In view of the clinical and biological significance of leukemic heterogeneity we studied the efficacy of spontaneous apoptosis and cell cycle distribution in CD34+ and CD34− leukemic subpopulations.
...Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) leukemic samples with CD34 heterogeneous expression were separated into CD34+ and CD34− fractions using fluorescence activated cell sorting. Cell cycle distribution, and apoptosis of the sorted subpopulations were estimated.
CD34+ leukemic subpopulations had lower ability to apoptosis than that of CD34− fractions in 6 out of 8 ALL samples and in 4 out of 5 AML samples. CD34+ fractions showed a higher percentage of proliferating cells compared to CD34− cells in T-lineage ALL. These differences may lead to a more resistant phenotype of one of the subpopulations and reappearance this population in relapse.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To improve the outcome of children with standard and intermediate risk acute lymphoblastic leukemia (ALL) by the addition of PEG-asparaginase (PEG) on day 3 of remission induction was one of the key ...aims of trial ALL MB-2008 (J. Roumjantseva et al, Pediatriya, 2009). Therapy was designed based on previous MB-group studies (A. Karachunsky et al, Leukemia, 2008). Aim of the present study was to evaluate the influence of day 3 PEG on the end-induction MRD-status which is one of the significant predictors of outcome in children with B-cell precursor ALL (BCP-ALL).
Patients and methods. In total, 474 patients (242 boys and 232 girls aged 1 to 17 years (median 5 years 8 months) were studied. Inclusion criteria were: BCP-ALL, treatment according to protocol ALL MB-2008, compliance with standard risk group (SRG, n=266) and intermediate risk group (ImRG, n=208) criteria according to the protocol stratification system, and availability of end-induction (day 36) bone marrow (BM) samples. Remission induction of ALL MB-2008 consisted of oral dexamethasone (6mg/m2 daily), 5 injections of vincristine (1,5 mg/m2), 0-2 injections of daunorubicin (DNR, 45 mg/m2) and 6 triple intrathecal injections. Patients were randomized in respect to PEG administration on day 3. SRG patients were divided into three groups (PEG+DNR+, PEG+DNR- and PEG-DNR+); ImRG patients were split into two (PEG+DNR+ and PEG-DNR+). MRD detection was performed by multicolor flow cytometry (MFC) in three reference laboratories. Samples with a tumor cell level above 0.01% were considered MRD-positive. Outcome parameters were event-free survival (EFS) and cumulative incidence of relapse (CIR). Median of follow up was 5 years.
Results. Outcome in 474 study group patients was not significantly different from outcome of 2310 children with BCP-ALL enrolled in SRG and ImRG of ALL MB-2008 trial in whom MRD was not studied: EFS was 0.86±0.02 and 0.84±0.01, respectively, p=0.14, and CIR was 0.12±0.02 and 0.09±0.01, p=0.38.
MFC MRD data obtained at day 36 had a significant impact on prognosis in SRG patients. EFS as well as CIR were worse for patients with MRD >0.1% (n=26; EFS 0.47±0.15, CIR 0.49±0.17) compared with 248 patients with low/negative MRD (EFS 0.94±0.2, CIR 0.05±0.02, p<0.001). Nevertheless, the distribution of MRD levels was completely different dependent on randomization arms: in arm PEG-DNR+ (n=92) 18.5% of patients belonged to the group with unfavorable MRD compared with only 6.2% in arm PEG+DNR- (n=80, p=0.017) and 4.3% in arm PEG+DNR+ (n=94, p=0.002). Interestingly, in two PEG+ arms the number of patients with high MRD was not significantly different (p=0.558). Despite the lower proportion of high MRD levels in PEG+ arms MRD maintained its prognostic significance also in these patients. EFS was 0.65±0.16 and CIR 0.35±0.18 in 9 children with MRD ≥0.1 compared with EFS 0.94±0.02 and CIR 0.05±0.02 in 165 patients of the low MRD group (p<0.001 for both EFS and CIR).
In ImRG, MFC MRD data disclosed a prognostic value, as well. EFS was 0.92±0.03 and CIR 0.06±0.02 for 143 patients with MRD-negativity on day 36; in contrast, outcome of 78 MRD-positive patients was significantly inferior (EFS 0.73±0.06, CIR 0.25±0.06, p<0.001 for both comparisons). Like in SRG, in ImRG the distribution of MRD levels was different between two randomization arms. In PEG-DNR+ group (n=93) only 54.8% of patients were MRD-negative at the end of remission induction. In contrast, in PEG+DNR+ group (n=115) the proportion of patients with favorable MRD results was significantly higher (76.5%, p=0.001). Again even with low number of MRD-positive cases in PEG+ arm, day 36 MRD disclosed a significant influence on outcome: In 88 MRD-negative patients EFS was 0.93±0.03 and CIR 0.05+0.03, significantly better than in 27 MRD-positive patients (EFS 0.80+0.08 and CIR 0.17+0.08, p=0.025 and p=0.019, respectively).
Conclusions. Administration of PEG at day 3 of remission induction leads to a more rapid MRD response in children both with standard-risk and intermediate-risk BCP-ALL. In SRG, rapidity of MRD clearance in PEG+ arms was independent of the additional administration of daunorubicin. We conclude that end-induction MRD data are independent predictors of outcome despite the small proportion of MRD-positive patients after the implementation PEG during induction.
No relevant conflicts of interest to declare.
Acute lymphoblastic leukemia (ALL) in infants is a relatively rare disease with peculiar biological features, high frequency of KMT2A gene rearrangements and grim prognosis. Even with new therapeutic ...approaches, event-free survival (EFS) in infants with ALL does not exceed 50%. Currently large cooperative studies of infant ALL have been promoted by the Interfant and MLL-Baby networks. Minimal residual disease (MRD) monitoring is considered a strong tool for optimizing management of childhood ALL. In contrast to older children in this age group the prognostic impact of MRD detected by multicolor flow cytometry (MFC) is still unclear. Aim of the present study was to evaluate the prognostic value of MFC MRD measurement during induction in infants with ALL treated with Interfant-99 and Interfant-06 protocols in AIEOP centers in Italy as well as MLL-Baby protocol in Russia and Belarus.
Patients and methods. Two independent groups of patients were investigated: study cohort of 139 consecutive infants with newly diagnosed ALL enrolled between September 2003 and April 2016 in Russian and Belorussian centers with MLL-Baby protocol and validation group of 146 ones enrolled in AIEOP centers in Interfant trials during the same period. By availability of MFC MRD data obtained at day 15 and/or end of induction (EOI), 81 and 86 patients of study and validation cohorts respectively were selected for outcome evaluation. In 61 MLL-Baby patients (75.3%) and 61 AIEOP cases (70.9%) different types of KMT2A gene rearrangements were identified. All patients were diagnosed as BCP-ALL, except one with cortical T-ALL. Overall, day 15 samples were studied in 64 MLL-Baby patients and 73 AIEOP cases while EOI samples in 75 and 63 cases respectively. MRD detection was performed in Reference Laboratories in Ekaterinburg, Minsk and Padua according the BFM AIEOP FLOW Network SOP. MRD negativity was defined as <0.01% of all bone marrow nucleated cells.
Results. Patients were stratified according to the AIEOP-BFM-ALL day 15 stratification usually used for older children into three risk groups: standard risk (SR: MRD<0.1%), intermediate risk (IR: MRD 0.1% to 10%) and high risk (HR: MRD≥10%). Patients' distribution was similar in both study and validation cohorts: 34.4% and 32.9% in SR, 53.1% and 54.5% in ImR, 12.5% and 12.3% in HR respectively. At EOI significant differences in MRD-positive and MRD-negative patients' distribution was observed in different protocols: 44 (58.7%) and 31 (41.3%) cases respectively for MLL-Baby, but 17 (27.0%) and 46 (73.0%) cases respectively for AIEOP group. It was observed that KMT2A-rearranged cases in both trials have slower MRD response compared to the children with wild type KMT2A. In study cohort the 22 SR patients had a 5-year EFS and cumulative incidence of relapse (CIR) significantly better than other ones, thus we considered two major groups of patients with different outcome: SR with 5-year EFS 67.4%, standard error (SE) 10.2; CIR 23.3%, SE 9.4 and non-SR with 5-year EFS 30.8%, SE 7.2; CIR 52.6%, SE 7.9, (p=0.0039 and p=0.0229, respectively). Difference between these groups was observed also in KMT2A-rearranged cases (n=49) both for 5-year EFS (60.0%, SE 12.7 and 23.2%, SE 7.3, p=0.0160) and in 5-year CIR (33.3%, SE 12.7 and 59.2%, SE 8.8, p=0.0881). Analysis of outcome in validation cohort confirmed these data. In study cohort outcome of children being MRD-negative at EOI (n=31, 5-year EFS 60.8%, SE 8.8 and CIR 29.3%, SE 8.4) was significantly better than that of MRD-positive patients (n=44, 5-year EFS 31.1%, SE 7.1 and CIR 57.6%, SE 7.8 with p=0.0153 and p=0.0267, respectively). Outcome by EOI MFC MRD in the validation cohort is generally in keeping with that of the study cohort. Interestingly, in AIEOP cohort MFC data showed a prognostic impact also in KMT2A-rearranged subgroup. In multivariate analysis with KMT2A-status, each MRD time-point data showed independent impact on the risk of relapse.
Conclusion. Our data was obtained by well-harmonized MFC MRD monitoring in a large group of infants with ALL treated in a multicenter setting with two different protocols. In spite of differences in therapy, we observed strong and independent prognostic impact of MFC MRD both at day 15 and at EOI regardless the protocol applied. We can conclude that MFC MRD can be used in combination with KMT2A-status to improve treatment allocation in future protocols.
Parasole:Servier: Honoraria; Baxalta: Honoraria; Eusapharma: Honoraria. Pieters:jazz farmaceuticals: Consultancy; medac: Consultancy.
Objectives: A decade after being licensed for treatment of CML in minors, the TKI imatinib (IMA) is well known for it’s inhibitory “off-target” effects on activity and proliferative capacity of ...osteoclasts and osteoblasts resulting in impaired bone remodeling (Vandyke K et al 2010 Blood 115:766; Tauer JT et al Blood 2011:118). This causes longitudinal growth retardation in not outgrown individuals (Millot F et al 2009 Blood 114:863; Shima H et al 2011 Pediatrics 159:676; Bansal D et al 2012 Ped Blood Cancer 59:481) which can be aggravated by a disrupted growth hormone:IGF-I axis as a possible additional off-target effect exerted by TKI treatment (Ulmer A et al 2013 Klin Padiatr 225:120; Bansal D et al 2012 Ped Blood Cancer 59:481). Starting a pediatric trial in the year 2006 which recruits approx. 15 pediatric patients (pts) with CML annually, we investigated to what extend growth is impaired depending on sex, age, and pubertal stage at start of IMA treatment in a pediatric cohort.
Methods: 102 pts (54 male / 48 female; median age 12 years, range: 1-18 years) at diagnosis of CML receiving IMA as upfront treatment were enrolled retrospectively in this analysis from centers in Germany and participating countries during 02/2006 to 06/2014. Height standard deviation scores (SDS) were derived from WHO-AnthroPlus, version 1.04 software, a global growth-monitoring tool providing normal range values for the age cohorts from birth till 19 years. 81 out of 102 pts fulfilled the criteria for continuous assessment of growth scheduled at three months intervals during IMA exposure. 21 pts were analyzed at intervals ≠ 3 month. Pts excluded comprised individuals shifted to a 2nd generation TKI, or cumulative interruptions of drug intake exceeding 4 weeks, or pts undergoing stem cell transplantation.
Results: The mean and median duration of IMA exposure was 12 months and 9 months, respectively (range: 0–98 month). 27/102 pts (13 male, 14 female) were prepubertal (age: <10 years) at initiation of IMA treatment while 46/102 pts were pubertal (age: 10-14 years; 23 male, 23 female), and 29/102 pts were in postpubertal stage (age: >14 years; 18 male, 11 female). In comparison to mean SDS at diagnosis a mean decrease in height of 0.48 SDS per year was observed in the total cohort during the first three years of treatment, being more pronounced in prepubertal pts. In pts diagnosed shortly before or at puberty a mean reduction of 0.75 SDS per year during the first three years were observed. Older teenagers revealed no change in body height z-score during TKI treatment compared to height z-score at diagnosis.
Discussion: Growth retardation is a significant adverse effect of IMA in children with CML affecting predominantly prepubertal children. Possible medical interventions still need to be investigated.
Acknowledgment: Supported by grant DFG SU122-3/1 to MS.
Display omitted
No relevant conflicts of interest to declare.
Purpose
Favorable outcomes were achieved for children with acute lymphoblastic leukemia (ALL) with the first Russian multicenter trial Moscow–Berlin (ALL-MB) 91. One major component of this regimen ...included a total of 18 doses of weekly intramuscular (IM) native
Escherichia coli
-derived asparaginase (
E. coli
-ASP) at 10000 U/m
2
during three consolidation courses. ASP was initially available from Latvia, but had to be purchased from abroad at substantial costs after the collapse of Soviet Union. Therefore, the subsequent trial ALL-MB 2002 aimed at limiting costs to a reasonable extent and also at reducing toxicity by lowering the dose for standard risk (SR−) patients to 5000 U/m
2
without jeopardizing efficacy.
Methods
Between April 2002 and November 2006, 774 SR patients were registered in 34 centers across Russia and Belarus, 688 of whom were randomized. In arm ASP-5000 (
n
= 334), patients received 5000 U/m
2
and in arm ASP-10000 (
n
= 354) 10 000 U/m
2
IM.
Results
Probabilities of disease-free survival, overall survival and cumulative incidence of relapse at 10 years were comparable: 79 ± 2%, 86 ± 2% and 17.4 ± 2.1% (ASP-5000) vs. 75 ± 2% and 82 ± 2%, and 17.9 ± 2.0% (ASP-10000), while death in complete remission was significantly lower in arm ASP-5000 (2.7% vs. 6.5%;
p
= 0.029).
Conclusion
Our findings suggest that weekly 5000 U/m
2
E. coli-
ASP IM during consolidation therapy are equally effective, more cost-efficient and less toxic than 10000 U/m
2
for SR patients with childhood ALL.
Acute lymphoblastic leukemia (ALL) in infants (less than 1 year old) is a unique tumor with distinct biological features and poor outcome even when modern treatment schemes are used. Rearrangementsof ...MLL-gene, located in 11q23 region, are detected in approximately 75-80% of infants with ALL and lead to treatment resistance and high relapse rate. Nevertheless patients with germline MLL also demonstrate inferior outcome compared to ALL in older children. Thus additional risk factors implementation is one of the crucial points in infant ALL management. Minimal residual disease (MRD) measurement by multicolor flow cytometry (FCM) or various PCR technics is a well-standardized method of treatment response evaluation in childhood ALL, although in infants MRD data is not so widely applied. The aim of the study was to evaluate the relapse prediction feasibility in infant ALL by FCM MRD assessment during remission induction of MLL-Baby protocol.
Methods. Totally 89 infants aged from 5 days to 11 months were enrolled in the present study. In 23 cases (25.8%) MLL gene was germline (MLL-g group), 33 patients (37.1%) had MLL-AF4 fusion while in remaining 33 cases (37.1%) other types of MLL-rearrangements were found. All patients were diagnosed as B-cell precursor ALL and all were treated by well established in Russia and Belarus MLL-Baby protocol, which is specially designed for infant ALL management. MRD was measured by 6-10-color FCM in bone marrow (BM) samples obtained at day 15 and at the end of remission induction (day 36). The availability of samples at at least one of these time-points was the only criteria for study group completion. In 43 patients with known types of MLL-rearrangements MRD was also assessed by fusion gene transcript (FGT) detection in RQ-PCR after first consolidation or first high risk block (for intermediate risk and high risk groups respectively). Relapse risk was investigated by cumulative incidence of relapse (CIR) estimation. Median of follow-up was 3 years 10 months.
Results. Finally at day 15 MRD was studied in 71 cases. 17 patients (23.9%) were tested MRD-negative while remaining ones displayed various levels of MRD-positivity: 8 cases (11.3%) - from 0.01% to 0.1%; 14 (19.7%) - from 0.1% to 1%; 22 (31.0%) - from 1% to 10%; 10 (14.1%) - more than 10%. Proportion of MRD-positivity was lower in MLL-g group compared to MLL-rearranged (MLL-r) patients (58.8% and 81.5% respectively, p=0.06). Prognostic impact of day 15 MRD differed due to MLL-status. In MLL-r group significant differences between MRD(+) and MRD(-) patients were observed (n=10, CIR 0.28(0.18) and n=37, CIR 0.67(0.08) respectively, p=0.025). At the same time in MLL-g group these outcome differences were not significant (n=7, CIR 0 and n=9, CIR 0.22(0.14) correspondingly, p=0.197). Interestingly, in patients carrying MLL-AF4 fusion, known to be one of the most adverse types of MLL-rearrangements, day 15 MRD-negativity predicted low relapse incidence (n=5, CIR 0 and n=16, CIR 0.68(0.12) respectively, p=0.045). Thus day 15 MRD-negativity allows to detect low-risk MLL-r infants but it is not applicable in MLL-g group. It was previously shown that any detectable level of FGT after first consolidation or first high risk block predicts very poor outcome in MLL-r cases (G. Tsaur et al, ASH-2011). In current series RQ-PCR data in patients, FCM MRD(+) at day 15, distinguished groups of intermediate and very high relapse risk (n=17, CIR 0.51(0.13) and n=18, CIR 0.84(0.09) respectively, p=0.017). At day 36 FCM MRD was assessed in 82 infants. Among them 35 (42.7%) were tested negative while remaining 47 (57.3%) were MRD(+) at various levels. Prognostic value of day 36 FCM MRD data in MLL-r group was not significant: MRD(+) patients (n=23) had CIR of 0.71(0.08) while in MRD(-) cases (n=33) CIR was 0.49(0.12), p=0.153. Conversely, in MLL-g group low end-induction MRD (less than 0.1%) lead to excellent outcome compared to patients with higher MRD (n=14, CIR 0 and n=7, CIR 0.22(0.20) respectively, p=0.010).
Conclusions. Thus FCM MRD data could distinguish infants with low risk of ALL relapse, but in MLL-r and MLL-g groups different time-points are prognosticaly significant. In MLL-g patients tandem application of FCM at early time-point and RQ-PCR later could help to define groups with low, intermediate and high relapse risk. MRD data could be added to MLL-Baby protocol risk group stratification, which is currently based on type of MLL-rearrangement.
No relevant conflicts of interest to declare.
Two patients with acute T-lymphoblastic leukemia showed heterogeneous expression of some immunophenotypic cell markers. Cell sorting was used to separate two CD34/CD117/TCRgammadelta and ...CD34/CD117/TCRgammadelta cell populations. The sorted TCRgammadelta population had more cells in S phase than the TCRgammadelta population. PCR analysis revealed identical TCR gene rearrangements in both populations. At relapse of one of the patients, only CD117, CD34, TCRgammadelta cells remained. The authors assume the presence of two immunophenotypically different cell subsets in different maturation stages at diagnosis.
Variants in recombination-activating genes (
) are common genetic causes of autosomal recessive forms of combined immunodeficiencies (CID) ranging from severe combined immunodeficiency (SCID), Omenn ...syndrome (OS), leaky SCID, and CID with granulomas and/or autoimmunity (CID-G/AI), and even milder presentation with antibody deficiency.
We aim to estimate the incidence, clinical presentation, genetic variability, and treatment outcome with geographic distribution of patients with the
defects in populations inhabiting South, West, and East Slavic countries.
Demographic, clinical, and laboratory data were collected from
-deficient patients of Slavic origin via chart review, retrospectively. Recombinase activity was determined
by flow cytometry-based assay.
Based on the clinical and immunologic phenotype, our cohort of 82 patients from 68 families represented a wide spectrum of
deficiencies, including SCID (
= 20), OS (
= 37), and LS/CID (
= 25) phenotypes. Sixty-seven (81.7%) patients carried
and 15 patients (18.3%) carried
biallelic variants. We estimate that the minimal annual incidence of
deficiency in Slavic countries varies between 1 in 180,000 and 1 in 300,000 live births, and it may vary secondary to health care disparities in these regions. In our cohort, 70% (
= 47) of patients with
variants carried p.K86Vfs
33 (c.256_257delAA) allele, either in homozygous (
= 18, 27%) or in compound heterozygous (
= 29, 43%) form. The majority (77%) of patients with homozygous
p.K86Vfs
33 variant originated from Vistula watershed area in Central and Eastern Poland, and compound heterozygote cases were distributed among all Slavic countries except Bulgaria. Clinical and immunological presentation of homozygous
p.K86Vfs
33 cases was highly diverse (SCID, OS, and AS/CID) suggestive of strong influence of additional genetic and/or epigenetic factors in shaping the final phenotype.
We propose that
p.K86Vfs
33 is a founder variant originating from the Vistula watershed region in Poland, which may explain a high proportion of homozygous cases from Central and Eastern Poland and the presence of the variant in all Slavs. Our studies in this cohort of
founder variants confirm that clinical and immunological phenotypes only partially depend on the underlying genetic defect. As access to HSCT is improving among RAG-deficient patients in Eastern Europe, we anticipate improvements in survival.
Leukocyte adhesion deficiency (LAD) is an autosomal recessive, primary immunodeficiency disorder, characterized by lethal deep tissue infections, leukocytosis with impaired pus-formation, delayed ...wound healing and hyper-inflammation. Life expectancy of affected patients is severely shortened. LAD-I is caused by defective expression of beta-2 integrin on immune cells, resulting in impaired leukocyte adhesion and migration. LAD-II is a metabolic disease with syndromal features. LAD-III is caused by mutations in the FERMT3 gene resulting in activation defect of all beta-integrins. LAD-III is characterized by leukocyte adhesion/migration deficiency and an additional Glanzmann's thrombasthenia-like syndrome.
In this retrospective multi-center study, data were collected from the European Group for Blood and Marrow Transplantation (EBMT) registry. This study included 65 children from 14 European and Asian centers with either LAD-I (n=60) or LAD-III (n=5), all receiving an allogeneic hematopoietic stem-cell transplantation (alloHSCT) from 2008 to 2015.
Median age at diagnosis was 0.3 years (yrs); interval from diagnosis to alloHSCT was 8.1 months (median; range 0.6 to 134.5 months). An HLA-identical sibling or matched non-sibling family donor was available for 35 (54%) patients; 28 (43%) received the allograft from a matched unrelated donor and 2 (3%) patients were transplanted from an HLA-haplo-identical family donor. Stem cell source was bone marrow in 37 (58%) patients, peripheral blood in 17 (26%) and cord blood in 10 (16%) of the cohort. Reduced intensity conditioning (RIC) was used in 19 (29%) and myeloablative conditioning (MAC) in 46 (71%) patients. Half of the cohort (n=33) received a busulfan-based conditioning regimen; treosulfan was used in 18 (28%) patients. Serotherapy was applied to 71% of the cohort and consisted primarily of ATG (85%).
The overall survival of the cohort was 80% (95%CI: 70-91%) at 24 months, and 77% (95%CI: 65-89%) at 60 months post alloHSCT. In total 12 patients died. The cause of death was GVHD (n=6) and infections (n=6).
Primary or secondary graft failure occurred in 9 patients. The incidence of graft failure at 24 months was 11% (95% CI: 7-24%) in RIC and 14% (95% CI: 3-24%) in MAC group. A second alloHSCT was performed in 8 patients; it was successful in 7 cases.
At 100 days, 10% (95% CI: 2-17%) and 16% (95% CI: 7-25%) of patients developed grade I-II and grade III+IV aGVHD, respectively . Extensive chronic GVHD was reported in 2 cases.
AlloHSCT restored leukocyte function in LAD-I and both leukocyte and thrombocyte function in LAD-III leading to complete remission of symptoms with hematological and immunological reconstitution. Transplantation should be performed early in the course of the disease and prior to the occurrence of life-threatening infectious complications.
Bader:Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.
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