Bloodstream infection in paediatric cancer centres--leukaemia and relapsed malignancies are independent risk factors.

UNLABELLED: In a prospective multicentre study of bloodstream infection (BSI) from November 01, 2007 to July 31, 2010, seven paediatric cancer centres (PCC) from Germany and one from Switzerland included 770 paediatric cancer patients (58% males; median age 8.3 years, interquartile range (IQR) 3.8-14.8 years) comprising 153,193 individual days of surveillance (in- and outpatient days during intensive treatment). Broviac catheters were used in 63% of all patients and Ports in 20%. One hundred forty-two patients (18%; 95% CI 16 to 21%) experienced at least one BSI (179 BSIs in total; bacteraemia 70%, bacterial sepsis 27%, candidaemia 2%). In 57%, the BSI occurred in inpatients, in 79% after conventional chemotherapy. Only 56 % of the patients showed neutropenia at BSI onset. Eventually, patients with acute lymphoblastic leukaemia (ALL) or acute myeloblastic leukaemia (AML), relapsed malignancy and patients with a Broviac faced an increased risk of BSI in the multivariate analysis. Relapsed malignancy (16%) was an independent risk factor for all BSI and for Gram-positive BSI. CONCLUSION: This study confirms relapsed malignancy as an independent risk factor for BSIs in paediatric cancer patients. On a unit level, data on BSIs in this high-risk population derived from prospective surveillance are not only mandatory to decide on empiric antimicrobial treatment but also beneficial in planning and evaluating preventive bundles. WHAT IS KNOWN: • Paediatric cancer patients face an increased risk of nosocomial bloodstream infections (BSIs). • In most cases, these BSIs are associated with the use of a long-term central venous catheter (Broviac, Port), severe and prolonged immunosuppression (e.g. neutropenia) and other chemotherapy-induced alterations of host defence mechanisms (e.g. mucositis). What is New: • This study is the first multicentre study confirming relapsed malignancy as an independent risk factor for BSIs in paediatric cancer patients. • It describes the epidemiology of nosocomial BSI in paediatric cancer patients mainly outside the stem cell transplantation setting during conventional intensive therapy and argues for prospective surveillance programmes to target and evaluate preventive bundle interventions.

Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria, and Switzerland. Probability of 10-year event-free survival (survival) improved from 65% (77%) in study ALL-BFM 81-78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: 1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months less maintenance therapy caused an increase in systemic relapses; 4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; 5) condensed induction therapy resulted in a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Allo-SCT using BU, CY and melphalan for children with AML in second CR.

Based on the results from the AML-BFM 98 trial, hematopoietic SCT (HSCT) is recommended for children with AML in second CR only. Here, we retrospectively analyze interphase data of children who underwent HSCT after myeloablative conditioning with BU, CY, and melphalan (BuCyMel) for AML in second remission (CR2) between 1998 and 2009. Out of 152 children, transplant data were available on 109 individuals. Sixty out of 109 children (55%) received BuCyMel. Median age at HSCT was 12.2 years (range 3.0; 18.3). GVHD prophylaxis mostly consisted of CsA and short term MTX with or without antithymocyte globulin. Matched-sibling donors were used for 6/60 analyzed recipients, the remainder either received grafts from matched unrelated (30/60) or mismatched donors. OS after 5 years was 62% (s.e. 6%), relapse incidence 35% (18/60 children) and treatment-related mortality accounted for 12% (7/60) of fatal events. In conclusion, even taking into account possible selection bias in this retrospective analysis, HSCT in CR2 using BuCyMel resulted in a respectable OS. Based on this data the prospective, controlled and centrally monitored AML SCT-BFM 2007 trial has started to recruit patients in January 2010 aiming to generate valid outcome data for further strategy decisions.

Very early/early relapses of acute lymphoblastic leukemia show unexpected changes of clonal markers and high heterogeneity in response to initial and relapse treatment.

Minimal residual disease (MRD) quantified after induction treatment of childhood acute lymphoblastic leukemia (ALL) predicts risk of relapse. It has been assumed that early relapses derive from a residual population of leukemic cells, which is still present after induction and that relapsed disease will consequently be more resistant to treatment. To test these hypotheses, we performed a prospective study on patients treated according to the frontline-trial ALL-BFM 2000, which used MRD response for risk-group stratification. Patients (n=45) showed a median time to relapse of 1.5 years. In 89% of patients at least one T-cell-receptor/immunoglobulin gene rearrangement chosen for initial MRD quantification remained stable; however, at least one of the preferred markers for MRD stratification at relapse was different to diagnosis in 50% of patients. A similar proportion of very early, early and late relapses appeared to gain a marker at relapse although backtracking-analysis revealed that in 77% of cases, the gained markers were present as small sub-clones at initial diagnosis. Comparing initial and relapse MRD response to induction, 38% of patients showed a similar, 38% a better and 25% a poorer response after relapse. These data demonstrate an unexpectedly high clonal heterogeneity among very early/early relapses and challenge some current assumptions about relapsed ALL.

Therapy of steroid-refractory acute GVHD with CD52 antibody alemtuzumab is effective.

The efficacy and safety of CD52 antibody alemtuzumab to treat severe acute GVHD in 18 consecutive patients refractory to standard high-dose corticosteroid therapy is reported. Patients (age range 13-68 years) had developed acute GVHD grade III and IV with gut and/or liver involvement after stem cell transplantation from family donors (n= 7) or HLA-matched unrelated donors (n=11), including five donors with one or two HLA mismatches. Initially, in three patients, start doses of alemtuzumab in the range of 70-80 mg were applied and repeated after 3 to 4 weeks. Impressive responses were seen, but virus reactivation and bacterial infections were frequent. In an attempt to reduce this complication, the next nine patients received a reduced starting dose of 20-33 mg, and the last six patients received 3-13 mg repeated every 2-3 weeks. Seventeen of 18 patients responded to alemtuzumab, six patients are alive with a median follow-up of 108 weeks. Chronic GVHD was observed frequently. Although pronounced lymphocyte depletion requiring close monitoring for signs of infections seems inevitable for efficacy, alemtuzumab given in moderate doses has a substantial activity not only in intestinal but also in severe acute GVHD of the liver.

Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008.

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

Long-term results of five consecutive trials in childhood acute lymphoblastic leukemia performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6609 children (<18 years of age) were treated in five consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria and Switzerland. Probability of 10-year event-free survival (EFS) (survival) improved from 65% (77%) in study ALL-BFM 81 to 78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: (1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk (HR) ALL patients, and eliminated in non- HR non-T-ALL patients, if it was replaced by high-dose and intrathecal (IT) MTX; (2) omission of delayed re-intensification severely impaired outcome of low-risk patients; (3) 6-month-less maintenance therapy caused an increase in systemic relapses; (4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; (5) condensed induction therapy resulted in significant improvement of outcome; (6) the daunorubicin dose in induction could be safely reduced in low-risk patients and (7) intensification of consolidation/re-intensification treatment led to considerable improvement of outcome in HR patients.

Re-transplantation from the same unrelated donor in three adolescents with severe aplastic anemia after graft rejection.

Hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD) has become the accepted salvage treatment for patients with severe aplastic anemia (SAA) lacking a matched sibling donor and failing immunosuppressive treatment. However, non-engraftment and early rejection remain main reasons for treatment related morbidity and mortality. We report on three adolescents who were grafted from MUD, rejected their graft and were re-grafted 47-51 days after first HSCT from the same donor. For conditioning, fludarabine, cyclophosphamide, ATG and/or OKT3 in combination with total lymphoid irradiation was used. Unmanipulated peripheral blood stem cells at a minimum dose of 8 x 10(6)/kg CD34+cells were infused. Acute toxicity was low. Two patients are alive and well for more than 3 years, one patient developed extended chronic graft-versus-host disease from which he died 34 months after second HSCT. Re-transplantation from MUD in the case of non-engraftment or rejection from the same donor is possible following immunoablation combined with intensive serotherapy in young patients with SAA.

Prognostic significance of minimal residual disease in infants with acute lymphoblastic leukemia treated within the Interfant-99 protocol.

Acute lymphoblastic leukemia (ALL) in infants younger than 1 year is a rare but relatively homogeneous disease ( approximately 80% MLL gene rearranged, approximately 70% CD10-negative) when compared with childhood and adult ALL. Several studies in children and adults with ALL have shown that minimal residual disease (MRD) status is a strong and independent prognostic factor. We therefore evaluated the prognostic significance of MRD in infant ALL. Ninety-nine infant patients treated according to the Interfant-99 protocol were included in this study. MRD was analyzed by real-time quantitative PCR analysis of rearranged immunoglobulin genes, T-cell receptor genes and MLL genes at various time points (TP) during therapy. Higher MRD levels at the end of induction (TP2) and consolidation (TP3) were significantly associated with lower disease-free survival. Combined MRD information at TP2 and TP3 allowed recognition of three patients groups that significantly differed in outcome. All MRD-high-risk patients (MRD levels > or =10(-4) at TP3; 26% of patients) relapsed. MRD-low-risk patients (MRD level <10(-4) at both TP2 and TP3) constituted 44% of patients and showed a relapse-rate of only 13%, whereas remaining patients (MRD-medium-risk patients; 30% of patients) had a relapse rate of 31%. Comparison between the current Interfant-06 stratification at diagnosis and the here presented MRD-based stratification showed that both stratifications recognized different subgroups of patients. These data indicate that MRD diagnostics has added value for recognition of risk groups in infant ALL and that MRD diagnostics can be used for treatment intervention in infant ALL as well.

Pharmacokinetic monitoring of intravenous cyclosporine A in pediatric stem-cell transplant recipients. The trough level is not enough.

In order to monitor CsA serum levels after SCT, trough levels (C0) are widely used. The aim of this study was to estimate the population and individual PK parameters for patients receiving intravenous CsA after SCT. In 27 pediatric patients after SCT receiving CsA (3 mg/kg/day) every 12 h, a total of 289 CsA concentrations was obtained. To describe the PK parameters of CsA, a two-compartment model with first order elimination was used. Covariate analysis identified body weight, age, and the co-administration with itraconazole and tobramycine as factors influencing the Cl. The statistical comparison of AUC, trough level, and C2 indicates a correlation between AUC and C2, but no correlation between the AUC and C0, r = 0.24 (p = 0.146) vs. r = 0.526 (p = 0.000692), respectively. Our results underscore the fact that CsA trough levels do not reflect the drug exposure in patients receiving intravenous CsA after SCT. By contrast, CsA blood levels measured 2-6 h after CsA infusion showed a better correlation with the AUC. Our data provide new information to optimize the balancing act between GvHD-prophylaxis, graft vs. leukemia effect, and CsA side-effects after SCT.

Cluster analysis of genomic ETV6-RUNX1 (TEL-AML1) fusion sites in childhood acute lymphoblastic leukemia.

Fusion between ETV6 and RUNX1 defines the largest genetic subgroup in childhood ALL. The genomic fusion site, unique to individual patients and specific for the malignant clone, represents an ideal molecular marker for quantification of minimal residual disease. Sequencing of DNA breakpoints has been difficult due to the extended size of the respective breakpoint cluster regions. We therefore evaluated a specially designed multiplex long-range PCR assay in 65 diagnostic bone marrow samples for its suitability in routine use. Resulting fusion sites and breakpoints derived from previous studies were subject to cluster analysis to identify potential sequence motifs involved in translocation initiation.

Allogeneic hematopoietic SCT in children with ALL: current concepts of ongoing prospective SCT trials.

The definition of indications for allogeneic SCT in children with high-risk (HR) ALL in the first remission or after the first or subsequent relapse depends on biological features, response to treatment and survival after chemotherapy alone. As the results of frontline and relapse protocols are improving over time, there is a strong need for prospective SCT trials, ensuring a well-standardized procedure regarding all relevant components that are potentially responsible for heterogeneity in post-SCT outcome. Therefore, in 2003, the ALL-BFM and the ALL-REZ BFM Study Group initiated a prospective, international, multicenter trial (ALL-SCT-BFM 2003). This trial will now be extended to a larger consortium, trial ALL-SCT-BFM-international (ALL-SCT-BFMi). Strict rules define HLA-typing, donor selection, conditioning regimen, GvHD prophylaxis and therapy as well as standards of supportive care to reduce treatment-related mortality and establish an early GVL effect. Moreover, comprehensive and closely reviewed documentation and serious adverse event reporting shall ensure high study quality. Case-by-case discussions of any fatal or critical course during annual meetings will improve the culture of failure management and lead to modifications of guidelines of supportive care. Finally, the results of these prospective trials will determine the current potential of the different SCT procedures in HR or relapsed childhood ALL.

Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia.

Detection of minimal residual disease (MRD) is the most sensitive method to evaluate treatment response and one of the strongest predictors of outcome in childhood acute lymphoblastic leukemia (ALL). The 10-year update on the I-BFM-SG MRD study 91 demonstrates stable results (event-free survival), that is, standard risk group (MRD-SR) 93%, intermediate risk group (MRD-IR) 74%, and high risk group (MRD-HR) 16%. In multicenter trial AIEOP-BFM ALL 2000, patients were stratified by MRD detection using quantitative PCR after induction (TP1) and consolidation treatment (TP2). From 1 July 2000 to 31 October 2004, PCR target identification was performed in 3341 patients: 2365 (71%) patients had two or more sensitive targets (< or =10(-4)), 671 (20%) patients revealed only one sensitive target, 217 (6%) patients had targets with lower sensitivity, and 88 (3%) patients had no targets. MRD-based risk group assignment was feasible in 2594 (78%) patients: 40% were classified as MRD-SR (two sensitive targets, MRD negativity at both time points), 8% as MRD-HR (MRD > or =10(-3) at TP2), and 52% as MRD-IR. The remaining 823 patients were stratified according to clinical risk features: HR (n=108) and IR (n=715). In conclusion, MRD-PCR-based stratification using stringent criteria is feasible in almost 80% of patients in an international multicenter trial.

Relapse, not regimen-related toxicity, was the major cause of treatment failure in 11 children with Down syndrome undergoing haematopoietic stem cell transplantation for acute leukaemia.

We report a retrospective analysis of 11 children with Down syndrome (DS) treated by SCT in eight German/Austrian SCT centres. Indications for transplantation were acute lymphoblastic leukaemia (N=8) and acute myeloid leukaemia (N=3). A reduced intensity conditioning (RIC) containing 2 Gy TBI was given to two patients, another five received a myeloablative regimen with 12 Gy TBI. Treosulphan or busulphan was used in the remaining four children. Four of eleven (36%) patients are alive. All of them were treated with a myeloablative regimen. One of the four surviving children relapsed 9 months after SCT and is currently receiving palliative outpatient treatment. The main cause of death was relapse (5/11). Two children died of regimen-related toxicity (RRT), one from severe exfoliative dermatitis and multiorgan failure after a treosulphan-containing regimen, the other from GvHD-related infections after RIC. Acute GvHD of the skin was observed in 10 of 10 evaluable patients, and chronic GvHD in 4 of 8. Our data show that DS patients can tolerate commonly used, fully myeloablative preparative regimens. The major cause of death is relapse rather than RRT resulting in an event-free survival of 18% and over all survival of 36%.

Childhood secondary ALL after ALL treatment.

Data on secondary acute lymphoblastic leukaemia (sALL) following ALL treatment are very rare. However, the incidence might be underestimated as sALLs without a significant lineage shift might automatically be diagnosed as relapses. Examination of immunoglobulin and T-cell receptor gene rearrangements brought a new tool that can help in discrimination between relapse and sALL. We focused on the recurrences of childhood ALL to discover the real frequency of the sALL after ALL treatment. We compared clonal markers in matched presentation and recurrence samples of 366 patients treated according to the Berlin-Frankfurt-Munster (BFM)-based protocols. We found two cases of sALL and another three, where the recurrence is suspicious of being sALL rather than relapse. Our proposal for the 'secondary ALL after ALL' diagnostic criteria is as follows: (A) No clonal relationship between diagnosis and recurrence; (B) significant immunophenotypic shift--significant cytogenetic shift--gain/loss of a fusion gene. For the sALL (A) plus at least one (B) criterion should be fulfilled. With these criteria, the estimated frequency of the sALL after ALL is according to our data 0.5-1.5% of ALL recurrences on BFM-based protocols. Finally, we propose a treatment strategy for the patients with secondary disease.

Immunobiological diversity in infant acute lymphoblastic leukemia is related to the occurrence and type of MLL gene rearrangement.

The aim of this study was to identify immunobiological subgroups in 133 infant acute lymphoblastic leukemia (ALL) cases as assessed by their immunophenotype, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement pattern, and the presence of mixed lineage leukemia (MLL) rearrangements. About 70% of cases showed the pro-B-ALL immunophenotype, whereas the remaining cases were common ALL and pre-B-ALL. MLL translocations were found in 79% of infants, involving MLL-AF4 (41%), MLL-ENL (18%), MLL-AF9 (11%) or another MLL partner gene (10%). Detailed analysis of Ig/TCR rearrangement patterns revealed IGH, IGK and IGL rearrangements in 91, 21 and 13% of infants, respectively. Cross-lineage TCRD, TCRG and TCRB rearrangements were found in 46, 17 and 10% of cases, respectively. As compared to childhood precursor-B-ALL, Ig/TCR rearrangements in infant ALL were less frequent and more oligoclonal. MLL-AF4 and MLL-ENL-positive infants demonstrated immature rearrangements, whereas in MLL-AF9-positive leukemias more mature rearrangements predominated. The immature Ig/TCR pattern in infant ALL correlated with young age at diagnosis, CD10 negativity and predominantly with the presence and the type of MLL translocation. The high frequency of immature and oligoclonal Ig/TCR rearrangements is probably caused by early (prenatal) oncogenic transformation in immature B-lineage progenitor cells with germline Ig/TCR genes combined with a short latency period.

Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

Acute lymphoblastic leukemia with t(4;11) in children 1 year and older: The 'big sister' of the infant disease?

The t(4;11)-positive acute lymphoblastic leukemia (ALL) is a rare disease in children above the age of 1 year. We studied the clinical and biological characteristics in 32 consecutively diagnosed childhood cases (median age 10.0 years, range 1.0-17.1 years). Immunophenotyping revealed a pro-B and a pre-B stage in 24 and eight cases, respectively. IGH genes were rearranged in 84% of leukemias with a predominance of incomplete DJ(H) joints. Whereas IGK-Kde and TCRD rearrangements were rare, TCRG rearrangements were present in 50% of cases and involved mainly Vgamma11 or Vgamma9 together with a Jgamma1.3./2.3 gene segment, an unusual combination among t(4;11)-negative B-cell precursor ALL. Oligoclonality was found in about 30% as assessed by heterogeneous IGH and TCRG rearrangements. Our data are in line with transformation of a precursor cell at an early stage of B-cell development but retaining the potential to differentiate to the pre-B cell stage in vivo. Although a distinct difference between infant and older childhood cases with t(4;11) became evident, no age-related biological features were found within the childhood age group. In contrast to infants with t(4;11)-positive ALL, childhood cases had a relatively low cumulative incidence of relapse of 25% at 3.5 years with BFM-based high-risk protocols.