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Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPKgene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK(CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPKalleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPKgene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.

Most relapses in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are not predicted using current prognostic features. Here, we determined the co-occurrence and independent prognostic relevance of 3 recently identified prognostic features: BCR-ABL1-like gene signature, deletions in IKZF1, and high CRLF2 messenger RNA expression (CRLF2-high). These features were determined in 4 trials representing 1128 children with ALL: DCOG ALL-8, ALL9, ALL10, and Cooperative ALL (COALL)-97/03. BCR-ABL1-like, IKZF1-deleted, and CRLF2-high cases constitute 33.7% of BCR-ABL1–negative, MLL wild-type BCP-ALL cases, of which BCR-ABL1-like and IKZF1 deletion (co)occurred most frequently. Higher cumulative incidence of relapse was found for BCR-ABL1-like and IKZF1-deleted, but not CRLF2-high, cases relative to remaining BCP-ALL cases, reflecting the observations in each of the cohorts analyzed separately. No relapses occurred among cases with CRLF2-high as single feature, whereas 62.9% of all relapses in BCR-ABL1–negative, MLL wild-type BCP-ALL occurred in cases with BCR-ABL1-like signature and/or IKZF1 deletion. Both the BCR-ABL1-like signature and IKZF1 deletions were prognostic features independent of conventional prognostic markers in a multivariate model, and both remained prognostic among cases with intermediate minimal residual disease. The BCR-ABL1-like signature and an IKZF1 deletion, but not CRLF2-high, are prognostic factors and are clinically of importance to identify high-risk patients who require more intensive and/or alternative therapies.

Bosutinib is a tyrosine kinase inhibitor (TKI), approved for adults with Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML); 400 mg in newly diagnosed (ND) patients (pts), and 500 mg in resistant/intolerant (R/I) pts, administered once daily (QD) with food. Its toxicity profile differs from other TKIs approved in children (imatinib, dasatinib and nilotinib), showing a higher incidence of gastrointestinal (GI) adverse events (AEs) but fewer toxicities such as musculoskeletal AEs (Cortes JE et al, 2016). Furthermore, in mice, bosutinib showed less growth impairment compared to other TKIs (Tauer JT et al, 2015).

Durairaj: Pfizer Inc: Current Employment, Current equity holder in publicly-traded company. Viqueira: Pfizer: Current Employment, Current equity holder in publicly-traded company. Ingrosso: Pfizer: Current Employment, Current equity holder in publicly-traded company. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy. Zwaan: SANOFI: Consultancy; NOVARTIS: Consultancy; ROCHE: Consultancy; INCYTE: Consultancy; PFIZER: Consultancy, Research Funding; JAZZ: Other: travel funding, Research Funding; BMS: Research Funding; Abbvie: Research Funding.

No relevant conflicts of interest to declare.

Malignant cells that infiltrate the bone marrow (BM) interfere with the normal cellular behavior of supporting cells, thereby creating an alternative malignant niche. This intercellular communication is mostly mediated by cytokines and their receptors. In this study, we find that expression of the CXCR4 receptor is significantly increased in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) cells compared with normal mononuclear hematopoietic cells derived of the bone marrow (p=0.016). Furthermore, we show that high CXCR4 expression is correlated with an unfavorable clinical outcome in BCP-ALL (5-yr CIR ±SE: 38.4% ±6.9% in CXCR4-high versus 12.0% ±4.6% in CXCR4-low expressing patients, p<0.001). Interestingly, BM serum levels of the CXCR4 ligand (CXCL12) are 2.7-fold lower (p=0.005) in samples taken at initial diagnosis of BCP-ALL compared with the levels in samples taken of non-leukemic controls. We show that induction chemotherapy restores CXCL12 levels in the BM to normal levels. Blocking the CXCR4 receptor with Plerixafor (FDA-approved drug) showed that the lower CXCL12 serum levels at initial diagnosis could not be explained by consumption by the leukemic cells, nor did we observe an altered CXCL12-production capacity of BM-MSC at this time-point. We rather observed that a very high density of leukemic cells negatively affected CXCL12 production by the BM-MSC while stimulating the secretion levels of G-CSF. These results suggest that highly proliferative leukemic cells are able to down-regulate the production of cytokines involved in homing (CXCL12), while simultaneously up-regulating the production of cytokines involved in hematopoietic mobilization (G-CSF). This disbalance may stimulate the spreading of BCP-ALL outside the BM.

The prognosis of childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) has improved enormously due to risk-adapted therapeutic stratification of patients. Still, most relapses occur in the group that is supposed to have an intermediate relapse risk. To overcome this problem, new prognostic markers are needed. Recently, new high-risk molecular features were identified in BCP-ALL: the BCR-ABL1-like signature, IKZF1 deletions and high CRLF2 expression (CRLF2-high). These features can occur together; however, whether they confer independent prognostic value is currently unknown. In this study we determined the frequency, co-occurrence and prognostic relevance of these new molecular features in four independent cohorts of children with ALL.BCR-ABL1-like gene signature (110-gene probe classifier), IKZF1 deletions (Multiplex Ligation-dependent Probe Amplification) and CRLF2 expression levels (Affymetrix U133 plus 2.0) were determined in leukemic cells (>90% blasts) from 1128 children with newly diagnosed ALL enrolled in three consecutive Dutch Childhood Oncology Group trials (DCOG ALL-8/-9/-10) and in the German COALL-97/03 trial. The cumulative incidence of relapse (CIR) was calculated using death as a competing event.BCR-ABL1-like, IKZF1-deleted and CRLF2-high cases were found in 16%, 17% and 10% of the BCP-ALL cases, respectively. Collectively, these three features constitute 34% of BCP-ALL cases for which 4% of those had all three, 29% had any two and 67% had only one these features. The BCR-ABL1-like signature and IKZF1-deletions co-occurred the most frequent of these three features (22%). The BCR-ABL1-like signature was only found in B-other cases negative for the genetic abnormalities ETV6-RUNX1, BCR-ABL1, hyperdiploid, TCF3- and MLL-rearrangement, whereas IKZF1-deleted and CRLF2-high cases were also found in other subtypes of BCP-ALL. The frequency of IKZF1- deletions was highest in BCR-ABL1- positive (70%) and BCR-ABL1-like (40%) cases. The BCR-ABL1-like signature, IKZF1 deletions and CLRF2-high were associated with prognosis in four, three and one out of 4 studied treatment protocols, respectively. Protocol-stratified analysis revealed that five-year CIR was higher in BCR-ABL1-like (32%, p<0.001) and IKZF1- deleted (34%, p<0.001) compared to other BCP-ALL cases (11% and 13%, respectively), whereas no unfavorable CIR was associated with the CRLF2-high feature. IKZF1- deleted cases also were at higher risk of relapse in the hyperdipoid (5-year CIR 34%, p=0.04) and non-BCR-ABL1- like B-other group (5-year CIR 40%, p=0.02) compared to 9% for non-deleted BCP-ALL cases whereas IKZF1-deletions had no (additive) prognostic value in ETV6-RUNX1- positive or BCR-ABL1-like cases. Cases with only the BCR-ABL1-like signature (HR 5.3) or only an IKZF1 deletion (HR 4.4) as well as those cases positive for both features (HR 3.7) were at higher risk to develop an adverse event than those cases negative for both features (p<0.001). Together these two features accounted for 62.9% of all relapses in BCP-ALL whereas no relapse occurred in the cases with high CRLF2 expression as single feature. The adverse prognosis associated with BCR-ABL1-like signature and/or IKZF1-deletions was independent of other risk factors including age and white blood cell count. Moreover, the BCR-ABL1-like signature (HR=3.7 p=0.026) and IKZF1- deletions (HR 2.7, p=0.043) were predictive of an unfavorable prognosis in the group of BCP-ALL cases with intermediate minimal residual disease levels at day 79 of treatment.High CRLF2 expression is the least predictive, whereas the BCR-ABL1-like signature and IKZF1- deletions –both individually and combined- are strong independent prognostic factors and have additional prognostic value on top of intermediate minimal residual disease levels in children with BCP-ALL. These findings demonstrate that these two molecular features are important clinically for identifying high-risk patients who may require more intensive therapy.No relevant conflicts of interest to declare.

Abstract 880

Leukemic cells of BCRABL1- positive patients enrolled in two consecutive treatment protocols of the Dutch Childhood Oncology Group (DCOG) were analyzed for IKZF1 and CRLF2 status. IKZF1 deletions were determined by Multiplex Ligation Probe-based Amplification assays (MLPA) and comparative genomic hybridization arrays (aCGH; Agilent, Sureprint G3 Human CG 180K). The p335 MPLA assay (MRC Holland) was used for discovery of IKZF1 deletions and data were validated by aCGH and a second MLPA assay (p202; MRC Holland) with higher resolution for affected regions. CRLF2 expression levels were determined by Affymetrix U133 plus 2.0 gene expression arrays. A large cohort of 459 newly diagnosed ALL cases served as reference. A high level of CRLF2 expression was assigned to 10% of all cases ranked according to CRLF2 expression level.Deletions in IKZF1 were found in 65% of the cases (n=11); 10 cases had a partial deletion between exon 2 and 7 of IKZF1 resulting in a dominant negative variant and 1 case had a deletion of the complete IKZF1 gene. Analysis of patient characteristics revealed that cases with deletions in IKZF1 have a median 4.5-fold higher white blood cell count compared to unaffected cases (p=0.009). Analysis of the cumulative incidence of relapse (pCIR) with death as a competing risk indicated that children with IKZF1 -deleted BCRABL1 -positive ALL had a significantly increased risk of developing a relapse compared with those with an unaffected IKZF1 gene (73% versus 17%, p=0.02). As a consequence, the 5-year event-free survival was also lower for the IKZF1- deleted group compared to the unaffected group (18% versus 67% p=0.04, Log-Rank). The expression level of CRLF2 in BCRABL1- positive cases was median 91.4 arbitrary units (p25-p75: 84.2–97.2) compared to 91.3 arbitrary units (p25-p75: 82.6–107.3) for BCRABL1 -negative cases (p>0.05). Expression of none of the BCRABL1 -positive ALL cases was ranked within the highest 10% percentile of the reference group. Hence, high expression of CRLF2 cannot explain a poor prognosis of BCRABL1- positive cases.our data show that a deletion of IKZF1 but not a high expression of CRLF2 is a strong discriminative prognostic factor in BCRABL1- positive pediatric precursor B-ALL. We therefore advocate to implement the IKZF1 status as a new prognostic factor in BCRABL1- positive pediatric ALL.No relevant conflicts of interest to declare.

Abstract 3528