Key Points• Although the risk of ALL relapse is significantly higher in children with DS, goodprognosis subgroups have been identified. • Patients with DS-ALL have higher treatment-related mortality throughout the treatment period independent of the therapeutic regimen.Children with Down syndrome (DS) have an increased risk of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). The prognostic factors and outcome of DS-ALL patients treated in contemporary protocols are uncertain. We studied 653 DS-ALL patients enrolled in 16 international trials from 1995 to 2004. Non-DS BCP-ALL patients from the Dutch Child Oncology Group and Berlin-Frankfurt-Münster were reference cohorts. DS-ALL patients had a higher 8-year cumulative incidence of relapse (26% 6 2% vs 15% 6 1%, P < .001) and 2-year treatment-related mortality (TRM) (7% 6 1% vs 2.0% 6 <1%, P < .0001) than non-DS patients, resulting in lower 8-year event-free survival (EFS) (64% 6 2% vs 81% 6 2%, P < .0001) and overall survival (74% 6 2% vs 89% 6 1%, P < .0001). Independent favorable prognostic factors include age <6 years (hazard ratio [HR] 5 0.58, P 5 .002), white blood cell (WBC) count <10 3 10 9 /L (HR 5 0.60, P 5 .005), and ETV6-RUNX1 (HR 5 0.14, P 5 .006) for EFS and age (HR 5 0.48, P < .001), ETV6-RUNX1 (HR 5 0.1, P 5 .016) and high hyperdiploidy (HeH) (HR 5 0.29, P 5 .04) for relapse-free survival. TRM was the major cause of death in ETV6-RUNX1 and HeH DSALLs. Thus, while relapse is the main contributor to poorer survival in DS-ALL, infection-associated TRM was increased in all protocol elements, unrelated to treatment phase or regimen. Future strategies to improve outcome in DS-ALL should include improved supportive care throughout therapy and reduction of therapy in newly identified good-prognosis subgroups. (Blood. 2014; 123(1):70-77)

Children with Down's syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL) and have a low frequency of established genetic aberrations. We aimed to determine which genetic abnormalities are involved in DS ALL. We studied the frequency and prognostic value of deletions in B-cell development genes and aberrations of janus kinase 2 (JAK2) and cytokine receptor-like factor 2 (CRLF2) using array-comparative genomic hybridization, and multiplex ligation-dependent probe amplification in a population-based cohort of 34 Dutch Childhood Oncology Group DS ALL samples. A population-based cohort of 88 DS samples from the UK trials was used to validate survival estimates for IKZF1 and CRLF2 abnormalities. In total, 50% of DS ALL patients had X1 deletion in the B-cell development genes: PAX5 (12%), VPREB1 (18%) and IKZF1 (35%). JAK2 was mutated in 15% of patients, genomic CRLF2 rearrangements in 62%. Outcome was significantly worse in patients with IKZF1 deletions (6-year eventfree survival (EFS) 45 ± 16% vs 95 ± 4%; P ¼ 0.002), which was confirmed in the validation cohort (6-year EFS 21 ± 12% vs 58 ± 11%; P ¼ 0.002). This IKZF1 deletion was a strong independent predictor for outcome (hazard ratio EFS 3.05; P ¼ 0.001). Neither CRLF2 nor JAK2 were predictors for worse prognosis. If confirmed in prospective series, IKZF1 deletions may be used for risk-group stratification in DS ALL.

BackgroundChildren with Down syndrome have an increased risk of developing acute lymphoblastic leukemia and a poor tolerance of methotrexate. This latter problem is assumed to be caused by a higher cellular sensitivity of tissues in children with Down syndrome. However, whether differences in pharmacokinetics play a role is unknown. Design and MethodsWe compared methotrexate-induced toxicity and pharmacokinetics in a retrospective casecontrol study between patients with acute lymphoblastic leukemia who did or did not have Down syndrome. Population pharmacokinetic models were fitted to data from all individuals simultaneously, using non-linear mixed effect modeling. ResultsOverall, 468 courses of methotrexate (1-5 g/m 2 ) were given to 44 acute lymphoblastic leukemia patients with Down syndrome and to 87 acute lymphoblastic leukemia patients without Down syndrome. Grade 3-4 gastrointestinal toxicity was significantly more frequent in the children with Down syndrome than in those without (25.5% versus 3.9%; P=0.001). The occurrence of grade 3-4 gastrointestinal toxicity was not related to plasma methotrexate area under the curve. Methotrexate clearance was 5% lower in the acute lymphoblastic leukemia patients with Down syndrome (P=0.001); however, this small difference is probably clinically not relevant, because no significant differences in methotrexate plasma levels were detected at 24 and 48 hours. ConclusionsWe did not find evidence of differences in the pharmacokinetics of methotrexate between patients with and without Down syndrome which could explain the higher frequency of gastrointestinal toxicity and the greater need for methotrexate dose reductions in patients with Down syndrome. Hence, these problems are most likely explained by differential pharmacodynamic effects in the tissues between children with and without Down syndrome. Although the number of patients was limited to draw conclusions, we feel that it may be safe in children with Down syndrome to start with intermediate dosages of methotrexate (1-3 g/m 2 ) and monitor the patients carefully.Key words: metrotrexate pharmacokinetics, Down syndrome, acute lymphoblastic leukemia, methotrexate.Citation: Buitenkamp TD, Mathôt RAA, de Haas V, Pieters R, and Zwaan CM. Methotrexateinduced side effects are not due to differences in pharmacokinetics in children with Down's syndrome and acute lymphoblastic leukemia. Haematologica 2010;95:01106-1113. doi:10.3324/haematol.2009

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Children with Down syndrome (DS) have an increased risk of developing acute myeloid and B-cell precursor acute lymphoblastic leukemia (BCP-ALL). 1 The prognosis of DS ALL is at best similar and often inferior to that of sporadic ALL (non-DS) patients. 2,3 DS ALL is characterized by unique biological features when compared with non-DS ALL. For instance, DS ALL has a lower frequency of the favorable genetic abnormalities t(12;21)(p13;q22) (ETV6-RUNX1) and the unfavorable t(9;22)(q34;q11) (BCR-ABL1), 3,4 but a higher frequency of JAK2 mutations and CRLF2 rearrangements. 5,6 Using genome-wide screening techniques, several (novel) genomic aberrations involved in the pathogenesis of (non-) DS ALL were identified. 7,8 The potential prognostic impact of most of these novel aberrations and whether these patients may benefit from specific therapies targeted to these unique genetic features needs to be investigated further. The B-cell translocation gene 1 (BTG1) was recently described as a recurrent lesion in pediatric BCP-ALL. 7,9,10 It was originally identified as a translocation partner of c-MYC in B-cell chronic lymphocytic leukemia. 11,12 BTG1 is a highly conserved gene and belongs to the family of BTG/TOB genes. 11,13 It has a role in several crucial cellular processes, such as proliferation, and apoptosis. Recently, Lundin et al. 14 reported on a high frequency of BTG1 deletions (B29%) in DS ALL. However, the study included a limited number of DS ALL patients (n ¼ 17) and hence may not be an accurate estimate of the frequency of BTG1 deletions in DS ALL. Therefore, we investigated the frequency of BTG1 deletions in a large series of DS ALL patients and in addition analyzed the prognostic significance of BTG1 abnormalities. We screened a population-based cohort of 116 DS ALL patients enrolled in consecutive DCOG and UK treatment protocols (DCOG ALL 8, 9 and 10 and UK ALL ALL97, ALL97/99 and ALL2003). 8 Clinical and cell-biological data, including cytogenetics, were available for all cases. DCOG and UK centrally reviewed diagnosis, classification and clinical follow-up of the patients. The Institutional Review Board approved the investigations, and informed consent was obtained according to local law and regulations. To identify BTG1 deletions, we performed multiplex ligationdependent probe amplification (MLPA) analysis using the SALSA MLPA kit P335-A3 ALL-IKZF1 (MRC Holland, Amsterdam, the Netherlands), which contains probes for selected B-cell development and differentiation genes. 8 The BTG1 gene is covered by four probes, probes to exon 1, exon 2 and two additional probes localized to the highly conserved promoter region of the gene (area 1 and 2). The full list and location of the MLPA probes can be downloaded from the MRC Holland website (http://www.mrc-holland.com). Peak heights o0.7 (0.75 for MRC UKALL samples) and 41.3 times the control peak height were considered abnormal, with those o0.7 (0.75) representing deletions, and those 41.3 representing duplications. The Kaplan-Meier method was used to estimate the ...

The development of a second malignancy after diagnosis of childhood acute lymphoblastic leukemia (ALL) is a rare event. Certain second malignancies have been linked with specific elements of leukemia therapy, yet the etiology of most second neoplasms remains obscure and their optimal management strategies are unclear. This is a first comprehensive report of non-Hodgkin lymphomas (NHL) following pediatric ALL therapy, excluding stem-cell transplantation. We analyzed data of patients enrolled in 12 collaborative pediatric ALL trials between 1980-2018, who developed NHL following ALL diagnosis. Eighty-five patients developed NHL, with mature B-cell lymphoproliferations as the dominant subtype (56/85). Forty-six of these 56 cases (82%) occurred during or within six months of maintenance therapy. The majority (65%) exhibited histopathological characteristics associated with immunodeficiency, predominantly evidence of Epstein-Barr virus (EBV)-driven lymphoproliferation. We investigated 66 cases of post-ALL immunodeficiency-associated lymphoid neoplasms, 52 from our study and 14 additional cases from a literature search. With a median follow-up of 4.9 years, five-year overall survival for the 66 patients with immunodeficiency-associated lymphoid neoplasms was 67.4% (95% CI, 56-81). Five-year cumulative risks of lymphoid neoplasm- and leukemia-related mortality were 20% (95% CI, 10.2-30) and 12.4% (95% CI, 2.7- 22), respectively. Concurrent hemophagocytic lymphohistiocytosis was associated with increased mortality (HR, 7.32; 95% CI, 1.62 to 32.98; p=0.01). A large proportion of post-ALL lymphoid neoplasms are associated with an immunodeficient state, likely precipitated by ALL maintenance therapy. Awareness of this underrecognized entity and pertinent diagnostic tests are crucial for early diagnosis and optimal therapy.

3579 Children with Down syndrome (DS) have an increased risk of developing B-cell precursor acute lymphoblastic leukemia (BCP-ALL), characterized by a low frequency of the common genetic aberrations, and a high frequency of CRLF2 and JAK aberrations. Because this disease is relatively rare, the clinical outcome, treatment-related mortality (TRM) and prognostic factors of DS-ALL patients treated in contemporary protocols are uncertain. Previous studies demonstrated poorer survival and a high rate of treatment related mortality (TRM), but most studies are small since DS ALL patients comprise only 1–2% of all protocol patients. We therefore conducted a large retrospective study of 653 children with DS-ALL treated in clinical trials of 16 collaborative study groups between 1995 and 2005. All genotypes obtained from conventional karyotyping, FISH or RT-PCR were centrally reviewed and assigned to specific cytogenetic groups. The 310 girls and 343 boys have a median age of 5.0 years (range, 1.2 – 17.9) and a median white blood-cell count (WBC) of 10.2 × 109/L (range, 0.2 – 459). The 827 non-DS BCP-ALL control patients from the Dutch Childhood Oncology Group treated in the same era had similar WBC (8.8 × 109/L; p=0.25) but were younger (4.6 years; p< 0.001). The median follow-up time was 6.8 years for DS-ALL survivors and 7.3 years for non-DS-ALL survivors. DS patients have a higher 8-year cumulative incidence of relapse (CIR) (26±2% vs. 18±1%; p=0.001) and higher 2-year TRM (7±1% vs. 1.0±<0.1%; p<0.0001) than controls, resulting in lower 8-year event free survival (EFS) (64±2% vs. 78±1%; p<0.0001) and lower 8-year overall survival (OS) (74±2% vs. 86±1%; p<0.0001). In the multivariate analysis, age ≥ 6 years and WBC ≥ 10 × 109/L were independent predictors for poor EFS (HR = 1.68, p = 0.003; WBC = 1.79, p = 0.001, respectively), and poor relapse-free survival (HR = 1.99; p = 0.001; HR = 1.55; p = 0.04, respectively). DS patients with age <6 years and WBC <10 × 109/L constituted a favourable risk-group when compared to the remaining DS patients but still had a relatively high relapse rate (EFS, 78±3% vs. 58±3%; p <0.001, TRM, 3±1% vs. 9±1%; p =0.002, CIR, 17±3% vs. 30±2%; p = 0.003). This criterion was a better predictor of outcome than that in classic NCI-criteria, even after exclusion of patients with TEL-AML1 rearrangement or trisomies 4 and 10. Of the 444 (68%) patients with available cytogenetic data, 40.3% had normal cytogenetics, 9.0% was high hyperdiploid (HeH) (34% in non-DS ALL; p<0.001), 8.3% had TEL-AML1 rearrangements (21.7% in non-DS ALL; p <0.001), and 2% had t(8;14)(q11.2)(q32). Remarkably, the 8-year EFS was nearly identical between TEL-AML1- rearranged DS and non-DS patients (95±4% vs.92±3%; p=0.77). HeH DS patients showed a trend towards lower EFS when compared to HeH non-DS patients (77±7% vs. 86±2%; p=0.06). Within HeH DS ALL patients, the number of patients with trisomy 4+10 was only 45%. The OS of these patients was 88±8%, and there were no relapses. Data on CRLF2 aberrations and JAK2 R683 mutations was available for 134 and 141 patients, respectively. Neither CRLF2 aberrations (OS 74±5%; p=0.29, CIR 29±6%; p=0.23) nor JAK2 mutations predict prognosis in DS ALL (OS 71±8%; p=0.21, CIR 25±9%; p=0.55). In total, 32% (n=16) of TRM's in DS patients occurred during remission induction, most (n=10) were caused by infection. The inclusion of anthracyclines in induction had no impact on TRM. When comparing NCI-SR patients of the CCG/POG studies (3-drug induction) with patients treated on AIEOP/BFM-studies (4-drug-induction) the incidence of TRM was 1.5±1% vs. 1.7±1%; p= 0.46). Also the EFS was similar (64±6% vs. 69±5%; p= 0.39). There was no distinct decrease of TRM after induction, it was not related to a specific treatment-phase or treatment-regimen and also occurred 2 years after diagnosis (∼1%), suggesting better supportive care throughout the treatment is needed. In conclusion, DS patients enrolled in contemporary protocols with curative intent continued to have a poor survival due to both high rates of relapse and TRM. Within DS ALL, patients age < 6 year, WBC <10 × 109/L and the presence of TEL-AML1 or trisomies 4 and 10 are favourable prognostic factors, which may be used to guide risk-directed treatment. Disclosures: No relevant conflicts of interest to declare.

Children with Down Syndrome (DS) have an increased risk of developing leukemia, including both acute myeloid (ML DS), as well as acute lymphoblastic leukemia (DSALL). Recently, Izraeli et al. reported on an activating mutation (R683) localized in exon 16 of the Janus Kinase 2 (JAK2) gene, in 18% of DS-ALL (n=16) patients collected from 9 European study groups (ASH 2007). Screening of other leukemia subsets showed that this mutation was exclusive for DS-ALL patients. This JAK2 mutation differs from the V617F exon 14 mutation found in myeloproliferative diseases. JAK2 is located on chromosome 9p24, and belongs to a family of intracellular non-receptor protein tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. It plays an important role in regulating the processes of cell proliferation, differentiation and apoptosis in response to cytokines and growth factors. Between 1991 and 2007, 45 children with DS ALL were treated in the Netherlands, according to the DCOG protocols ALL 7–10. Of 36 children samples were available in the DCOG cell bank, on which we performed JAK2 mutation screening of the pseudokinase and kinase domains of JAK2 by direct sequencing. All 36 patients were classified as BCP-ALL. Mutations in JAK2 exon 16 were identified in 6 (16.6%) DS-ALL patients. In five patients a point mutation resulted in substitution of Arginine at position 683, the same as was described by Izraeli et al. In one patient an insertion was found. JAK2 mutated patients did not differ in age at diagnosis (3.3 vs. 5.1 years, p=0.08) or in sex (p= 0.8) compared to non-mutated DSALL patients. The diagnostic WBC for DS-ALL patients with a JAK2 exon 16 mutation was lower than for non-mutated patients (3.6×109/L vs. 12.1×109/L; p=0.04). Ploidy status based on karyotyping was known in 29/36 patients. None of the JAK2 mutated samples was hyperdiploid (&gt;52 chromosomes) vs. one in the non-mutated samples (p=0.89). TEL/AML rearrangements were screened in 23/36 samples, and 3/23 (13%) samples showed a TEL-AML rearrangement. None of the JAK2 mutated samples was TEL-AML rearranged (p= 0.76). One JAK2 mutated patient had a normal karyotype, the other JAK2 mutated patients had random cytogenetic abnormalities. We next analyzed the prognostic significance of JAK2 mutated DS-ALL children versus the other patients. The median follow up time for all patients was 3.1 years (range 0.1–15.1 years). Interestingly, none of the JAK2 mutated patients relapsed, versus 4/30 wild type JAK2 patients. The differences between pOS (100% vs. 83.3% p=0.41), pEFS (100% vs. 80%, p=0.38) and pDFS (100% vs. 82.8%, p=0.44) were not statistically significant, probably due to small numbers. Since DS-ALL children are more sensitive to the side effects of chemotherapy, and have relatively high toxic mortality rates, reduction of therapy intensity might be an option for DS-ALL children with a JAK2 exon 16 mutation, if our results could be confirmed in larger series. The development of specific JAK2 inhibitors may allow further reduction of chemotherapy.