients, 26% T2b and 52% T3. Initial PSA was = 10 ng/ml in 22% of the patients and >10 ng/ml in 78%. Median follow-up was 28 months (range: 12–79). The 5-year overall survival and actuarial biochemical control were 99% and 87% respectively. No chronic severe complications were noted.ConclusionsThe good results of local control, disease-free survival and few complications that the external radiotherapy combined with HDR brachytherapy have shown suggest that the method should be considered as first-choice in the treatment of prostate tumours of high-and intermediate-risk.