Intrachromosomal amplification of chromosome 21 (iAMP21) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children is a high-risk subtype for which targeted drugs are lacking. In this study, we determined the frequency of secondary lesions in 28 iAMP21 BCP-ALL patient samples and investigated cellular sensitivity for candidate-targeted drugs. iAMP21 was enriched in FLT3 aberrations (10.7% vs. 50.0%, p = 0.003) and SH2B3 inactivation (7.14% vs. 46.4%, p = 0.002), compared with 28 B-other cases, and these alterations co-occurred in 21.4%. The occurrence of lesions in CRLF2 and IL7R was similar between iAMP21 and B-other cases (25% vs. 17.9%, p = 0.746 and 7.14% vs. 0%, p = 0.491 respectively) as were mutations in JAK1 and JAK2 (3.57% vs. 0% and 10.7% vs. 10.7%, p = 1 for both). Sensitivity to the FLT3 inhibitor gilteritinib did not differ between iAMP21 and B-other cases irrespective of FLT3 status. However, iAMP21 samples harboring both FLT3-ITD and SH2B3 lesions showed the highest sensitivity. CRLF2-rearranged iAMP21 samples were slightly more sensitive to JAK inhibitor ruxolitinib than those without, although a lack of sensitivity was present in 50% of iAMP21 cases. Trametinib sensitivity varied among iAMP21 samples with over half of iAMP21 samples being sensitive irrespective of RAS-pathway mutation status or other secondary lesions. In summary, iAMP21 leukemias were enriched in FLT3 and in SH2B3 lesions, which when co-occurring affected sensitivity to FLT3 inhibition by gilteritinib but not JAK inhibition by ruxolitinib. Together, our results suggest that FLT3 and RAS signaling inhibitors are of interest for further (pre)clinical evaluation in iAMP21 BCP-ALL.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and adolescents and in recent decades, the survival rates have risen to >90% in children largely due the introduction of risk adapted therapy. Therefore, knowledge of factors influencing risk of relapse is important. The transcription factor IKAROS is a regulator of lymphocyte development and alterations of its coding gene, IKZF1, are frequent in ALL and are associated with higher relapse risk. This concise review will discuss the normal function of IKAROS together with the effect of gene alterations in ALL such as relieved energy restriction and altered response to anti-leukemic drugs. Besides the biology, the clinical impact of gene alterations in the different subtypes of ALL will be discussed. Finally, possibilities for treating ALL with IKZF1 alterations will be considered including novel therapies like cell signaling inhibitors and immunotherapy.

Recent trials show 5-year survival rates >95% for ETV6::RUNX1 Acute Lymphoblastic Leukemia (ALL). Since treatment has many side effects, an overview of cumulative drug doses and intensities between eight international trials is presented to characterize therapy needed for cure. A meta-analysis was performed as a comprehensive summary of survival outcomes at 5 and 10 years. For drug dose comparison in non-high risk trial arms, risk group distribution was applied to split the trials into two groups: trial group A with ~70% (range: 63.5-75%) of patients in low risk (LR) (CCLSG ALL2004, CoALL 07-03, NOPHO ALL2008, UKALL2003) and trial group B with ~45% (range: 38.7-52.7%) in LR (AIEOP-BFM ALL 2000, ALL-IC BFM ALL 2002, DCOG ALL10, JACLS ALL-02). Meta-analysis did not show evidence of heterogeneity between studies in trial group A LR and medium risk (MR) despite differences in treatment intensity. Statistical heterogeneity was present in trial group B LR and MR. Trials using higher cumulative dose and intensity of asparaginase and pulses of glucocorticoids and vincristine showed better 5-year event-free survival but similar overall survival. Based on similar outcomes between trials despite differences in therapy intensity, future trials should investigate, to what extent de-escalation is feasible for ETV6::RUNX1 ALL.

Introduction Intrachromosomal amplification of chromosome 21 (iAMP21) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children is a high-risk subtype for which targeted drugs are lacking. In this study we aimed to determine the frequency of secondary lesions and investigated the cellular sensitivity for candidate targeted drugs. Methods We performed total RNA sequencing on 28 iAMP21 and 28 B-other (negative for sentinel fusion genes) pediatric samples to determine the frequency of secondary lesions in newly diagnosed patients. A panel of 18 patient derived xenografts (PDX) of 8 primary iAMP21 ALL samples was generated, and secondary lesions were validated by PCR, RT-PCR, and whole exome sequencing. To test sensitivity, primary or PDX cells were exposed ex vivo to a concentration range of gilteritinib (FLT3 inhibitor), trametinib (MEK1/2 inhibitor), or ruxolitinib (JAK1/2 inhibitor). Results Secondary lesions in the cytokine receptor gene FLT3 were enriched in iAMP21 compared with B-other ALL including internal tandem duplications (ITD) and other activating lesions (50.0% vs. 10.7%, p=0.003). Lesions in genes encoding cytokine receptors CRLF2 and IL7R had a similar frequency between iAMP21 and B-other cases (25% vs. 17.9%, p=0.75 and 7.1% vs. 0%, p=0.49 respectively). Inactivating lesions in SH2B3, the downstream negative regulator of JAK/STAT and FLT3 signalling, were more frequent in iAMP21 cases vs. B-other (46.4% vs. 7.1%, p=0.002), while the frequency of JAK1 and JAK2 mutations did not differ (3.6 vs. 0% and 10.7 vs. 10.7% p = 1 for both). All SH2B3, CRLF2 and JAK lesions were retained in PDX samples, whereas in contrast FLT3-ITD was retained in only 2 of 5 PDX. Gilteritinib sensitivity did not differ between iAMP21 and B-other cases (median LC50 1.24 µM, range 0.33 to 4.85 µM vs. median LC50 1.21 µM, range 1.20 to 1.3 µM, p=0.95). Grouping iAMP21 cases by FLT3 and SH2B3 status, samples with both FLT3-ITD and SH2B3 lesion had the highest sensitivity to gilteritinib (median LC50 0.39 µM, range 0.35 to 0.43 µM). Samples with only FLT3 or SH2B3 lesion did not show increased sensitivity compared to those without a lesion (p>0.5 for both). Median ruxolitinib sensitivity did not statistically differ between iAMP21 and B-other cases (median LC50 8.38 µM, range 1.00 to >10 µM, vs. median LC50 0.48, range 0.21 to >10 µM; p=1) although extreme resistance to ruxolitinib seemed more frequent in iAMP21 cases (6 out of 12 cases) and was not related to FLT3 or SH2B3 status. CRLF2-rearranged ( CRLF2r) iAMP21 cases were slightly more sensitive to ruxolitinib than those lacking CRLF2r, although this difference did not reach significance (median LC50 4.72 µM, range 1.0-4.85 vs. median LC50 >10 µM, range 2.66 to >10 µM; p=0.08). The highest sensitivity was found in the only case with both a CRLF2r and a JAK1 mutation (LC50 of 1.00 µM). A large variation was present in trametinib sensitivity amongst both iAMP21 and B-other cases (median LC50 0.11 µM, range 0.017 to >5 µM vs. median LC50 0.18, range 0.018 to >5 µM; p=0.72). More than half of iAMP21 cases were sensitive irrespective of RAS-pathway lesion status. Conclusion iAMP21 leukemias are enriched in FLT3 and in SH2B3 lesions which, when co-occurring, affect sensitivity to FLT3 inhibition by gilteritinib but do not affect JAK-inhibition by ruxolitinib. This suggests these lesions act synergistically and might bypass downstream JAK/STAT signalling. This might also explain the observed sensitivity to RAS-pathway inhibition irrespective of secondary lesions. These results suggest that further research into FLT3 and RAS signalling inhibitors might lead to better treatment options for pediatric iAMP21 BCP-ALL.

IKZF1 deletions are an established prognostic factor in childhood acute lymphoblastic leukemia (ALL). However, their relevance in patients with good risk genetics, namely ETV6 :: RUNX1 and high hyperdiploid (HeH), ALL remains unclear. We assessed the prognostic impact of IKZF1 deletions in 939 ETV6 :: RUNX1 and 968 HeH ALL patients by evaluating data from 16 trials from 9 study groups. Only 3% of ETV6 :: RUNX1 cases (n = 26) were IKZF1 -deleted; this adversely affected survival combining all trials (5-year event-free survival [EFS], 79% versus 92%; P = 0.02). No relapses occurred among the 14 patients with an IKZF1 deletion treated on a minimal residual disease (MRD)-guided protocols. Nine percent of HeH cases (n = 85) had an IKZF1 deletion; this adversely affected survival in all trials (5-year EFS, 76% versus 89%; P = 0.006) and in MRD-guided protocols (73% versus 88%; P = 0.004). HeH cases with an IKZF1 deletion had significantly higher end of induction MRD values ( P = 0.03). Multivariate Cox regression showed that IKZF1 deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (hazard ratio of relapse rate [95% confidence interval]: 2.48 [1.32-4.66]). There was no evidence to suggest that IKZF1 deletions affected outcome in the small number of ETV6 :: RUNX1 cases in MRD-guided protocols but that they are related to higher MRD values, higher relapse, and lower survival rates in HeH ALL. Future trials are needed to study whether stratifying by MRD is adequate for HeH patients or additional risk stratification is necessary.

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and recent cure rates approach 90% on first line therapy. IKZF1 deletions have been reported as an unfavorable prognostic factor and are rare in ETV6::RUNX1 and high hyperdiploid (HeH) ALL, the two cytogenetic subgroups with the most favorable prognosis. Due to its rarity, the prognostic effect of IKZF1deletions within this subset of patients remains unclear. Therefore, we assessed the prognostic impact of IKZF1deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients. We performed this retrospective analysis on data of children and adolescents of 1-18 years with B-cell precursor ALL diagnosed in 1991-2016 and treated on one of 16 trials from 9 study groups, of which 6 were minimal residual disease (MRD) guided. Cytogenetic, fluorescence in-situ hybridization, and RT-PCR analyses of pre-treatment bone marrow samples to determine ploidy and fusion gene status were performed locally. Trials using MRD for risk stratification applied either PCR or flow cytometry analyses. The IKZF1 status was determined by multiplex ligation-dependent probe amplification by each individual study group. 3% of ETV6::RUNX1 (n=26) cases had an IKZF1 deletion; this affected survival adversely overall (5-year event free survival [EFS]: 79% vs. 92%, p = 0.02) (Table 1). However, in minimal residual disease (MRD) guided protocols, IKZF1 did not affect outcome in ETV6::RUNX1 cases ( 5-year EFS: 100% vs. 93%, p = 0.34) (Table 1). 9% of HeH cases had an IKZF1 deletion (n=85); this adversely affected survival overall (5-year EFS: 76% vs. 89%, p = 0.006), and in MRD-guided protocols (73% vs. 88%, p = 0.004) (Table 1). Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (Hazard ratio of EFS [95% CI]: 2.00 [1.20-3.30]) (Table 1). As the prognostic effect of IKZF1 deletions has been shown to vary by MRD risk group, we examined the prognostic effect of IKZF1deletions in HeH cases with detectable MRD <5% at end of induction in more detail. Of these cases, 10% carried an IKZF1deletion (n=34) which negatively affected outcome (5-year EFS: 78% vs. 92%, p = 0.01) Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in this intermediate MRD HeH ALL (Hazard ratio of EFS [95% CI]: 2.90 [1.20-6.80]). However, cases with an IKZF1 deletion had significantly higher MRD values than their wild-type counterparts (p = 0.03, Figure 1). In conclusion, our analysis of a large composite cohort consisting of 16 trials shows that IKZF1deletions do not affect survival in ETV6::RUNX1 ALL when treated on MRD-guided protocols. In contrast, our data shows that in HeH ALL IKZF1deletions are associated with lower survival rates, higher relapse rates, and higher MRD values. Future results of current trials such as the ALLTogether will likely reveal whether risk stratification predominantly reliant on MRD is adequate for HeH patients or whether stratification by copy number alteration profile, including IKZF1 status, or by other methods would be more suitable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background Socioeconomic differences in survival among children with acute lymphoblastic leukemia (ALL) have been reported in high-income countries and there is an unmet need for strategies to identify vulnerable patient subgroups. Reported differences in survival for children from families with different socioeconomic positions seem to arise when starting maintenance therapy. This could reflect reduced physician's compliance or family adherence to maintenance therapy. Methods This nationwide cohort study with extensive monitoring of systemic methotrexate (MTX)/6-mercaptopurine (6MP) dosing and metabolite levels, retrospectively investigated 173 Danish children treated according to The Nordic Society for Pediatric Hematology and Oncology ALL2008 protocol from 2008 to 2016. Results Significantly lower prescribed doses of MTX and 6MP were seen in the children in families with short parental education (short vs. medium vs. higher education: mMTX: 13.8, 16.2, and 18.6 mg/m(2)/week; p < .01; m6MP: 47.4, 64.9, and 66.1 mg/m(2)/day; p = .03) or parents unemployed/not in workforce (unemployed/not in workforce vs. mixed vs. at work: mMTX: 15.0, 19.9, and 17.2 mg/m(2)/week; p < .01; m6MP: 54.8, 72.0, and 65.1 mg/m(2)/day; p < .01). When assessing family adherence by analyzing MTX and 6MP metabolite levels, including per prescribed dose of MTX and 6MP, we found no significant differences by levels of parental education, affiliation to work market, or income (p > .05 for all comparisons). Conclusions These results indicate that inferior physician compliance to protocol recommendations on drug dosage rather than families' adherence to therapy may contribute to the association between socioeconomic position and cure rates in childhood ALL, although precise mechanisms remain to be explored.