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Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

New drugs are needed for the treatment of relapsed acute lymphoblastic leukemia and preclinical evaluation of the MEK inhibitor, selumetinib, has shown that this drug has excellent activity in those leukemias with RAS pathway mutations. The proapoptotic protein, BIM is pivotal in the induction of cell death by both selumetinib and glucocorticoids, suggesting the potential for synergy. Thus, combination indices for dexamethasone and selumetinib were determined in RAS pathway-mutated acute lymphoblastic leukemia primagraft cells in vitro and were indicative of strong synergism (combination index

Purpose To determine the prevalence and prognostic association of immunoglobulin heavy chain (IGH@) translocations in acute lymphoblastic leukemia (ALL). Patients and Methods The cohort comprised 3,269 patients treated on either the UKALL2003 trial for children and adolescents (1 to 24 years old) or the UKALLXII trial for adolescents and adults (15 to 59 years old). High-throughput fluorescent in situ hybridization was used to detect IGH@ translocations. Results We identified IGH@ translocations in 5% of patients with ALL (159 of 3,269 patients), in patients with both B-cell (148 of 2,863 patients) and T-cell (11 of 408 patients) disease. Multiple partner genes were identified including CRLF2 (n = 35), five members of the CEPB gene family (n = 17), and ID4 (n = 11). The level of the IGH@-positive clone varied and indicated that some IGH@ translocations were primary events, whereas others were secondary aberrations often associated with other established aberrations. The age profile of patients with IGH@ translocations was distinctive, with a median age of 16 years and peak incidence of 11% among 20- to 24-year-old patients. Among patients with B-cell precursor ALL who were Philadelphia chromosome negative, those with an IGH@ translocation had an inferior overall survival compared with other patients (UKALL2003: hazard ratio, 2.37; 95% CI, 1.34 to 4.18; P = .003; UKALLXII: hazard ratio, 1.73; 95% CI, 1.22 to 2.47; P = .002). However, this adverse effect was not independent of age or minimal residual disease status and did not seem to be driven by an increased risk of relapse. Conclusion IGH@ translocations define a genetic feature that is frequent among adolescents and young adults with ALL. Although associated with an adverse outcome in adults, it is not an independent prognostic factor in children and adolescents.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that has a dismal outcome when treated with standard therapy. For improved diagnosis and risk stratification, the initiating genetic events need to be elucidated. To investigate the genetic basis of BCP-ALL, genomes of 94 iAMP21 patients were interrogated by arrays, FISH, and multiplex ligation-dependent probe amplification. Most copy number alterations targeted chromosome 21, reinforcing the complexity of this chromosome. The common region of amplification on chromosome 21 was refined to a 5.1-mb region that included RUNX1, miR-802, and genes mapping to the Down syndrome critical region. Recurrent abnormalities affecting genes in key pathways were identified: IKZF1 (22%), CDKN2A/B (17%), PAX5 (8%), ETV6 (19%), and RB1 (37%). Investigation of clonal architecture provided evidence that these abnormalities, and P2RY8-CRLF2, were secondary to chromosome 21 rearrangements. Patient outcome was uniformly poor with standard therapy irrespective of the presence or absence of these changes. This study has provided evidence that chromosome 21 instability is the only anomaly among those so far investigated that is common to all iAMP21 patients, and therefore the initiating event is likely to be found among the complex structural rearrangements of this abnormal chromosome.

Deregulated expression of CRLF2 (CRLF2-d) arises via its juxtaposition to the IGH@ enhancer or P2RY8 promoter. Among 865 BCP-ALL children treated on MRC ALL97, 52 (6%) had CRLF2-d, but it was more prevalent among Down syndrome patients (54%). P2RY8-CRLF2 (n = 43) was more frequent than IGH@-CRLF2 (n = 9). CRLF2-d was not associated with age, sex, or white cell count, but IGH@-CRLF2 patients were older than P2RY8-CRLF2 patients (median 8 vs 4 years, P = .0017). Patients with CRLF2-d were more likely to present with enlarged livers and spleens (38% vs 18%, P < .001). CRLF2-d was not seen in conjunction with established chromosomal translocations but 6 (12%) cases had high hyperdiploidy, and 5 (10%) had iAMP21. Univariate analysis suggested that CRLF2-d was associated with an inferior outcome: (event-free survival [EFS] hazard ratio 2.27 [95% confidence interval 1.48-3.47], P < .001; OS 3.69 [2.34-5.84], P < .001). However, multivariate analysis indicated that its effect was mediated by other risk factors such as cytogenetics and DS status (EFS 1.45 [0.88-2.39], P = .140; OS 1.90 [1.08-3.36], P = .027). Although the outcome of IGH@-CRLF2 patients appeared inferior compared with P2RY8-CRLF2 patients, the result was not significant (EFS 2.69 [1.15-6.31], P = .023; OS 2.86 [1.15-6.79], P = .021). Therefore, we concluded that patients with CRLF2-d should be classified into the intermediate cytogenetic risk group.

The results of this study suggest that certain eucalyptus essential oils may be of use as an alternative to synthetic acaricides in the management of the poultry red mite, Dermanyssus gallinae. At a level of 0.21 mg/cm², the essential oil from Eucalyptus citriodora achieved 85% mortality in D. gallinae over a 24 h exposure period in contact toxicity tests. A further two essential oils from different eucalyptus species, namely E. globulus and E. radiata, provided significantly (P < 0.05) lower mite mortality (11 and 19%, respectively). Notable differences were found between the eucalyptus essential oils regarding their chemical compositions. There appeared to be a trend whereby the essential oils that were composed of the fewer chemical components were the least lethal to D. gallinae. It may therefore be the case that the complexity of an essential oil’s chemical make up plays an important role in dictating the toxicity of that oil to pests such as D. gallinae.

Bloodstream infections caused by bacteria and fungi are a major problem worldwide. These bloodstream infections can affect both people and livestock, placing a significant burden upon developed and developing economies. In this paper we describe a multiplexed testing format, which can identify a range of bacteria and fungi within a single blood sample. Key to this technique is the specificity and sensitivity of the nucleotide probes that capture the sample. The sensitivity and specificity of the probes may allow detection of disease-causing microorganisms without the need for polymerase chain reaction amplification if the dynamics of probe binding can be observed in real time.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is a heterogeneous disease in which patient outcome is influenced by genetic lesions. Cytogenetic classification has improved survival through risk stratification for treatment. Translocations involving the Immunoglobulin Heavy Chain Locus (IGH) comprise 5% of BCP-ALL and lead to overexpression of juxtaposed genes, due to the powerful IGH enhancer elements. Multiple IGH partner genes have been described in BCP-ALL, including five members of the Ccaat Enhancer-Binding Protein (CEBP) transcription factor family: CEBPA, CEBPB, CEBPD, CEBPE and CEBPG, which comprise 12% of the IGH-translocation subgroup. Patients with potential IGH-CEBP translocations were identified by mining cytogenetic data from UK ALL trials. The translocations were confirmed by fluorescence in situ hybridisation (FISH), followed by screening for commonly occurring BCP-ALL lesions using Multiplex Ligation-dependent Probe Amplification (MLPA) (SALSA P335 kit, MRC Holland), and detection of novel genetic lesions using SNP6.0 arrays. A total of 33 IGH-CEBP patients were identified; CEBPD (n=11, 34%); CEBPA (n=10, 30%); CEBPB (n=8, 24%); CEBPE (n=3, 9%); CEBPG (n=1, 3%). Cohort median age was 15 years (range, 2-65 years). Interestingly the majority of IGH-CEBPD patients were under the age of 10 years, while CEBPA and CEBPB patients were older, all over the age of 10 years (p=0.005). The median white blood cell (WBC) count of the cohort was low at 8.24x109/l (range, 0.9-430 109/l), the highest WBCs were observed in the CEBPB subgroup (p=0.04). Associated abnormalities were mixed: 5 Down syndrome (DS) patients were identified in the cohort all exclusive to the IGH-CEBPD subgroup, an observation which has been previously reported, BCR-ABL1 translocation (n=1), high hyperdiploidy (n=1) and hypodiploidy (n=1). Although numbers were small, some trends were observed: 27% (n=9) of patients were deceased: CEBPB (n=4, 44%), CEBPA (n=2, 22%), CEBPD (n=2, 22%) and CEBPE (n=1, 13%). Overall 15% (n=5) relapsed: CEBPD (n=3, 60%), CEBPA and CEBPB (each n=1, 20%). Minimum residual disease data (MRD) at day 28 were available for 8 (24%) patients, CEBPD (n=3, 38%), CEBPA (n=2, 25%), CEBPB, CEBPE and CEBPG (n=1, 13% each). Although numbers were low, all 3 MRD negative patients were IGH-CEBPD while the remaining patients, all MRD positive, comprised the remaining CEBP subgroups. MLPA screening of 28 patients identified that 25% had whole gene deletions of CDKN2A/CDKN2B (n=7): the most common copy number abnormality (CNA) observed in IGH-CEBP. IKZF1 deletions of exons 4-7, a deletion resulting in the formation of the dominant negative isoform IK6, were present in 21% (n=6) patients: CEBPB (n=4, 67%) and CEBPD (n=2, 33%) (P=0.04). Four CNAs of the PAX5 gene were observed: individual gains of exon 5 and exon 7 (n=1 each, 50%); single exon gains of PAX5 have been predicted to lead to altered protein activity, whole gene deletion (n=1), deletion of exon 1 along with gain of exons 7 and 8 (n=1). Using SNP6.0 arrays (n=13) a recurrent focal deletion of the ABL2 gene was discovered (n=4, 31%). These patients had a common region of deletion covering exon 2 of the gene. This deletion covers the Src Homology 2 domain (SH2), which allows for the recognition of phosphorylated tyrosine residues, vital for cell signalling. Patients spanned three subgroups; CEBPB (n=2, 50%), CEBPA (n=1, 25%) and CEBPD (n=1, 25%). In this study, we show that in the IGH-CEBP subgroup, the CEBP partner gene influences disease phenotype. CEBPB patients showed more aggressive BCP-ALL characteristics, with the highest WBC, death rate (50%), older age and highest levels of associated abnormalities, with all patients showing one or more genetic lesions. The CEBPD subgroup comprises the youngest patients, a statistically significant finding both with and without inclusion of DS status. This subgroup also had the highest number of relapses in the cohort, and was the only CEBP subgroup to be MRD negative at day 28, albeit based on low patient numbers. A focal deletion within ABL2, a paralog of the ABL1 gene, is an interesting new abnormality in this subgroup, which may contribute to disease development. Disclosures No relevant conflicts of interest to declare.

Abstract 1274 Translocations involving the IGH@ locus have recently been characterised as a novel cytogenetic subgroup in BCP-ALL, involving unique partner genes including: CEBP family of transcription factors; cytokine receptors, EPOR and CRLF2; and the inhibitory transcription factor, ID4. As for the mature B-cell malignancies, their expression is always deregulated by juxtaposition of transcriptional enhancers within the locus. We have developed a high throughput FISH screening approach to ascertain the true incidence of IGH@ translocations in BCP-ALL. We screened approximately 80% (2603/3194) of patients entered to the childhood clinical trial (UKALL2003) using an IGH@ break-apart rearrangement probe with automated scanning and capture (CytoVision scanning system, Leica Microsystems). We identified IGH@ translocations in 4% (104/2603) of patients tested. This included an incidence of 4% (96/2286) in BCP-ALL and the novel observation of 2% (8/317) in T-ALL patients. The median age at diagnosis of BCP- and T-ALL IGH@ translocation patients was significantly higher at 12 and 14 years, respectively, compared to the median age of 5 years for the whole trial. The median white cell count (WCC) was low in IGH@ positive BCP-ALL (median 9.05×109/L) and higher in T-ALL (median 72.8×109/L). This study confirmed CRLF2 to be the most frequent IGH@ partner gene, observed in 20% (22/104) of BCP-ALL patients. ID4 and the CEBP family were found in 8% (8/104) and 9% (10/104), respectively. CEBPD was most common (n=6), while CEBPA (n=2), CEBPE (n=1) and CEBPG (n=1) were rare. Single patients were identified with involvement of BCL2, IGF2BP1, IGK@ and the TCRA/D locus. Novel involvement of TAL1 was identified in one T-ALL patient. In 57% of children (59/104: BCP-ALL n=52, T-ALL n=7) the IGH@ translocation partner gene remains unknown. Patients with a known partner gene did not differ from those with an unknown partner gene with respect to gender, age, WCC and National Cancer Institute (NCI) risk. However, patients with a known partner gene were strongly associated with an intermediate cytogenetic risk group while patients with an unknown partner gene were mixed (good risk, 7% v 19%, intermediate risk, 93% v 69% and poor risk, 0% v 12% p=0.01). There was no difference seen for gender, age, WCC, cytogenetic risk and NCI risk when each partner gene was investigated independently, apart from those with CRLF2 involvement who had a higher WCC (50×109/l 41% v 12%, p=0.005) and were solely within the intermediate cytogenetic risk group (100% v 74%, p=0.03). We investigated copy number aberrations of genes commonly altered in BCP-ALL by Multiplex Ligation-dependent Probe Amplification (MLPA) using the Salsa P335-A1 IKZF1 kit (MRC Holland) in 60 IGH@ translocation patients. The genes investigated were IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, as well as the rearrangement, P2RY8-CRLF2. Deletions of none (31%), one (18%), two (17%), three (22%) or four (8%) genes were found. Deletions of CDKN2A, CDKN2B and IKZF1 were the most frequent, in 18%, 21% and 20%, respectively. Whilst the incidence of CDKN2A/B deletions was similar to that seen in childhood BCP-ALL, IKZF1 deletions were more prevalent (20% v 14%) and PAX5 deletions occurred at a lower incidence in IGH@ rearranged patients (13% v 19%). In the two T-ALL patients with DNA available, both showed deletions of CDKN2A with one patient also showing deletion of CDKN2B. In conclusion, IGH@ translocations are present in 4% of childhood ALL with involvement in both B- and T-cell disease. Patients belong to the intermediate cytogenetic risk group with an increased incidence of IKZF1 deletions. This differs from our recent report on adult BCP-ALL, where patients with an IGH@ translocation were associated with a worse outcome, although the increased incidence of IKZF1 deletions remained consistent across all age ranges. In over 50% of IGH@ positive children, the translocation partner remains to be elucidated, indicating the presence of as yet unidentified cryptic rearrangements. Characterisation of these partners may identify additional oncogenes involved in leukemogenesis or provide potential novel therapeutic targets, as recently demonstrated for CRLF2 in BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 581 Patients with iAMP21 (intrachromosomal amplification of chromosome 21) represent a distinct cytogenetic subgroup of childhood B cell precursor ALL (BCP-ALL; incidence ∼2%) with a poor prognosis on standard therapy (29% EFS at 5 years). From cytogenetic studies the amplification of chromosome 21 appears to be the primary genetic change, however, whether this or associated abnormalities provide the initiating event of leukaemogenesis remains to be elucidated. Currently fluorescence in situ hybridisation (FISH) with probes directed to the RUNX1 gene provides the only reliable detection method. Knowledge of the underlying genetic mechanism will lead to improved diagnosis and determine those patients at high risk of relapse who require more intensive therapy. Our previous study using genomic arrays (1Mb aCGH, n=10; NimbleGen chromosome 21 custom oligonucleotide array, n=5) identified a common region of amplification (CRA) on 21q of 6.6 Mb and a common region of deletion (CRD) of 3.3 Mb within the telomeric region in 100% and 70% of patients, respectively. However, expression profiling (Affymetrix U133A, n=8) indicated that no important genes within the CRA or CRD were differentially expressed. We now have clinical, cytogenetic and FISH data on 94 iAMP21 patients (44 F, 50 M; median age 9.5y, range 2-30); the largest iAMP21 cohort investigated to date. Higher resolution analysis of 17/94 diagnostic samples by aCGH (Agilent 185K platform) have refined these regions to 5.1 Mb and 2.5 Mb in 100% and 88% of patients, respectively. Recent genome mapping has highlighted the presence of the microRNA miR-802 within the CRA and shown this region overlaps with the Down Syndrome Critical Region at 21q22.3 encompassing the genes DSCR1, DSCR3 and DYRK1. Detailed analysis of chromosome 21 identified recurrent breakpoints centromeric of the CRD within three genes: PDE9A (n=2), COL6A2 (n=2) and DSCAM (n=2). The role of these genes in the initiation of the chromosomal instability associated with iAMP21 is currently under investigation. Global analysis of the iAMP21 genome identified an average of 13 copy number alterations (CNA) per case with recurrent deletions of the leukaemia associated genes PAX5 (n=2), IKZF1 (n=4) and CDKN2A (n=4). To determine the incidences of PAX5, IKZF1 and CDKN2A deletions among the cohort of 94 iAMP21 patients, FISH using probes targeting these genes was carried out on availabel samples. IKZF1 (exons 3-6) was deleted at the highest incidence of 21% (13/62), while CDKN2A and PAX5 were deleted at a lower incidence of 17% (12/71) and 8% (4/48), respectively. The high incidence of IKZF1 deletions found in this patient group correlates with their association with high risk ALL. We have recently reported a deletion within the PAR1 region of the sex chromosomes, centromeric of the CRLF2 gene involving CSF2RA and IL3RA, which leads to deregulated expression of CRLF2. This deletion occurred at an incidence of 23% (17/73), as determined by FISH. Among pairs of matched diagnostic and relapse samples; one had this deletion at both diagnosis and relapse while the second had a deletion in the diagnostic sample only. Gain of an additional × chromosome was identified in 28% (18/65). Of note is that the 3 aberrations: abnormalities of chromosome 21, gain of the × chromosome and high incidence of deletions centromeric of CRLF2 in this patient group mirror the findings in Down Syndrome (DS) ALL, although we have not identified any mutations of the JAK2 kinase domain among this cohort of iAMP21 patients to date. This genome-wide study provides further support for the proposal that amplification of genes within the CRA on chromosome 21 is the primary event driving leukaemogenesis in iAMP21 patients. It has also highlighted that the 3 abnormalities in common with DS ALL and/or deletions of IKZF1, which all occur at a high incidence, contribute towards the progression of this high risk subtype of childhood BCP-ALL. Disclosures: No relevant conflicts of interest to declare.