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Inotuzumab Ozogamicin is a CD22-directed antibody conjugated to calicheamicin, approved in adults with relapsed or refractory (R/R) B cell acute lymphoblastic leukemia (BCP-ALL). Patients aged 1-18 years, with R/R CD22 + BCP-ALL were treated at the RP2D of 1.8 mg/m 2 . Using a single-stage design, with an overall response rate (ORR) ≤ 30% defined as not promissing and ORR > 55% as expected, 25 patients needed to be recruited to achieve 80% power at 0.05 significance level. Thirty-two patients were enrolled, 28 were treated, 27 were evaluable for response. The estimated ORR was 81.5% (95%CI: 61.9-93.7%), and 81.8% (18/22) of the responding subjects were minimal residual disease (MRD) negative. The study met its primary endpoint. Median follow up of survivors was 16 months (IQR: 14.49-20.07). One year Event Free Survival was 36.7% (95% CI: 22.2-60.4%), and Overall Survival was 55.1% (95% CI: 39.1-77.7%). Eighteen patients received consolidation (with HSCT and/or CAR T-cells therapy). Sinusoidal obstructive syndrome (SOS) occurred in seven patients. MRD negativity seemed correlated to calicheamicin sensitivity in vitro, but not to CD22 surface expression, saturation, or internalization. InO was effective in this population. The most relevant risk was the occurrence of SOS, particularly when InO treatment was followed by HSCT., (© 2022. The Author(s).)

Deficiency of the transcription factor GATA2 is a highly penetrant genetic disorder predisposing to myelodysplastic syndromes (MDS) and immunodeficiency. It has been recognized as the most common cause underlying primary MDS in children. Triggered by the discovery of a recurrent synonymous GATA2 variant, we systematically investigated 911 patients with phenotype of pediatric MDS or cellular deficiencies for the presence of synonymous alterations in GATA2. In total, we identified nine individuals with five heterozygous synonymous mutations: c.351C>G, p.T117T (N = 4); c.649C>T, p.L217L; c.981G>A, p.G327G; c.1023C>T, p.A341A; and c.1416G>A, p.P472P (N = 2). They accounted for 8.2% (9/110) of cases with GATA2 deficiency in our cohort and resulted in selective loss of mutant RNA. While for the hotspot mutation (c.351C>G) a splicing error leading to RNA and protein reduction was identified, severe, likely late stage RNA loss without splicing disruption was found for other mutations. Finally, the synonymous mutations did not alter protein function or stability. In summary, synonymous GATA2 substitutions are a new common cause of GATA2 deficiency. These findings have broad implications for genetic counseling and pathogenic variant discovery in Mendelian disorders.

Infants <1 year of age have a high prevalence of prognostically unfavorable leukemias and a presumed susceptibility to treatment-related toxicities. A total of 125 infants with acute myeloid leukemia (AML) were treated in studies AML-BFM-98 (n = 59) and -2004 (n = 66). Treatment regimens of both studies were comparable, consisting of intensive induction followed by four courses (mainly high-dose cytarabine and anthracyclines). Allogeneic-hematopoietic stem-cell-transplantation (allo-HSCT) in 1st remission was optional for high-risk (HR) patients. Most infants (120/125=96%) were HR patients according to morphological, cytogenetic/molecular genetic and response criteria. Five-year overall survival was 66 ± 4%, and improved from 61 ± 6% in study-98 to 75 ± 6% in study-2004 (P(logrank) 0.14) and event-free survival rates were 44 ± 6% and 51 ± 6% (P(logrank) 0.66), respectively. Results in HR infants were similar to those of older HR children (1-<2- or 2-<10-year olds, P(logrank) 0.90 for survival). Survival rates of HSCT in 1st remission, initial partial response and after relapse were high (13/14, 2/8 and 20/30 patients, respectively). The latter contributes to excellent 5-year survival after relapse (50±8%). Despite more severe infections and pulmonary toxicities in infants, treatment-related death rate was identical to that of older children (3%). Our data indicate that intensive frontline and relapse AML treatment is feasible in infants, toxicities are manageable, and outcome is favorable.

We report on the outcome of children with advanced primary myelodysplastic syndrome (MDS) transplanted from an HLA-matched sibling (MSD) or an unrelated donor (UD) following a preparative regimen with busulfan, cyclophosphamide and melphalan. Ninety-seven patients with refractory anemia with excess blasts (RAEB, n=53), RAEB in transformation (RAEB-T, n=29) and myelodysplasia-related acute myeloid leukemia (MDR-AML, n=15) enrolled in the European Working Group of MDS in Childhood (EWOG-MDS) 98 study and given hematopoietic stem cell transplantation (HSCT) were analyzed. Median age at HSCT was 11.1 years (range 1.4-19.0). Thirty-nine children were transplanted from an MSD, whereas 58 were given the allograft from a UD (n=57) or alternative family donor (n=1). Stem cell source was bone marrow (n=69) or peripheral blood (n=28). With a median follow-up of 3.9 years (range 0.1-10.9), the 5-year probability of overall survival is 63%, while the 5-year cumulative incidence of transplantation-related mortality (TRM) and relapse is 21% each. Age at HSCT greater than 12 years, interval between diagnosis and HSCT longer than 4 months, and occurrence of acute or extensive chronic graft-versus-host disease were associated with increased TRM. The risk of relapse increased with more advanced disease. This study indicates that HSCT following a myeloablative preparative regimen offers a high probability of survival for children with advanced MDS.

Juvenile myelomonocytic leukemia (JMML) is a clonal myeloproliferative disorder of early childhood. In all, 21 patients with JMML who received donor leukocyte infusion (DLI) after allogeneic hematopoietic stem cell transplantation (HSCT) for either mixed chimerism (MC, n=7) or relapse (n=14) were studied. Six patients had been transplanted from an HLA-matched sibling and 15 from other donors. Six of the 21 patients (MC: 3/7 patients; relapse: 3/14 patients) responded to DLI. Response rate was significantly higher in patients receiving a higher total T-cell dose (> or =1 x 10(7)/kg) and in patients with an abnormal karyotype. None of the six patients receiving DLI from a matched sibling responded. Response was observed in five of six patients who did and in one of 15 children who did not develop acute graft-versus-host disease following DLI (P=0.01). The overall outcome was poor even for the responders. Only one of the responders is alive in remission, two relapsed, and three died of complications. In conclusion, this study shows that some cases of JMML may be sensitive to DLI, this providing evidence for a graft-versus-leukemia effect in JMML. Infusion of a high number of T cells, strategies to reduce toxicity, and cytoreduction prior to DLI may improve the results.

The International Prognostic Scoring System (IPSS) for myelodysplastic syndrome (MDS) is based upon weighted data on bone marrow (BM) blast percentage, cytopenia, and cytogenetics, separating patients into four prognostic groups. We analyzed the value of the IPSS in 142 children with de novo MDS and 166 children with juvenile myelomonocytic leukemia (JMML) enrolled in retro- and prospective studies of the European Working Group on childhood MDS (EWOG-MDS). Survivals in MDS and JMML were analyzed separately. Among the criteria considered by the IPSS score, only BM blasts <5% and platelets >100 x 10(9)/l were significantly associated with a superior survival in MDS. In JMML, better survival was associated with platelets >40 x 10(9)/l, but not with any other IPSS factors including cytogenetics. In conclusion, the IPSS is of limited value in both pediatric MDS and JMML. The results reflect the differences between myelodysplastic and myeloproliferative diseases in children and adults.

The clinical significance of WT1 gene expression at diagnosis and during therapy of AML has not yet been resolved. We analysed WT1 expression at presentation in an unselected group of 47 childhood AML patients using real-time quantitative reverse-transcription PCR. We also showed that within the first 30 h following aspiration RQ-RT-PCR results were not influenced by transportation time. We observed lower levels of WT1 transcript in AML M5 (P = 0.0015); no association was found between expression levels and sex, initial leukocyte count and karyotype-based prognostic groups. There was significant correlation between very low WT1 expression at presentation and excellent outcome (EFS P = 0.0014). Combined analysis of WT1 levels, three-colour flow cytometry residual disease detection and the course of the disease in 222 samples from 28 children with AML showed remarkable correlation. Fourteen patients expressed high WT1 levels at presentation. In eight of them, who suffered relapse or did not reach complete remission, dynamics of WT1 levels clearly correlated with the disease status and residual disease by flow cytometry. We conclude that very low WT1 levels at presentation represent a good prognostic factor and that RQ-RT-PCR-based analysis of WT1 expression is a promising and rapid approach for monitoring of MRD in approximately half of paediatric AML patients.

Pre-school acute lymphoblastic leukemia (ALL) peak is consistent in developed but not in developing countries and its magnitude apparently correlates with the socioeconomic status. A population-based study describing ALL incidence during socioeconomic transition has been lacking. Central European post-communist countries (with very low foreign migration and centralized statistics) offer reliable data for the period before and during major socioeconomic changes. Population-based data on Czech ALL patients younger than 18 years were taken from two independent Czech national registries partially overlapping in time (1980-1998, n = 1236 and 1991-1999, n = 570). During the 1980s and 1990s, ALL incidence among children 1-4 years increased 1.5 times (P = 0.01). This increase was more prominent in females than in males (slopes 0.13 and 0.09, P values 0.03 and >0.05, respectively). No significant change was observed in other age groups (0, 5-9, 10-14, 15-17 years or all others combined). We discuss possible underlying socioeconomic factors including infant care and breast-feeding, hygiene, birth order, industry and pollution. Moreover, we try to pinpoint the immunophenotypic/molecular-genetic subsets of ALL that might be socioeconomically affected. Selective increase of ALL in children 1-4 years old provides epidemiological evidence that etiology and/or trigger mechanisms are different for a considerable proportion of these children and that these mechanisms are exogenous.

We reviewed the clinical features, treatment, and outcome of 100 children with myelodysplastic syndrome (MDS), juvenile myelomonocytic leukemia (JMML), and acute myeloid leukemia (AML) associated with complete monosomy 7 (-7) or deletion of the long arm of chromosome 7 (7q-). Patients with therapy-induced disease were excluded. The morphologic diagnoses according to modified FAB criteria were: MDS in 72 (refractory anemia (RA) in 11, RA with excess of blasts (RAEB) in eight, RAEB in transformation (RAEB-T) in 10, JMML in 43), and AML in 28. The median age at presentation was 2.8 years (range 2 months to 15 years), being lowest in JMML (1.1 year). Loss of chromosome 7 as the sole cytogenetic abnormality was observed in 75% of those with MDS compared with 32% of those with AML. Predisposing conditions (including familial MDS/AML) were found in 20%. Three-year survival was 82% in RA, 63% in RAEB, 45% in JMML, 34% in AML, and 8% in RAEB-T. Children with -7 alone had a superior survival than those with other cytogenetic abnormalities: this was solely due to a better survival in MDS (3-year survival 56 vs 24%). The reverse was found in AML (3-year survival 13% in -7 alone vs 44% in other cytogenetic groups). Stable disease for several years was documented in more than half the patients with RA or RAEB. Patients with RA, RAEB or JMML treated with bone marrow transplantation (BMT) without prior chemotherapy had a 3-year survival of 73%. The morphologic diagnosis was the strongest prognostic factor. Only patients with a diagnosis of JMML fitted what has previously been referred to as the monosomy 7 syndrome. Our data give no support to the concept of monosomy 7 as a distinct syndrome.

The presence of TEL/AML1 fusion gene in childhood acute lymphoblastic leukaemia (ALL) defines a subgroup of patients with better than average outcome. However, the prognostic significance of this aberration has recently been disputed by the Berlin-Frankfurt-Münster (BFM) study group due to its relatively high incidence found in relapsed patients (19.6% and 21.9%, in two cohorts). In contrast, only four out of 45 (8.9%) unselected relapsed patients (all of whom had been treated according to BFM protocols) in the Czech Republic carry this fusion. From March 1995 to June 1998, 41 out of 190 (21.6%) newly diagnosed children with ALL were TEL/AML1-positive. There is a statistically significant difference between the incidence of TEL/AML1 fusion at diagnosis and at relapse within our group (P = 0.035). Interim analysis of the minimal residual disease (MRD) detection shows heterogeneity within the group of newly diagnosed TEL/AML1-positive leukaemias--10 out of 24 patients tested at the end of induction therapy had detectable levels of MRD. However, only one of these patients reached relapse-predictive level (10(-3)) of MRD. In conclusion, we corroborate low frequency of TEL/AML1 positivity among relapsed patients with ALL among Czech children who are treated by the BFM protocols. Moreover, we demonstrate different patterns of bone marrow clean-up in TEL/AML1-positive patients.

Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assessing the amount of transcripts of the BCR/ABL gene, the molecular marker of chronic myeloid leukemia (CML), is the only method sensitive enough for monitoring of minimal residual disease (MRD) in CML patients after bone marrow transplantation (BMT). In this study we present a simple modification of competitive Q-RT-PCR using natural competitors from cell lines K562 and BV173. The competitors were used in the form of unpurified RNA in cell lysates which ensured their high stability. Mixing competitors and samples before RNA extraction eliminated problems with quantification of cDNA or RNA entering the competitive reaction and with checking for the RNA quality and reverse transcription (RT) efficiency. The bulk of the malignant clone was expressed as the number of leukemic cells in 10(6) leukocytes when the overproduction of the BCR/ABL mRNA in the cell lines we used as competitors was taken into account. It was found to be 82-fold and 14-fold in K562 and BV173, respectively, in comparison with 100% Ph-positive CML standard. The assay reliability was verified by comparison of results with the mathematical model of competitive PCR. The assay is highly reproducible and sensitive (10(-5)). Its accuracy was proved to be excellent in a wide range of malignant cell concentrations. The method is demonstrated on three CML patients suffering from MRD after BMT. In conclusion, this method fulfills all criteria of competitive Q-RT-PCR. Because of its simplicity it is suitable for clinical laboratories and due to the high stability of the lysates used it may serve in the standardization of results between different laboratories.

The antigen KOR-SA3544 is physiologically expressed exclusively on granulocytes. Aberrant expression of KOR-SA3544 has been invariably found in BCR/ABL-positive acute lymphoblastic leukemia (ALL) and in some BCR/ABL-negative ALL. In an interim analysis of a prospective clinical and cytometric study data of 73 children with newly diagnosed or relapsed ALL with and without TEL/AML1 fusion are presented. KOR-SA3544 expression over 3% was detected in the majority of TEL/AML1-negative patients with newly diagnosed common or preB ALL (19 of 31) and not in TEL/AML1-positive patients (0 of 18, P < 0.0001). The level of expression of KOR-SA3544 was 0.02-90% (median 6.0%) and 0.03-2.4% (median 0.23%) in TEL/AML1-negative and TEL/AML1-positive patients, respectively. All five newly diagnosed patients with DNA index > or =1.16 and <1.6 exhibited high levels of KOR-SA3544 expression. Membrane expression of CD79a was found to correlate with TEL/AML1 negativity, although less significantly than KOR-SA3544 (P = 0.03). Furthermore, our data confirm that TEL/AML1 positivity correlates with non-hyperdiploidy and low presenting age. In conclusion, KOR-SA3544 correlated strongly with TEL/AML1 negativity, it was a better predictor of TEL/AML1 status than other factors tested and was found at high levels in hyperdiploidy. In combination with age, KOR-SA3544 predicted TEL/AML1 status in 86% newly diagnosed preB/cALL patients.