ALBERT

Description: Abstract 70 Intrachromosomal amplification of chromosome 21 (iAMP21) represents a distinct cytogenetic subgroup of BCP-ALL, in which patients experience a high-risk of relapse on standard treatment protocols. The abnormal chromosome 21 defining iAMP21 has a heterogeneous, complex profile at the genomic level. This complexity has made it difficult to elucidate target genes or the initiating mechanism giving rise to iAMP21 using standard genomic approaches. In this study, detailed genomic and mutational analysis has highlighted potential novel targets in the development of iAMP21 BCP-ALL. DNA was available from 45 iAMP21 patient samples. Patient 1 was a 10 year old female; her diagnostic karyotype was 47,XX,+10,der(21)dup(21)(q?)r(21)(q?). Fluorescence in situ hybridization detected multiple copies of RUNX1, thus defining iAMP21. SNP 6.0 arrays indicated the characteristic genomic profile of chromosome 21, comprising a ∼30Mb (from 17–47Mb) region of copy number gain/amplification. Many of the breakpoints occurred within the Down Syndrome Critical Region (DSCR), specifically within the gene, DSCAM at 41.4Mb, and a telomeric deletion was identified with a breakpoint within the gene, TSPEAR at 45.9Mb. Chromosome 7 abnormalities were frequent, with one deletion including IKZF1 at 50.3Mb. The IKZF1, ETV6 and RB1 deletions seen by SNP 6.0 arrays were confirmed by Multiplex Ligation Probe-dependent Amplification (MLPA) and quantitative PCR. Whole-exome sequencing of the diagnostic and remission DNA of patient 1 identified 44 somatic mutations; 21 were computationally predicted to be potentially damaging to the function of the protein. The variant frequency of the individual somatic mutations ranged from 1.1% − 65.2%, indicating heterogeneity within the iAMP21 genome. The majority of the variants were detected at a frequency of

Description: Key Points EBF1-PDGFRB fusion accounts for ∼0.5% of B-cell precursor acute lymphoblastic leukemia and 2.7% of the B-other subtype. EBF1-PDGFRB-positive patients are MRD positive and are slow early responders who respond to imatinib.

Description: BCR-ABL1-like acute lymphoblastic leukaemia (ALL) is a subgroup of B cell precursor (BCP) ALL, which has a similar gene expression profile to BCR-ABL1 positive ALL and shares the same high risk of relapse. BCR-ABL1-like ALL is genetically heterogeneous and no single abnormality defines them. However a number of novel fusion genes have been reported in this subgroup, which involve the kinase genes: PDGFRB, CSF1R, ABL1, ABL2 and JAK2. Studies have shown that patients with these fusions may also respond to tyrosine kinase inhibitors (TKI), such as imatinib. Here we present a subset of patients with the SSBP2-CSF1R fusion, including a patient treated with imatinib after relapse. Five patients with BCP-ALL were identified with cytogenetically visible abnormalities of chromosome 5, which resulted in fusion of the SSBP2 at 5q14 to CSF1R at 5q33. Three patients showed balanced translocations, t(5;5)(q14;q33) and 2 showed duplication of the long arm of chromosome 5, dup(5)(q14q33). FISH analysis using in-house dual colour break-apart probes confirmed rearrangement of the CSF1R and SSBP2 genes in 4 patients. In the two cases showing dup(5)(q14q32) the duplication was confirmed by single nucleotide polymorphism (SNP) array analysis with the breakpoints occurring within SSBP2 and CSF1R. Paired end sequencing in 3 cases confirmed that the breakpoints within SSBP2 and CSF1R with the predicted transcriptional consequence being an in-frame fusion of SSBP2 exon 5 or 6 to CSF1Rexon 12. Genome wide SNP array analysis was performed in 4 cases, which revealed few copy number abnormalities (CNA) at diagnosis, with less than 5 CNA per patient. The only recurrent CNA was loss of IKZF1, seen in 2 patients; one had an intragenic deletion of exons 4-7 and the other a large deletion of approximately 22.5 Mb, spanning 7p11 to 7p14.2 and including biallelic loss of IKZF1exons 2-3. The clinical and demographic data for the five patients are shown in Table 1. Complete remission (CR) was achieved in all cases. Two patients, who were 10 years. The oldest patient was a 40 year old female who died due to graft versus host disease following a bone marrow transplant. Patients 4 and 5 were treated as high risk due to age, high WCC (〉50 x109/L) and minimal residual disease (MRD) risk. Despite receiving intensive therapy, both patients suffered relapses. Patient 4, who relapsed while receiving consolidation therapy, failed to achieve CR2 and subsequently died. Patient 5 suffered an isolated bone marrow relapse one month after the end of treatment. She was treated according to the ALLR3 trial high risk arm and achieved CR2 and MRD negativity by day 35. The detection of the SSBP2-CSF1R fusion prompted the addition of imatinib (400 mg/d) to her regimen with the intention of maintaining remission until unrelated donor stem cell transplant. Unfortunately the patient died 11 weeks after relapse from infection (E. coli septicaemia). Although these cases were identified by cytogenetics, unbiased screening of a single childhood trial, UKALL2003 was carried out. Among 276 BCP-ALL patients without any of the established cytogenetic changes, a single case (Patient 4) with the SSBP2-CSF1Rfusion was identified. This equates to less than 0.1% of childhood BCP-ALL. The incidence and outcome in adult BCP-ALL remains to be determined. This study highlights the rarity and variable outcome for paediatric patients with SSBP2-CSF1R fusions. Two young children treated as low risk achieved long-term event free survival, however 2 older children classified as high risk suffered early relapses. It is possible that children with ALL who are SSBP2-CSF1Rpositive may benefit from the incorporation of TKI into their treatment regime in the early stages of their disease. Given the rarity of this abnormality, it may not be necessary to screen all children, however those with refractory or high risk ALL should be investigated for lesions potentially responsive to TKI. Table 1 Patient no. Age Sex Trial WCC(x109/L) Karyotype Follow up 1 2 M ALL97 50.3 46,XY,t(5;5)(q14q33) CR1 〉10yrs 2 4 F ALL97 18.2 47,XX,t(5;5)(q14;q33),+21 CR1 〉10yrs 3 40 F UKALLXII 12.1 Failed. arr [hg19] 5q14q33(80721553-149443298)x3 Remission death 4 10 M UKALL2003 301.8 46,XY,t(5;5)(q14;q33)/46,XY,idem,t(3;20)(p21;q13) Relapsed and died 5 11 F Non-trial 8 46,XX,dup(5)(q14q33)† Relapsed and died in CR2 † karyotype at relapse Disclosures No relevant conflicts of interest to declare.

Description: Incorporating cytogenetics into risk stratification for the treatment of childhood ALL has contributed to increased survival rates. However 25% patients, the B-other subgroup, do not have any of the known major chromosomal abnormalities. Within this group, BCR-ABL1-like cases show a similar gene expression profile to those with BCR-ABL1 fusion and share the same high risk of relapse. BCR-ABL1-like cases are genetically heterogeneous and some harbour tyrosine kinase activating gene fusions e.g. EBF1-PDGFRB. Recent reports suggest patients with EBF1-PDGFRB ALL who are refractory to conventional therapy respond to the tyrosine kinase inhibitor (TKI), imatinib. Here we present 14 patients treated on ALL97 (n=3), UKALL2003 (n=10) or UKALL2011 (n=1) who were EBF1-PDGFRB positive including 1 patient treated with imatinib. Involvement of PDGFRB and EBF1 was confirmed using dual colour break-apart probes (normal signal pattern=0R0G2F). Eleven patients showed signal patterns (1R0G1F) consistent with a deletion of 5q33 having breakpoints within EBF1 and PDGFRB, as seen in previously reported cases of the fusion. The presence of the deletion was confirmed by single nucleotide polymorphism (SNP) array in 6 cases. Three cases showed signal patterns (1R1G1F) consistent with a balanced rearrangement resulting in the EBF1-PDGFRB fusion and a balanced t(5;5)(q33.1;q33.3) was seen in one of these cases. SNP array analysis for 2 of these cases showed no copy number abnormalities of chromosome 5. However, in the third case a series of deletions were seen along the long arm of chromosome 5, indicating that the EBF1-PDGFRB fusion was generated by a complex rearrangement such as chromothripsis. Multiplex Ligation-dependent Probe Amplification (MLPA) using the P335 IKZF1 kit (MRC Holland) was carried out in 11 cases. Deletions of the probe for exon 16 of EBF1 were seen in all cases with the 5q33 deletion. Loss of PAX5 was detected in 4 patients, while IKZF1 and CDKN2A/B were deleted in 3 cases each. Among the 14 patients there was a predominance of females (n=10). The median age was 12 years with 9 patients 〉10 years at diagnosis. The median white cell count (WCC) was 34.4 x109/L with 5 patients presenting with a WCC of 〉50×109/L. Two of 3 patients treated on ALL97 remain alive (one after relapse); both patients received regimen C and a transplant. The third patient, who was not treated as high risk, relapsed and died within 5 years. An unselected cohort of 276 B-other UKALL2003 patients revealed 8 (3%) cases of EBF1-PDGFRB (~0.6% of BCP-ALL). All 8 patients achieved CR but were MRD positive at day 29. Overall 4 patients relapsed, including 2/3 patients who received standard chemotherapy (regimen A/B) and 2/5 patients who received high risk chemotherapy (regimen C). Three relapsed patients were salvaged and remain in second CR 1-7 years post relapse. Targeted screening of 13 UKALL2003 patients who failed induction revealed 2 more patients with EBF1-PDGFRB; one subsequently died while the second remains in 1st CR 7 years later. None of the UKALL2003 or ALL97 EBF1-PDGFRB patients received TKI therapy. As a result of these findings, patients entered on UKALL2011 are screened for EBF1-PDGFRB fusion if they fail to achieve CR by day 29 or remain MRD positive (〉0.5%) at week 14. A 5 year old girl who failed induction (50% blasts at day 35) tested positive for EBF1-PDGFRB. This prompted her withdrawal from UKALL2011 and she was treated according to EsPhALL with standard BFM consolidation plus imatinib (300mg/m2) followed by 3 intensive BFM HR blocks. After 4 weeks consolidation therapy her MRD levels had fallen to 6% and she was MRD negative by flow cytometry after 8 weeks. She continues in molecular remission 2 months after a transplant. In this largest cohort of EBF1-PDGFRB patients reported to date, we demonstrate the genetic mechanisms by which the fusion occurs and the spectrum of cooperating mutations. EBF1-PDGFRB fusion is associated with female sex, older age and a mixed outcome. Although these patients had high levels of MRD and a high relapse rate there was also evidence of durable remissions; especially with intensive chemotherapy. Evidence from this study and others suggest patients with this abnormality who fail standard chemotherapy may respond to imatinib. It is possible that treatment with imatinib might avoid the need for intensive chemotherapy to achieve a cure in these patients but that would need to be tested within a prospective trial. Disclosures No relevant conflicts of interest to declare.