Dose individualization of intravenous busulfan in pediatric patients undergoing bone marrow transplantation: impact and in vitro evaluation of infusion lag-time.

OBJECTIVES: To apply therapeutic drug monitoring and dose-individualization of intravenous Busulfan to paediatric patients and evaluate the impact of syringe-pump induced Busulfan infusion lag-time after in vitro estimation. METHODS: 76 children and adolescents were administered 2 h intravenous Busulfan infusion every 6 h (16 doses). Busulfan plasma levels, withdrawn by an optimized sampling scheme and measured by a validated HPLC-PDA method, were used to estimate basic PK parameters, AUC, Cmax, kel, t1/2, applying Non-Compartmental Analysis. In vivo infusion lag-time was simulated in vitro and used to evaluate its impact on AUC estimation. KEY FINDINGS: Mean (%CV) Busulfan AUC, Cmax, clearance and t1/2 for pediatric population were found 962.3 µm × min (33.1), 0.95 mg/L (41.4), 0.27 L/h/kg (33.3), 2.2 h (27.8), respectively. TDM applied to 76 children revealed 6 (7.9%) being above and 25 (32.9%) below therapeutic-range (AUC: 900-1350 µm × min). After dose correction, all patients were measured below toxic levels (AUC < 1500 µm × min), no patient below 900 µm × min. Incorporation of infusion lag-time revealed lower AUCs with 17.1% more patients and 23.1% more younger patients, with body weight <16 kg, being below the therapeutic-range. CONCLUSIONS: TDM, applied successfully to 76 children, confirmed the need for Busulfan dose-individualization in paediatric patients. Infusion lag-time was proved clinically significant for younger, low body-weight patients and those close to the lower therapeutic-range limit.

Low-dose alemtuzumab for GvHD prevention followed by prophylactic donor lymphocyte infusions in high-risk leukemia.

We analyzed the use of low-dose alemtuzumab in a cohort of 158 consecutive patients who underwent allogeneic PBSC transplantation. Patients with high-risk acute leukemia were prospectively screened for prophylactic donor lymphocyte infusion (pDLI). Lymphocytes were administered repeatedly at low and non-escalating doses (0.5-1 × 106/kg). Low-dose alemtuzumab was effective in prevention of acute GvHD after sibling or well-matched unrelated transplantation, whereas a more intensified approach was needed after mismatched transplantation. The cumulative incidence of chronic moderate/severe chronic-GvHD (cGvHD) was 15.6%. In total, 63 high-risk leukemia patients were eligible for pDLI. Only 1 out of the 39 pDLI recipients relapsed as compared with 7 out of the 24 recipients, who did not receive pDLI due to logistical hurdles. In multivariate analysis, the use of adjuvant lymphocyte therapy was significantly associated with reduced incidence of relapse and improved disease-free survival. In summary, low-dose alemtuzumab confers to a low cGvHD incidence and the administration of pDLIs in this context is very likely to reduce relapse risk in high risk leukemia patients. This is translated in an estimated 5-year probability of GvHD-free and relapse-free survival of 43.3% for the 136 leukemia patients.

Brentuximab vedotin in combination with or without donor lymphocyte infusion for patients with Hodgkin lymphoma after allogeneic stem cell transplantation.

In our study, we evaluated the safety and efficacy of Brentuximab vedotin (BV) with or without the addition of donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (allo-SCT) in 16 patients with advanced Hodgkin lymphoma (HL). Thirteen patients with relapsed HL after allo-SCT received BV as treatment for active disease. Three patients without progression of HL after allo-SCT received BV as consolidation. Twelve patients had been previously exposed to BV for treatment of relapse after autologous-SCT. Ten out of 16 patients received BV in combination with DLI. Among the 13 patients treated for active disease, CR and PR was observed in 7 and 2 patients, respectively. With a median follow-up of 13 months, 13 out of 16 patients are alive, while 3 died because of disease progression. The median PFS was 6 months. DLI-associated GVHD occurred in seven patients. Five patients with GVHD required immunosuppression, and in all cases, GVHD resolved after a short course of low dose steroids, implying that an anti-GVHD modulating effect could be induced by the concurrent administration of BV. No serious adverse event was observed in any of the patients.

Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey.

Despite major improvements in allogeneic hematopoietic cell transplantation over the past decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Preclinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grades III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGVHD (1-10). The overall response rate was 81.5% (44/54) in SR-aGVHD including 25 complete responses (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3-90.7%, 95% confidence interval (CI)) and 97.4% (92.3-100%, 95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and cytomegalovirus-reactivation were observed during ruxolitinib treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial.

Specific abnormalities versus number of abnormalities and cytogenetic scoring systems for outcome prediction after allogeneic hematopoietic SCT for myelodysplastic syndromes.

Newer cytogenetic scoring systems for myelodysplastic syndromes (MDSs), like cytogenetic stratification of the revised international prognostic scoring system (IPSS-R) or monosomal karyotype, may also improve outcome prediction after hematopoietic SCT (HCT). We compared the prognostic value of specific cytogenetic abnormalities, IPSS-R karyotype and monosomal karyotype for HCT outcome in 98 patients with MDS and AML post MDS. Higher-risk IPSS-R karyotype, 3q21q26 and transformation to AML before HCT were associated with increased cumulative incidence of relapse (CIR), whereas OS was adversely influenced by del 5q/-5, abnormalities of chromosomes 11 and 17 and cytogenetic IPSS-R very poor category. Karyotype with ⩽2 abnormalities and no abnormalities of chromosomes 3, 5, 7, 11 and 17 was an independent prognostic factor of lower CIR (hazard ratio (HR)=0.2, P=0.01) and longer OS (HR=0.5, P=0.03). In conclusion, some specific cytogenetic abnormalities and high cytogenetic complexity, as reflected by IPSS-R very poor karyotype, rather than monosomal karyotype, were associated with higher CIR and shorter OS after HCT. Conversely, results were encouraging in patients lacking those abnormalities, who may be very good candidates for HCT.

A prospective, cohort, multicentre study of candidaemia in hospitalized adult patients with haematological malignancies.

Invasive candidiasis is a life-threatening infection in patients with haematological malignancies. The objective of our study was to determine the incidence, microbiological characteristics and clinical outcome of candidaemia among hospitalized adult patients with haematological malignancies. This is a population-based, prospective, multicentre study of patients ≥ 18 years admitted to haematology and/or haematopoietic stem cell transplantation units of nine tertiary care Greek hospitals from January 2009 through to February 2012. Within this cohort, we conducted a nested case-control study to determine the risk factors for candidaemia. Stepwise logistic regression was used to identify independent predictors of 28-day mortality. Candidaemia was detected in 40 of 27,864 patients with haematological malignancies vs. 967 of 1,158,018 non-haematology patients for an incidence of 1.4 cases/1000 admissions vs. 0.83/1000 respectively (p <0.001). Candidaemia was caused predominantly (35/40, 87.5%) by non-Candida albicans species, particularly Candida parapsilosis (20/40, 50%). In vitro resistance to at least one antifungal agent was observed in 27% of Candida isolates. Twenty-one patients (53%) developed breakthrough candidaemia while receiving antifungal agents. Central venous catheters, hypogammaglobulinaemia and a high APACHE II score were independent risk factors for the development of candidaemia. Crude mortality at day 28 was greater in those with candidaemia than in control cases (18/40 (45%) vs. 9/80 (11%); p <0.0001). In conclusion, despite antifungal prophylaxis, candidaemia is a relatively frequent infection associated with high mortality caused by non-C. albicans spp., especially C. parapsilosis. Central venous catheters and hypogammaglobulinaemia are independent risk factors for candidaemia that provide potential targets for improving the outcome.

Outcomes and prognostic factors of adults with acute lymphoblastic leukemia who relapse after allogeneic hematopoietic cell transplantation. An analysis on behalf of the Acute Leukemia Working Party of EBMT.

To describe outcomes, treatment and prognostic factors that influence survival of adult patients with acute lymphoblastic leukemia (ALL), who relapsed after allogeneic hematopoietic cell transplantation (HCT), we retrospectively analyzed 465 ALL adult patients from European Group for Blood and Marrow Transplantation (EBMT) centers who relapsed after a first HCT performed in complete remission (CR1 65%, CR2/3 35%). Salvage treatments were: supportive care (13%), cytoreductive therapy (43%), donor lymphocyte infusion without or with prior chemotherapy (23%) and second HCT (20%). Median time from HCT to relapse was 6.9 months, median follow-up was 46 months and median survival after relapse was 5.5 months. Estimated 1-, 2- and 5-year post-relapse survival was 30 ± 2%, 16 ± 2% and 8 ± 1%, respectively. In a multivariate analysis, adverse factors for survival were: late CR (CR2/3) at transplant (P<0.012), early relapse after transplant (<6.9 months, P <0.0001) and peripheral blast percent at relapse (P <0.0001). On the basis of multivariate model for survival, three groups of patients were identified with estimated 2 year survival of 6 ± 2, 17 ± 3 and 30 ± 7%. Outcome of ALL patients relapsing after HCT is dismal and there is a need for new therapies. Our study provides the standard expectations in ALL relapse and may help in the decision of post-relapse therapy.

CT60 single-nucleotide polymorphism as a surrogate marker for donor lymphocyte infusion outcome after allogeneic cell transplantation for acute leukemia.

The benefit of survival at the expense of new GVHD after DLI for acute leukemia following human allogeneic hematopoietic cell transplantation (allo-HCT) remains a matter of controversy. The detection of biological markers predicting this outcome would be an enormous breakthrough. The purpose of this study was the analysis of CT60 single-nucleotide polymorphism (SNP) of the CTLA-4 T-regulatory gene as a surrogate marker for DLI outcome in this difficult setting. Using Pyrosequencing, we genotyped the alleles of the CT60 SNP of 79 DLI donors and correlated them with the post-DLI outcome of their matching recipients. The presence of a donor 'AA' or 'AG' CT60 genotype vs a 'GG' genotype was an independent factor for remaining in complete chimerism/remission post-DLI (odds ratio (OR) 2.61 vs 0.42, respectively, P=0.05). Further, in cases with evident post-DLI allo-reactivity the importance of an 'AA' or 'AG' vs a 'GG' genotype gained significance for ongoing complete chimerism (OR 4.35 vs 0.32, P=0.03). Neither alterations in cumulative DLI dose nor any other clinical parameter significantly weakened the importance of CT60 SNP. Our results provide evidence for the necessity of genotyping CT60 SNP prior to DLI administration in patients with acute leukemia.

Pharmacokinetics and clinical activity of very low-dose alemtuzumab in transplantation for acute leukemia.

The optimal dose of in vivo-administrated alemtuzumab in the allogeneic transplantation setting has not been defined. We report our experience on 37 patients with high-risk diseases, mainly acute leukemia (AML 23, ALL 10 patients), who underwent sibling (49%) or unrelated (51%) PBSCT (35 patients), and received a total dose of only 10-20 mg Campath-1H as part of the conditioning, and post-transplant CYA without MTX. The neutrophil and especially the platelet engraftment were rapid. There were only two grade III-IV acute GvHD cases, which occurred in unrelated transplants in the Campath-10 cohort. Chronic GvHD developed in six cases (17%) and was limited to skin in five of them. After a median follow-up of 371 days (59-1191), 70% patients are alive and in CR (Karnofsky 100%), and 11 died (TRM n=6, relapse n=5). From the five patients relapsed, three were at advanced stage at transplant and four underwent sibling HCT with the higher (20 mg) alemtuzumab dose. With the 10 mg alemtuzumab schedule (5 mg/day at days -2 and -1) we achieve at day of transplantation low but still lymphotoxic alemtuzumab serum concentrations (176 ng/mL), whereas levels declined fast thereafter, and at engraftment nearly no Campath antibody remained in the patient's serum.

Human herpesvirus 6-related pure red cell aplasia, secondary graft failure, and clinical severe immune suppression after allogeneic hematopoietic cell transplantation successfully treated with foscarnet.

Human herpesvirus 6 (HHV-6) is frequently detected after allogeneic hematopoietic cell transplantation (allo-HCT); however, the clinical interpretation of HHV-6 viremia in a transplant patient is challenging as it may signify asymptomatic reactivation, chromosomal integration of the virus genome in the donor or recipient with no clinical significance, or severe HHV-6 disease. Here we present a case of HHV-6 disease after allo-HCT presenting as pure red cell aplasia, secondary graft failure, and severe immunosuppression causing multiple severe bacterial super-infections. Examination of pre-transplant patient and donor samples as well as serial determination of HHV-6 DNA copy numbers after transplantation were necessary to definitively interpret HHV-6 viremia as active HHV-6 infection with a causative role in pancytopenia and immune suppression. Foscarnet treatment resulted both in viral load decline and disappearance of HHV-6-related bone marrow suppression and predisposition to severe infections. Clinicians should be aware of the wide array of clinical manifestations and the diagnostic pitfalls of post-transplant HHV-6 disease. These issues are extremely challenging, as they may result either in dangerous underestimation of HHV-6 disease or in the institution of unnecessary antiviral therapy. Late bone marrow aplasia and late severe infections after allo-HCT without other obvious causes may be HHV-6 related.

Alloreactive microenvironment after human hematopoietic cell transplantation induces genomic alterations in epithelium through an ROS-mediated mechanism: in vivo and in vitro study and implications to secondary neoplasia.

We hypothesized that chronic tissue stress due to interaction of alloreactive donor cells with host epithelium after allogeneic hematopoietic cell transplantation (allo-HCT) may cause genomic alterations. We therefore analyzed 176 buccal samples obtained from 71 unselected allotransplanted patients for microsatellite instability (MSI). MSI was observed in 52% of allotransplanted patients but never in 31 healthy or autotransplanted controls. The patient age, the donor age, a female-to-male transplantation and a low number of CD34(+) cells in the graft were significantly correlated with genomic instability. There was a trend for increasing risk of MSI for patients who experienced severe graft-vs-host disease. Secondary malignancy was diagnosed in five (14%) of the MSI(+) and only in one (3%) MSI(-) patient. In an in vitro model of mutation analysis we found significant induction of frameshift mutations and DNA strand breaks in HaCaT keratinocytes co-cultured with mixed lymphocyte cultures (MLCs) but not after their exposure to interferon-gamma, tumor necrosis factor-alpha, transforming growth factor-beta (TGF-beta), MLC supernatant, peripheral blood mononuclear cells (PBMCs) or phytohemagglutinin-stimulated PBMC. A reactive oxygen species-mediated mechanism is implicated. The in vivo and in vitro data of our study show that alloreactions after allo-HCT may induce genomic alterations in epithelium. Progress in understanding DNA damage and repair after allo-HCT can potentially provide molecular biomarkers and therapeutic targets.

Microchimerism in peripheral blood and urine in renal transplant recipients: preliminary results.

The role of microchimerism in peripheral blood and urine of renal transplant recipients remains a matter of debate, depending on the sensitivity of the methods used for detection. We studied 17 female renal transplant recipients who had received renal allografts from male donors. Polymerase chain reaction (PCR) was applied to blood and urine for the microsatellite markers D1S80, DYZ1, TH01, and kalphai SE33. Detection of DYZ1 that is present only on the Y chromosome was considered proof for microchimerism. No microchimerism was detected in peripheral blood, whereas it could be detected in the urine of 8/17 (48%) patients. There were no differences between patients with and without microchimerism regarding patient age, dialysis vintage, immunosuppression, time post-transplantation, and allograft function as measured using serum creatinine, creatinine clearance, and proteinuria. Two patients in each group showed chronic allograft dysfunction. These findings raise questions regarding the role of microchimerism in renal transplantation.

c-Jun N-terminal kinase activation failure is a new mechanism of anthracycline resistance in acute myeloid leukemia.

Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug resistance in AML. c-Jun N-terminal kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

A fludarabine, thiotepa reduced toxicity conditioning regimen designed specifically for allogeneic second haematopoietic cell transplantation after failure of previous autologous or allogeneic transplantation.

We present a phase II study of fludarabine 5 x 30 mg/m(2), thiotepa 3 x 5 mg/kg as preparative regimen specifically for allogeneic second haematopoietic stem cell transplantation (HCT) after failure of previous HCT. Forty-nine patients (median age 52 years, range 27-68) received an allogeneic second HCT after failed autologous (n=29) or allogeneic (n=20) HCT. Diagnoses were AML (n=18), ALL (n=3), multiple myeloma (n=11), lymphoma (n=16) and CML (n=1). GVHD prophylaxis consisted of CYA and mainly low dose alemtuzumab (40 mg). The median follow-up for patients alive is 528 days (range 217-1344). In 43 of 49 (88%) evaluable patients response rates were CR=19, PR=14 and SD=10 at one month. At one year, the probability (95% confidence interval) of relapse is 55.1 (38.2-72)% and the nonrelapse mortality (NRM) is 29 (14.2-44.4)%. Estimated survival at one year is 42.6 (28.7-56.6)% and event free survival is 38.1 (24.4-51.8)%. Survival was significantly better for patients experiencing relapse beyond one year, than for patients relapsing within one year from first transplantation (51.2 (33.5-68.9)% vs 27 (7-48.5)%; P=0.013). We conclude that this regimen is feasible and well tolerated for allogeneic second HCT.

Lymphocyte reconstitution following allogeneic hematopoietic stem cell transplantation: a retrospective study including 148 patients.

Here we investigated the influence of parameters known before hematopoietic stem cell transplantation (HSCT) as well as the relevance of graft-versus-host disease (GvHD) and cytomegalovirus (CMV) reactivation on post transplant lymphocyte reconstitution in 148 patients treated in our institution between 1996 and 2003. Median patient age was 42 (19-68) years, HSCT followed standard high dose (n=91) or reduced-intensity conditioning regimens (n=57) with bone marrow (BM, n=67) or peripheral blood stem cells (PBSC, n=81) from related (n=71) or unrelated (n=77) donors. In the first months, we observed a partially faster reconstitution of CD3+4+, CD3+8+ and CD4+45RA+ T cells in patients following peripheral blood stem cell transplantation when compared to bone marrow transplantation. Prolonged CD3+4+ and CD4+45RA+ lymphopenia was noted after unrelated donor HSCT and GvHD prophylaxis containing anti-T-lymphocyte globulin. Lymphocyte subset counts in patients older than the median age were comparable to those in patients transplanted at a younger age and not influenced by the conditioning regimen. CD3+8+ T cell reconstitution was strongly correlated with CMV reactivation, but not significantly affected by CMV serostatus before HSCT. Incidence or extent of GvHD did not significantly influence lymphocyte reconstitution. Therefore, the source of graft is the most predictive parameter in early lymphocyte reconstitution, but the differences in lymphocyte recovery completely resolved within the first year after HSCT.

Evaluation of immunomodulatory treatment based on conventional and lineage-specific chimerism analysis in patients with myeloid malignancies after myeloablative allogeneic hematopoietic cell transplantation.

Both conventional chimerism analysis (CCA) and lineage-specific chimerism analysis (LCA) have potential pitfalls as diagnostic means for the detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). Therefore, the present study examines the results of both methods in order to determine how predictive consecutive evaluations were, with respect to the risk that the patient would relapse during post-transplant follow-up and with respect to responsiveness to immunomodulatory treatment. A total of 168 individuals with acute myeloid leukemia (AML) (n = 137) and myelo dysplastic syndrome (n = 31) were investigated with CCA and LCA at mean intervals of 24 days (range: 11-116). The median follow-up after myeloablative aHCT was 22 months (range: 4-49). Of 168 patients, 65 experienced a clinical relapse after aHCT. CCA and LCA were comparatively sensitive and specific for relapse at the intervals of chimerism testing employed in this study. Of 32 patients, 10 who were offered donor lymphocyte infusions (DLI) treatment for increasing (n = 29) or stable (n = 3) mixed chimerism (MC) achieved at least transitory CC. The observation that all patients with increasing MC relapsed despite DLI treatment (54%) or withdrawal of immune suppression (24%) indicates that novel strategies to deal with rapidly evolving relapse in AML patients, such as shortening of chimerism monitoring intervals, need to be evaluated.