ALBERT

Description: Abstract 2521 Purpose: The outcome for Teenagers and Young Adults (TYA) with T-cell Acute Lymphoblastic Leukaemia (T-ALL) has improved significantly using paediatric based chemotherapy protocols. Event free survival figures from the recently closed UKALL2003 highlight this observation. It remains poorly understood however why their outlook remains inferior to children aged 1 – 15 years whilst receiving identical treatment. Possible explanations for this phenomenon are distinct tumour biology, host factors and treatment adherence. The primary aim of this study is to characterise the tumour associated genetic events leading to TYA T-ALL and establish whether T-ALL in Teenagers and Young Adults is a distinct disease entity. The secondary aims are to identify new prognostic markers and potential targets to optimise therapy. Methods: DNA and RNA were extracted from 60 TYA T-ALL (15 – 25 years) historic samples. The DNA was derived from blasts at diagnosis or relapse, and matched with germ line DNA where available. The DNA was examined at high resolution for Copy Number Alterations (CNAs) and Loss of Heterozygosity (LOH) using the Affymetrix SNP6.0 platform and analysed by CNAG, dCHIP and Partek. The most frequent CNAs were confirmed by MLPA (P-383-T-ALL). In addition the DNA was screened for mutations in NOTCH1/FBXW7/PTEN using conventional Sanger sequencing and for mutations in NRAS, KRAS, CBL, FLT3 and SHP2 using denaturing high performance liquid chromatography. In cases without CDKN2A gene deletion we initially performed CDKN2A gene promoter methylation by SABiosciences EpiTect Methyl qPCR Arrays, followed up by Methylation Specific PCR (MSP). Where available, RNA was extracted from viable cells and the quality confirmed by Bioanalyser. Next the RNA was reverse transcribed to cDNA and subjected to real-time PCR gene expression analysis for CDKN2A and T-ALL related oncogenes. Results: The most frequent CNA was deletion of CDKN2A in 72.7% of patient samples (91.6% homozygous deletions). Other frequent CNAs were loss of MLLT3 (31%), STIL (27%), PTEN (19%), LEF1 (15%) and gain of MYB (12%). These CNAs were confirmed by MLPA. The STIL-TAL fusion occurred in 27% of patients. The only recurrent region of Copy Number Neutral LOH encompassed 9p24.3-p13.3, including the gene CDKN2A (27%). NOTCH1, FBXW7 and PTEN mutations occurred at expected frequencies. Rare mutations were identified in NRAS and CBL, and these mutations represented known variants. Bisulphite modified DNA was subjected to CDKN2A gene promoter methylation analysis. All samples examined were negative by Epitect Methyl Array, including the positive control cell line Raji. Promoter methylation was positive by MSP for the Raji cell line and some TYA T-ALL cases without CDKN2A gene deletion. Conclusions: This data identifies the frequency of recurrent mutations in T-ALL of Teenagers and Young Adults but reveals no striking differences with those observed in T-ALL in younger children. We are in the process of analysing additional cases to complete a total of 75 patients, including whole exome sequencing of a subset of cases. This sample size will allow us to detect genomic aberrations with a 5% incidence within this cohort (〉90% probability) and to determine any prognostic significance. The most frequently observed aberration is CDKN2A gene deletion. In some cases without CDKN2A deletion we identified CDKN2A gene promoter methylation, offering an alternative mechanism of CDKN2A inactivation. To be able to appreciate potential cooperation between genetic abnormalities, we are correlating aberrant expression of the oncogenes TAL1, LYL1, LMO1, LMO2, TLX1, TLX3, NKX2, ERG and MEF2C with the genomic abnormalities identified. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Merck: Honoraria; Mundipharma: Honoraria; Pharmacyclics: Honoraria.

Description: For most children who relapse with acute lymphoblastic leukaemia (ALL), the prognosis is poor and there is a need for better prognostic biomarkers and novel therapies to improve outcome. Relapse samples from children with B lineage ALL entered into the ALL-REZ BFM 2002 clinical trial were screened for somatic mutations which activate the RAS pathway using DHPLC and Sanger sequencing. Mutations were found in 78 patients, giving an incidence of 37.9% and were made up of NRAS (n=30), KRAS (n=30), FLT3 (n=10) and PTPN11 (n=9); one patient had both a KRAS and FLT3 mutation. Clinically, mutated cases were associated with early relapse which was particularly significant for NRAS/KRAS mutated patients (p=0.001), with a 5 year probability of event free survival of 46.9% versus 60.1% for wild type (p=0.21). In addition, there was an over representation of NRAS/KRAS mutated patients with CNS involvement, 23.3% compared to 10.3% for the mutation negative group (p=0.014). Mutation screening of the matched diagnostic samples found many to be wild type (26 from 54) but using more sensitive allelic specific assays, low level mutated subpopulations were found in 57% of cases (8 from 14), suggesting these cells survived up front therapy and subsequently emerged at relapse. Assessment of p-ERK levels by western blotting in a large cohort of diagnostic ALL samples confirmed that RAS pathway mutated cases almost always had constitutive activation of the pathway (27 from 32; 84.3%), while relatively few wild type patients were p-ERK positive (9 from 48; 18.7%) (p8 after 30 drug doses compared to a mean fold increase of 〉5 for CV. Mice treated with selumetinib and sacrificed at the end of the study showed a significant decrease in spleen size compared to CV treated mice. There was minimal effect of selumetinib in wild type ALLs. Histological analysis of post-mortem brains from mice engrafted with an NRAS mutant primagraft found extensive meningeal leukaemic infiltration in CV-treated, but not selumetinib-treated mice. Pharmacodynamic assessment in spleens of mice engrafted with mutant primagrafts showed absence of p-ERK and increased levels of the apoptotic biomarkers, Bim and cleaved PARP. In summary, we show that RAS pathway mutations are a common genetic abnormality in relapsed ALL and are associated with high risk features and are often relapse-drivers. Targeted MEK inhibition with selumetinib shows excellent activity in RAS mutated ALL both in vitro and in vivo and may offer clinical benefit for a substantial proportion of children with relapsed ALL. Given our findings, clinical trials of selumetinib in RAS pathway positive relapsed patients are warranted. Disclosures: No relevant conflicts of interest to declare.

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: Although balanced translocations often result in the formation of chimaeric fusion genes, the analysis of unbalanced translocations has largely failed to identify target loci. This is due to the heterogeneity of the chromosomal breakpoints and the multiplicity of partner chromosomes. One such example is that of dicentric chromosomes found in patients with acute lymphoblastic leukaemia (ALL). We investigated patients with dic(7;9)(p11;p11~13) [n=13], dic(9;12)(p11~13;p13) [n=38] and dic(9;20)(p11~13;q11) [n=59] to determine the breakpoints of these translocations. FISH with probes for 9p showed that breakpoints were heterogeneous [n=54]. Apart from two cases with dic(9;20), all showed either deletion of the entire PAX5 gene [53.7%, n=29] or a breakpoint within the gene [42.6%, n=23]. For those cases with breakpoints within PAX5, FISH identified ETV6 as the partner in dic(9;12) cases (18/19 cases tested). Sequencing showed a novel breakpoint within intron 1 of ETV6 in a one case of dic(9;12). Molecular copy number counting (MCC) and long distance inverse PCR (LDI-PCR) identified five novel fusion sequences in which PAX5 partnered the LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21) and C20orf112 (20q11.1) loci. The PAX5-ASXL1 and PAX5-KIF3B fusion sequences were in opposing orientation, the remaining three fusions were in the same orientation but out of frame, implicating loss of PAX5 function. To confirm the significance of PAX5 disruption, we demonstrated that both the deletion and gene fusion events resulted in the same under-expression of PAX5 exons 1–2 (p

Description: Dendritic cells (DC) play a key role in the pathogenesis of Graft versus Host Disease (GvHD), a complication of haematopoietic stem cell transplantation and offer an attractive target for therapy. Regulatory T cells (Treg) have a potent immunoregulatory effect on the maturation and the antigen-presenting cell (APC) function of DC and adoptive transfer of Treg is highly efficacious in the induction of tolerance in an experimental model of GvHD and has entered Phase I clinical trials. Several mechanisms of suppression have been proposed, including Treg acting directly on DCs, attenuating their antigen-presenting and co-stimulatory functions by arresting their maturation. However, the molecular basis underpinning these effects in DCs remains ill-defined. We investigated the effect of Treg treatment on DCs by conducting gene expression profiling and confirmed the functional consequences using downstream assays. Immature, mature and Treg-treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC, respectively) and moDC populations were generated using a well-established 6 day culture with GM-CSF and IL-4, followed by 24 hour LPS maturation. Treg were added on day 3 of culture at a 3:1 ratio. All cell populations were harvested on day 7 and sorted via FSC/SSC/CD3neg gating to remove Treg present in the co-culture and control for any changes in gene expression caused by shear stress. Gene expression profiling was carried out using the Illumina HumanHT12 microarray platform. Data was processed using R/Bioconductor workflows and the functional significance of differentially expressed genes was evaluated using Ingenuity Pathway Analysis software. Mat-DC and Treg-DC expression profiles were compared relative to the im-DC for data analysis. Upon LPS treatment, the levels of 1834 unique genes were differentially regulated in mat-DC by at least twofold (862 genes upregulated/972 downregulated) compared to the im-DC counterparts. In the Treg-DC, 1326 unique genes were differentially modulated (633 genes upregulated/693 genes downregulated). Microarray analysis of the CD markers identified a higher expression of the previously identified surface markers CD80, CD83 and CD86 in the mat-DC compared to the Treg-treated counterpart (validated by flow cytometry), confirming the semi-mature phenotype. Novel findings from the dataset include the reduction of the endocytotic-related genes, CD206 and CD209, in the Treg-DCs compared to the im-DC and this reduction manifested functionally in an impaired antigen uptake, as assessed by FITC-Dextran. Additionally, the surface marker, CD38, was downregulated in the Treg-DC compared to the mat-DC, confirmed by flow cytometry. CD38 has been shown to be NFκB-dependent and a marker of maturation in monocyte-derived DCs, further supporting the semi-mature phenotype. Furthermore, CD38 is functionally involved in CD83 expression and IL-12 induction. We assessed IL-12 cytokine secretion by Treg-treated DCs and showed a significantly reduced level of induction compared to mat-DC (p=0.0079). Pathway analysis revealed NFκB-related genes to be downregulated in the Treg-DC compared to the mat-DC. These differentially expressed genes included the TLR-adaptor protein, MYD88, the NFκB subunit, RELB and an inhibitor of NFκB, NFκB1A. This finding, coupled to the importance of NFκB signalling pathway in DC function, prompted us to investigate it at the functional level by measuring levels of phosphorylation of serine 536 of the RelA subunit as a marker of activity in response to LPS stimulation. DC cultured in the absence of Tregs (mat-DC) showed significantly higher levels of Ser536 phosphorylation when compared to those unstimulated cells (im-DC) (p= 0.0018). Concordant with the gene expression data, Treg-treated DCs (Treg-DC), showed a significantly attenuated NFκB activation when compared to their LPS-stimulated DCs counterparts (p = 0.0191), however, signalling was not completely abolished compared to those unstimulated DCs (p= 0.0003). In conclusion, gene expression profiles of Treg-treated DCs are significantly different to their mat-DC and im-DC counterparts. Here, we present the novel finding that Tregs modulate DC function, in part, by attenuation of the NFkB signalling pathway, arresting the DCs at a semi-mature phenotype, as evidenced by expression arrays and functional assays. Disclosures No relevant conflicts of interest to declare.

Description: ATM was identified as the gene mutated in Ataxia Telangiectasia (AT), a degenerative neurological disorder that is characterized by, amongst others, an increased risk to develop a hematomalignancy at young age. Striking is the high incidence of T cell derived lymphoid malignancies amongst AT patients. As sporadic T-lineage Acute Lymphoblastic Leukemia (T-ALL) is particularly found in childhood, we have investigated the occurrence and frequency of mutations in 62 coding exons of the ATM gene in 41 children with T-ALL at initial diagnosis and their relation to susceptibility for T-ALL. Moreover, as ATM is implicated in cellular signaling events in response to DNA damaging agents, we have also investigated whether genetic variants of ATM are possibly related to cellular resistance to drugs and clinical outcome in childhood T-ALL. We have identified 18 different ATM alterations, of which 9 were not previously reported, in 68% of T-ALL patients. None of the alterations co-segregated with loss of heterozygosity of the ATM gene. The simultaneous presence of more than one genetic variant was observed in 32% of T-ALL patients compared to only 12% of healthy controls (P = 0.006). An increased frequency of ATM alterations was of no influence on the level of ATM mRNA and did not associate with cellular resistance to daunorubicin, prednisolone, vincristine and L-asparaginase nor with age at diagnosis, white blood cell count or long-term clinical outcome. However, the presence of specific genetic variants, i.e. S707P, F858L, T896A, P1054R and L1472W was related to an age younger than 10 years (P = 0.034) and an increased relapse-risk (P = 0.025). In addition, an increased frequency of carriers of these genetic variants was observed amongst T-ALL children (29%) compared to controls (11%) (P = 0.028). We conclude that ATM gene alterations might pre-dispose carriers to develop T-ALL in childhood, in particular involving specific genetic variants of ATM as their occurrence is significantly associated with a young age.

Description: Key Points Chromosomal abnormalities predict outcome after relapse in BCP-ALL, and high-risk cytogenetics takes precedence over clinical risk factors. Patients with mutations or deletions targeting TP53, NR3C1, BTG1, and NRAS were associated with clinical high risk and an inferior outcome.

Description: 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.

Description: Abstract 1736 Poster Board I-762 Introduction Glucocorticoids (GC) are pivotal agents used in the treatment of childhood acute lymphoblastic leukaemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome in childhood ALL, but the underlying molecular basis remains unclear. Previous studies using cell lines have identified mutation/deletion of the glucocorticoid receptor (GR) as a mechanism of GC-resistance. However, genetic aberration of the GR is rare in clinical samples1. This disparity may be due to the mismatch repair deficient status of many ALL cell lines which consequently have a greater likelihood of acquiring mutations under GC-selection. We have used a discovery proteomics approach for hypothesis generation on potential mechanisms for resistance. To achieve this, we compared a well-characterized mismatch repair proficient GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9) both bearing wildtype GR, in a comparative proteomics experiment using 4-channel isobaric tagging for relative and absolute quantitation (the iTRAQ approach). Methods Cells were treated with either vehicle control or 0.1μM dexamethasone for 24 hours and subjected to subcellular fractionation to prepare a nuclear fraction. Each sample was labelled with a distinct isobaric tag for relative quantification and analysed by 2-dimensional liquid chromatography/ tandem mass spectrometry. The proteins were identified and relatively quantified using Protein Pilot software (Applied Biosystems). Ratios were calculated for dexamethasone-treated ‘versus’ control vehicle for each cell line and an ITRAQ ratio of greater than or equal to ± 1.2 or less than 0.8 fold change were considered to be differentially expressed. Results The comparative dataset highlighted two transcription factors which are involved in B-cell differentiation, PAX5 and IRF4, to be differentially expressed in the PreB 697 compared to the R3F9 cell line. The GC-resistant R3F9 cell line had reduced PAX5 and IRF4 protein expression compared to the parental cell line and this was further validated in other GC-resistant sub-clones derived from the PreB 697 cell line by western blot analysis. The reduced PAX5 level in the GC-resistant cell lines was not due to monoallelic loss, as measured by a QRT-PCR method or mutation as determined by DHPLC analysis of ‘hot-spot’ exons. In addition, PAX5 mRNA levels were not significantly altered, thus suggestive of a post-transcriptional mechanism for PAX5 protein reduction. To test the direct role of PAX5 in GC-resistance, we reduced PAX5 mRNA and protein levels using RNA interference in the parental GC-sensitive, PreB 697 cell line. PAX5 protein levels were reduced by at least 80% and were maintained for 48 hours post-transfection. The PreB 697 cell line was transfected with siRNA directed to PAX5 using electroporation, the cells were allowed to recover for 24 hours and the levels of cell kill were assessed in response to a 48 hour incubation with 1 μM dexamethasone by Annexin V staining and the MTS assay. Paradoxically, PAX5 knockdown increased GC-sensitivity (mean 60.4% apoptosis, S.D. 16.8, N=3) in comparison to a non-specific siRNA (mean 31.0% apoptosis, S.D. 5.2, N=3) but did not influence sensitivity to either vincristine or daunorubicin. Thus, this response was specific to glucocorticoids. Conclusion Using a proteomic approach we have shown alterations in PAX5 protein levels are associated with a GC-resistant phenotype which an mRNA-based technology would fail to detect. Modulation of PAX5 in ALL cells may influence the response to GC-therapy. It is known that GC-sensitivity alters during B-cell development, with early lymphoid precursors being highly sensitive and more mature B cells being highly resistant to GC-induced apoptosis. We propose that reduced PAX5 protein levels may reflect an altered differentiation state of the sub-clones of PreB 697 which are associated with a GC resistant phenotype. 1Irving et al, Cancer Res, 2005 2Schmidt et al, FASEB, 2006 Disclosures No relevant conflicts of interest to declare.

Description: Key Points RAS pathway mutations are prevalent in relapsed childhood ALL, and KRAS mutations are associated with a poorer overall survival. RAS pathway mutations confer sensitivity to mitogen-activated protein kinase kinase inhibitors.