ALBERT

Description: Expression of BCR-ABL1 is the hallmark of chronic myelogenous leukemia (CML) and a subset of de novo acute lymphoblastic leukemia (ALL), but the factors determining disease lineage, and progression of CML to myeloid or lymphoid blast crisis, are incompletely understood. We recently reported deletion of IKZF1 (encoding the lymphoid transcription factor Ikaros) in 85% of de novo pediatric and adult BCR-ABL1 ALL, and in lymphoid blast crisis in a small cohort of CML cases (Nature2008;453:110), suggesting that IKZF1 deletion is important in the pathogenesis of BCR-ABL1 lymphoid leukemia. To identify genetic determinants of disease stage and blast crisis lineage in CML, we have now performed high-resolution, genome wide analysis of DNA copy number abnormalities (CNA) and loss-of heterozygosity (LOH) and candidate gene resequencing in a cohort of 90 CML patients that included 64 samples obtained at chronic phase (CP), 15 samples at accelerated phase (AP), 9 lymphoid blast crisis (LBC) and 22 myeloid blast crisis (MBC) samples. Importantly, 25 patients had sequential samples (CP and/or AP, as well as blast crisis samples) enabling analysis of lesions acquired at progression to blast crisis. All blast crisis samples were flow sorted to at least 90% purity prior to DNA extraction. Germline samples for 28 cases obtained at remission or by flow sorting of blast crisis samples were also examined. Affymetrix SNP 6.0 arrays, interrogating over 1.87 million genomic loci, were used for 85 samples, and 500K arrays for the remainder. Identification of tumor-specific (somatic) copy number analysis was performed by directly comparing CML samples to matched germline samples were available, or by filtering results against databases of inherited copy number variants for samples lacking germline material. Genomic resequencing of IKZF1, PAX5 and TP53 was performed for all AP, LBC and MBC samples. There were few CNAs in CP-CML (mean 0.27 deletions and 0.07 gains per case), with no recurring lesions identified apart from deletions or gains at the chromosomal breakpoints of BCR and ABL1 (3 cases each). Notably, the size of these translocation associated deletions was highly variable, ranging from 6kb (one ABL1 deletion) and 15 kb (one BCR deletion) to deletions extending to the telomeres of chromosomes 9 and 22. No significant increase in lesion frequency was identified in AP cases (0.14 deletions and 0.9 gains per case), however the number and cumulative extent of genomic aberrations was significantly higher in both lymphoid and myeloid blast crisis samples. LBC cases had a mean of 8.1 deletions/case (P

Description: Investigations into seasonal trends for the onset of leukemia have produced conflicting results. The majority of studies demonstrating seasonal variation have done so for childhood ALL and have suggested a role for an infectious agent. Few investigations have studied adult AML and none have looked at the potential differences between various AML subtypes. An infectious etiology for adult AML will more likely be reflected by cases that are truly de novo and that have not arisen by transformation from a pre-leukemic phase or from prior exposure to a carcinogenic agent. The Victorian Cancer Cytogenetics Service is the central reference laboratory for cytogenetic analysis of all new adult leukemias in the state of Victoria. We have investigated seasonal trends in the diagnosis of 635 cases of new adult AML (≥15 yrs) received for cytogenetic analysis during a five-year period, March 1999 – Feb 2004. Cases of new AML that were secondary in nature were excluded. Cases were subdivided into three recognised prognostic risk groups (favourable, intermediate and adverse) based on cytogenetic findings according to the published criteria of the UK Medical Research Council. The favourable risk group (FR) consisted of t(15;17), inv(16)/t(16;16) and t(8;21) (n = 95). The adverse risk group (AR) included complex karyotypes, abnormalities of 3q, monosomy 7 and loss of chromosome 5 or part of 5q (n = 118). The intermediate risk group (IR) consisted of all other cytogenetic findings including normal results (n = 422). The cytogenetic abnormalities comprising the FR group are rarely associated with secondary AML whereas the converse is true of the AR group. Initial descriptive statistics demonstrated that the greatest trend towards seasonal variation was in the FR group with a peak occurrence in Spring. Spectral analysis was subsequently performed on the data in order to detect if any underlying pattern was genuinely present for the three prognostic groups. The outcome of this analysis supported the possibility of an approximately 12 month cycle for the FR group with the potential peak around the month of October, the middle month of a southern hemisphere Spring. The relatively short time frame of only 5 years limited the interpretation of the analysis. However, these results suggest a possible seasonal trend in the incidence of new adult AML presenting with cytogenetic abnormalities associated with a favourable prognosis. Analysis of data covering a greater time period is proposed to further investigate these preliminary findings.

Description: Abstract 2922 The myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by ineffective hematopoiesis and a highly variable clinical course, for which novel treatments are beginning to emerge. Conventional cytogenetic studies (CCS) of bone marrow (BM) are routinely used in clinical practice to detect abnormal clones in proliferating (metaphase) cells, identifying clonal aberrations in ∼50% of de novo MDS cases. Cytogenetics is also one of the key International Prognostic Scoring System (IPSS) components used to estimate overall survival and leukemia-free survival in MDS. Chromosome abnormalities may be quantified at presentation and during treatment by the use of fluorescence in situ hybridization (FISH) with DNA probes for specific chromosome loci (e.g., chromosomes 5, 7, 8 and 20) in non-proliferating (interphase) nuclei. However, it is not clear whether or not peripheral blood CCS will yield the same diagnostic and prognostic data as bone marrow CCS at disease presentation or whether patients without apparent chromosome abnormalities by CCS have “hidden” abnormalities that can be identified by interphase FISH. To answer these questions, 15 members of the International Working Group on MDS Cytogenetics agreed to perform CCS and FISH in parallel on both peripheral blood and bone marrow samples collected from MDS patients. To be certain that all participating sites scored and interpreted their individual FISH data in a similar fashion, a quality assurance (QA) study was completed with each site studying two identical test (proficiency) samples. Concordance among sites was very good to excellent allowing for the establishment of a standardized protocol with clear scoring criteria before patient samples were processed. In the second phase of the study, a total of 77 MDS patients were accrued to the study with 61% showing an abnormal karyotype. A FISH panel consisting of eight probe sets [-5/5q-, -7/7q-/der(1;7), +8/8q-, -11/+11/11q-/add(11q), 12p-/+21/t(12;21), -13/13q-, 17p- and 20q-/i(20q)/i(20p), Abbott Molecular, Inc.] was performed on both specimen types. While CCS was frequently unsuccessful (57.5%) in the PB specimens, FISH was informative (concordant with BM/PB CCS) in 92% of cases, with 49% of PB FISH demonstrating an abnormal clone. FISH was discordant in 4 of 77 BM and PB samples (5%), while CCS and FISH on BM and PB were discordant in 6 of 77 BM specimens (8%) and 6 of 73 PB specimens (8%). The data suggest that evaluation of interphase nuclei from PB on follow-up (non-diagnostic) FISH studies on MDS patients will be equally informative (and less costly and stressful) as a BM sample. Disclosures: Slovak: PerkinElmer: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Ohyashiki:Nippon Shinyaku Co., Ltd.: Research Funding.

Description: The vast majority of acute promyelocytic leukemia (APL) cases are characterized by the formation of a PML/RARA fusion gene. Disruptions of retinoic acid receptor α (RARα) function have also been described in four types of variant APL in which an alternative partner gene (PLZF, NPM, NUMA, or STAT5B) is fused to RARA. We describe a novel variant APL with a RARA fusion formed by a complex gene rearrangement which is undetectable by conventional cytogenetics. A 66 yr old male with a history of mild thrombocytopenia was diagnosed with APL based on the blood and marrow morphology, the coagulopathy, and a microspeckled PML immunofluorescence pattern. The bone marrow immunophenotype was negative for CD2, CD19, CD34, CD56, CD117 and HLA-DR, and with weak expression of CD13, CD33 and CD11b, a pattern atypical for APL. The diagnostic bone marrow karyotype was 47,XY,+22[5]/46,XY[30] with no t(15;17)(q22;q21). FISH with the Vysis LSI PML/RARA dual fusion translocation probe did not show any fusion signals but there was splitting of an RARA signal on one 17q. A second probe, the Vysis LSI RARA break apart probe, showed deletion of the 5′ RARA probe and the 3′ RARA probe appeared to localize more distally than normal. The Cytocell PML/RARA ES probe also showed no fusion signals but one RARA signal appeared smaller. The diagnostic marrow was negative for PML/RARA transcripts by RT-PCR using PML and RARA specific primers, but an atypical product was observed. Sequencing of this product showed partial homology to the PRKAR1A gene that maps to 17q24 and encodes the regulatory subunit type I-alpha (RIα) of cyclic AMP-dependent protein kinase A. RT-PCR using PRKAR1A and RARA specific primers amplified two transcript splice variants of a PRKAR1A/RARA fusion gene. The shorter out-of-frame fusion transcript lacked PRKAR1A exon 3 and encoded a carboxy-truncated RIα protein. The longer in-frame fusion transcript resulted from cryptic splicing of the first 100 bases of PRKAR1A exon 3 to RARA exon 3, and encoded a chimeric RIα-RARα fusion protein that contained the dimerization domain of RIα and the same carboxy terminal domains of RARα that are found in all other known RARA rearrangements in APL. FISH using a BAC probe (RP11–120M18) encompassing the PRKAR1A gene identified signals on both copies of 17q; a strong signal on the normal 17 and a weaker signal on der(17). Before cytogenetic, FISH and molecular results were available, the patient was registered on the Australasian Leukaemia and Lymphoma Group’s APML4 treatment protocol which includes ATRA, age-adjusted idarubicin and arsenic trioxide. Arsenic was ceased on day 22 due to toxicity. Morphological and cytogenetic FISH complete remission was documented on day 35. A bone marrow biopsy eleven months from original diagnosis showed no evidence of leukemia and PRKAR1A/RARA RT-PCR was indicative of molecular remission. This novel PRKAR1A/RARA gene rearrangement identified in a variant APL is the fifth variant APL in which the RARA partner gene has been identified and the second known rearrangement of PRKAR1A in a malignant disease.

Description: Background There are conflicting data on the impact of monosomal karyotype (MK) on overall survival (OS) in patients with myelodysplastic syndrome (MDS) and in particular whether MK is an independent predictor of survival in the subset of MDS patients with a complex karyotype (CK). We aimed to determine whether MK was associated with inferior survival independent of number of cytogenetic abnormalities. Methods Patients and data sources: A retrospective cohort study was performed using three population-based datasets from the Australian state of Victoria (population approximately 5.3 million). All incident cases of MDS notified to the Victorian Cancer Registry (VCR) from 2000 to 2010 were included. VCR data was linked with cytogenetic testing results performed by The Victorian Cancer Cytogenetic Service, the single provider of cytogenetic services for hematological malignancies in Victoria, and with hospital admission records held within the Victorian Admission Episode Database (VAED), a database of all hospital admissions within the state of Victoria. Cytogenetics: Metaphase cytogenetic analysis was performed according to standard techniques and karyotypes were described using the International System for Cytogenetic Nomenclature (ISCN). Statistical analysis: MK was defined as the presence of 2 or more autosomal monosomies or one monosomy with at least one additional structural abnormality. CK was defined as 3 or more cytogenetic abnormalities (CA). Overall survival (OS) was defined from date of diagnosis until death, censoring surviving patients at study end. OS was estimated using Kaplan-Meier curves, with survival between groups compared with the log rank test. Multivariable analysis was performed using the Cox proportional regression method, including all those variables associated with OS with a p-value less than 0.2 on univariate analysis. Results 1476 incident MDS cases notified to the VCR had cytogenetic testing performed. Metaphase cytogenetic testing was successful in 1407 cases (95%). The median age at diagnosis was 77 (interquartile range [IQR] 69-83) and 866 (62%) were male. The most common MDS subtypes were refractory anemia (RA) with multi-lineage dysplasia (n=482; 34%), RA with excess blasts (RAEB, n=243, 17%), RA (n=136, 9%), RA with ring sideroblasts (n=127, 9%) and not specified in 334 (24%). Hospital admission records were available for 1197 (85%), of whom 676 (56%) had one or more co-morbidity and 150 (12%) were red cell transfusion dependent (RC-TD) at diagnosis. 908 (65%) had a normal karyotype and 499 (34%) had at least one cytogenetic abnormality. CK was present in 129 (9%) and more than 5 CA in 96 (7%). MK was present in 90 (6%), of whom 82 (91%) also had CK and 74 (82%) also had 5 or more CA. Patients with a MK had worse overall survival compared to patients who did not have a MK (non-MK) (median OS 7 months vs. 41 months, Figure 1, p

Description: Abstract 4065 Background: Amplification of chromosome 1q occurs in 40% of myeloma and predicts lower response to induction regimens. We assessed if a PAD regimen overcomes effects of 1q amplification on overall response rate (ORR), event-free (EFS) and overall survival (OS) in newly-diagnosed, transplant-eligible myeloma. Methods: This phase II trial enrolled 107 patients, stratified by 1q amplification. All could receive 4× 21-day cycles of PAD induction: bortezomib 1.3mg/m2 D 1,4,8,11; doxorubicin 20mg/m2 D1,4; dexamethasone 20mg D1,2,4,5,8,9,11,12. Responders proceeded to melphalan 200mg/m2 and ASCT. ORR after PAD was primary endpoint. Secondary endpoints included ORR 3 monthspost-ASCT, and 2-year EFS and OS. Results: 22% (20/93 evaluable cases) had amplified 1q. Median PAD cycles was4. ORRs were similar in amplified and non-amplified cases (100% vs. 87% post-PAD; 100% vs. 93% post-ASCT). After median 2 years, 2-year EFS was similar in amplified and non-amplified cases (70% vs. 75%; log-rank p=0.86), but with a trend to lower 2-year OS in amplified cases (86% vs. 94%; log-rank p=0.28). Common grade 3 or 4 adverse events included constipation (0% vs. 8% of patients), neutropenia (12% vs. 4% of patients), anaemia (4% vs. 6% of patients), back pain (4% vs. 6% of patients), neuralgia (0% vs. 6% of patients) and neuropathy (8% vs. 4%). Conclusions: Four cycles of PAD induced high response rates in both 1q amplified and non-amplified myeloma with acceptable toxicity. After 2 years, EFS was also similar, but with a non-significant trend to lower OS in patients with 1q amplification. Funding: This study is sponsored by Janssen-Cilag Pty Ltd, Australia. Trial Registration: ACTRN12609000296235. Disclosures: Joshua: Janssen-Cilag Pty Ltd: Research Funding. Hertzberg:Janssen-Cilag Pty Ltd: Research Funding. Auguston:Janssen-Cilag Pty Ltd: Research Funding. Spencer:Novartis: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria. Horvath:Janssen-Cilag Pty Ltd: Research Funding. Bashford:Janssen-Cilag Pty Ltd: Research Funding. Campbell:Janssen-Cilag Pty Ltd: Research Funding. Ashurst:Janssen-Cilag Pty Ltd: Employment. Wade:Janssen-Cilag Pty Ltd: Employment. Copeman:Janssen-Cilag Pty Ltd: Employment. Butcher:Janssen-Cilag Pty Ltd: Consultancy. Prince:Janssen-Cilag Pty Ltd: Research Funding.

Description: Deletion of the long arm of chromosome 20 [del(20q)] is a common recurrent chromosomal abnormality in acute myeloid leukaemia (AML) and myeloproliferative neoplasms (MPN). The abnormal chromosome 20 has a “Common Deleted Region” (CDR) that contains Protein Tyrosine Phosphatase Receptor T (PTPRT), a tyrosine phosphatase that is mutated in many human cancers. We have previously reported (MacKinnon et al, Genes, Chromosomes and Cancer 2010) that del(20q) also harbours an amplified “Common Retained Region,” (CRR) which contains multiple copies of Haemopoietic Cell Kinase (HCK). HCK is an oncogenic Src tyrosine kinase and its aberrant activation has been shown to contribute to the pathogenesis of some haematological malignancies. Our hypothesis is that the amplification of HCK in the CRR cooperated with the loss of PTPRT in the CDR. Indeed, our results strongly suggested that HCK amplification with PTPRT loss led to a myeloproliferative phenotype seen in MPN. Our model proposed that MPN occurred either through the consequence of direct interaction between HCK and PTPRT, or through aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3). Constitutively activated STAT3 has been shown to be oncogenic in several haematological malignancies. STAT3 is a direct target of both HCK and PTPRT. It is phosphorylated (hence activated) by HCK, and dephosphorylated (hence inactivated) by PTPRT. This provides a downstream leukaemogenic pathway for our model.The ultimate aim of our experiments is to prove this hypothesis using mouse models. Murine haemopoietic stem cells (HSC) were isolated from the bone marrows of wild type C57BL/6 (WT) and PTPRT-null mice by Fluorescence Activated Cell Sorting for Lineage negative, C-kit and Sca-1 positive (LKS+) cells. Retroviral constructs of HCK were generated by cloning it into the retroviral vector pMSCViresEGFP(MIG), with GFP as reporter. Murine HSC were transduced with either retroviral HCK or MIG vector control. Hence wild type (WT) and PTPRT-null murine HSC transduced with either MIG or HCK were used in our experiments (WTMIG, WTHCK, PTPRTMIG, PTPRTHCK). We have previously presented that PTPRTHCK showed a significant increase in methylcelluose colony numbers and colony sizes compared to PTPRTMIG, while there was no difference between WTMIG and WTHCK. In addition, an intracellular anti-phosphoSTAT3 antibody assay demonstrated that in both WTHCK and PTPRTHCK, significant STAT3 hyperphosphorylation, and hence overactivation, occurred. This response was more exaggerated in the PTPRTHCK. Finally, direct interaction between HCK and PTPRT was shown with an interaction assay using recombinant proteins. We now present the in vivo data in the murine recipients. In Vivo Reconstitution LKS+ HSC transduced with either MIG or HCK were transplanted into lethally irradiated SJL/PTPRCa murine recipients to assess AML or MPN formation in a reconstitution study over 12 months. The recipients from the four cohorts (WTMIG, WTHCK, PTPRTMIG and PTPRTHCK) were regularly analysed by full blood counts, peripheral blood GFP%, and blood films. At 12 months, they were culled and the organs harvested and analysed by flow cytometry, cytospin, and paraffin-embedded histology (H + E and immunohistochemistry). Recipents of PTPRTHCK HSC had a significantly higher proportion of nucleated erythroid populations in the bone marrow by flow and larger splenic size by weight. They also showed a higher haemoglobin (Hb) at 12 months, although this did not reach statistical significance. Fig 1. Haemoglobin level of the recipient mice. PTPRTHCK recipients had a higher average Hb compared to the others, although only reaching statistical significance when compared to PTPRTMIG recipients. Fig 1. Haemoglobin level of the recipient mice. PTPRTHCK recipients had a higher average Hb compared to the others, although only reaching statistical significance when compared to PTPRTMIG recipients. Fig 2. %Nucleated erythroid population in bone marrow by flow. PTPRTHCK recipients had a higher % of nucleated erythroid cells compared to the others Fig 2. %Nucleated erythroid population in bone marrow by flow. PTPRTHCK recipients had a higher % of nucleated erythroid cells compared to the others Fig 3. Splenic size of recipient mice at 12 months. PTPRTHCK recipients had a larger splenic size compared to others, although this was just outside statistical significance when compared to WTHCK. Fig 3. Splenic size of recipient mice at 12 months. PTPRTHCK recipients had a larger splenic size compared to others, although this was just outside statistical significance when compared to WTHCK. Conclusion The data reveal a likely new oncogenic-signalling cascade: that HCK amplification and loss of PTPRT in del(20q) may cooperate to cause MPN directly, or by aberrant activity of hyperphosphorylated STAT3. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1324 Deletion of the long arm of chromosome 20 [del(20q)] is a common recurrent chromosomal abnormality in acute myeloid leukaemia (AML). It is a key step in AML development and a better understanding of the associated molecular events is important. The abnormal chromosome 20 in del(20q) AML has been shown to have lost a “Common Deleted Region” (CDR) that contains Protein Tyrosine Phosphatase Receptor T (PTPRT), a tyrosine phosphatase that is mutated in many human cancers such as AML. We have previously reported (MacKinnon et al, Genes, Chromosomes and Cancer 2010) that del(20q) also harbours an amplified “Common Retained Region,” (CRR) which contains Haemopoietic Cell Kinase (HCK). HCK is anoncogenic Src tyrosine kinase and its aberrant activation has been shown to contribute to the pathogenesis of some haematological malignancies. We hypothesize that the amplification of HCK in the CRR cooperates with the loss of PTPRT in the CDR to cause AML. Our model proposes that AML occurs either through direct interaction between HCK and PTPRT, or through aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3), a cytoplasmic second messenger that is important in cellular signalling. Constitutively activated STAT3 has been shown to be oncogenic in several malignancies, including AML. STAT3 is a direct target of both HCK and PTPRT. It is phosphorylated (hence activated) by HCK, and dephosphorylated (hence inactivated) by PTPRT. This provides a downstream leukaemogenic pathway for our model. The ultimate aim of our experiments is to prove this hypothesis using mouse models. Murine haemopoietic stem cells (HSC) were isolated from the bone marrows of wild type C57BL/6 (WT) and PTPRT-null mice by Fluorescence Activated Cell Sorting for Lineage negative, C-kit and Sca-1 positive (LKS+) cells. Retroviral constructs of HCK were generated by cloning it into the retroviral vector pMSCViresEGFP(MIG), with GFP as reporter. Murine HSC were transduced with either retroviral HCK or MIG vector control and Phoenix cell system was used for retroviral packaging. Experiments using isolated LKS+ HSC were performed to examine for features of AML. Examination of bone marrow cells from del(20q) AML patients by quantitative PCR revealed an increase in HCK mRNA expression and a reduction in PTPRT expression. Wild type (WT) and PTPRT-null murine HSC transduced with either MIG or HCK were cultured in methylcellulose media. Colony forming units (CFU) were enumerated on day7 and day12. We found that both WT and PTPRT-null HSC transduced with HCK showed a significant increase in colony numbers compared to MIG transduced HSC. Furthermore, the fold increment in colony number was higher in the PTPRT-null genotype as shown in figure 1. Moreover, an intracellular anti-phosphoSTAT3 assay was performed to assess STAT3 phosphorylation levels in the transduced HSC. It demonstrated that in both WT and PTPRT-null HSC that have been transduced with HCK, STAT3 hyperphosphorylation, and hence overactivation, occured. This response was again more exaggerated in the PTPRT-null HSC, as seen in figure 2. We are currently transplanting transduced LKS+ HSC (either MIG or HCK) into lethally irradiated murine recipients to assess AML formation in a reconstitution study. The recipient mice will be assessed for evidence of engraftment and subsequent AML. The preliminary data reveals a likely new oncogenic-signalling cascade: that HCK amplification and loss of PTPRT in del(20q) AML may cooperate to cause AML directly, or by aberrant activity of hyperphosphorylated STAT3. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3824 Myelodysplastic syndromes (MDS) are clonal stem cell disorders characterised by ineffective blood cell production and increased leukaemia risk. Disordered regulation of the epigenome has been implicated in the pathogenesis of MDS and treatment with the hypomethylating agent azacitidine has a proven survival benefit in MDS cohorts. Other novel agents under investigation for the treatment of MDS include histone deacetylase inhibitors (HDACi) and small molecule inhibitors that target the PI3K/AKT/mTOR signal transduction pathway. PI3K/AKT/mTOR pathway inhibitors modify cellular translational capacity and translational efficiency providing a complementary mechanism of action to agents that act through epigenetic modification such as hypomethylating agents and HDACi. We hypothesized that addition of an mTOR inhibitor (mTORi) would enhance the sensitivity of MDS clones to therapy with HDACi or hypomethylating agents and that responses would be karyotype dependent. Accordingly we sought to stratify in vitro responses to treatment with rapamycin (mTORi) alone, sodium valproate (HDACi) alone, decitabine (hypomethylating agent) alone, combination rapamycin plus sodium valproate or combination rapamycin plus decitabine. Residual bone marrow from patients with a new diagnosis of MDS was salvaged after completion of the diagnostic work-up (n=18). Patients had a diagnosis of RA (n=2), RARS (n=2), RCMD (n=11) and RAEB-2 (n=3). 7/18 (39%) patients had a karyotypic abnormality detected by metaphase cytogenetics. IPSS-R cytogenetic risk categories represented were: very good (n=3), good (n=11), intermediate (n=2) and poor (n=2). Genomic DNA was hybridized to a 300K single nucleotide polymorphism array (SNP-A). A karyotype abnormality score was assigned for the total number of independent cytogenetic abnormalities detected by combined use of metaphase cytogenetics and SNP-A. Bone marrow mononuclear cells were plated in duplicate in methylcellulose containing SCF, GM-CSF, IL-3 and EPO together with vehicle control, 1mM sodium valproate, 100nM rapamycin, 300uM decitabine, 1mM sodium valproate plus 100nM rapamycin or 300uM decitabine plus 100nM rapamycin. Colony formation was scored on day 14. Drug responses were classified as sensitive (colony numbers

Description: Abstract 2690 Cytogenetic aberrations are key diagnostic and prognostic markers in ALL; however, suboptimal chromosome morphology, low lymphoblast mitotic activity in cell culture and the inability to detect subtle (≤ 5∼10 Mb) abnormalities often preclude a rapid and accurate ALL clinical cytogenetic workup. Recent genome-wide microarray studies have reported submicroscopic DNA copy number alterations (CNAs) in ALL that target key genes involved in B-cell and T-cell cellular processes (cell cycle regulation, differentiation, proliferation and survival) and alterations of these genes are rapidly being associated with clinical treatment and outcome (e.g., IKZF1, NUP214/ABL1, NUP214/SET, PTEN, PTPN2). To improve the cytogenetic diagnosis of ALL, we analyzed DNA samples from 22 newly-diagnosed, pediatric T-cell and 22 B-cell (16 newly diagnosed and 6 relapse) ALL patients (pts) using a 133K targeted oligo-based microarray. The aCGH results were compared to their cytogenetic, FISH and clinic-pathological findings. When sufficient material was available, locus-specific FISH studies were performed to confirm the submicroscopic CNAs. The 22 pediatric T-cell samples, obtained from the COG Cell Bank, showed the following karyotypes: del(6q) alone (n=10), del(6q) with one or two additional abnormalities (n=8), or normal karyotypes (n=4). The B-cell ALL karyotypes showed one to three abnormalities (n=10), ≥5 abnormalities (n=10), including hypodiploidy/hyperdiploidy/near-triploid/near tetraploidy, or normal karyotypes (n=2). Twelve B-cell ALL pts showed prognostically-significant translocations. By aCGH, CNAs were observed in all 44 cases, ranging from five to14 (median, 8) CNAs in T-cell and three to 35 (median, 10) CNAs in B-cell ALL. aCGH detected del(6q) in all 18 known T-cell pts (size range, 16.8 kb to 106 Mb) and in six B-cell pts. Submicroscopic aberrations detected in T-cell ALL included: CDKN2A (mono or bi-allelic) deletions (n=19), ranging in size from 24 kb to 6.76 Mb, including 6 focal deletions under 200 kb, a ∼77 Kb deletion in 1p33, resulting in a STIL/TAL1 fusion (n=8), other TAL1 or STIL deletions (n=3), PTEN deletions (n=6) ranging from 15 kb to 1 Mb (n=6), 49 kb biallelic GSTT1 deletions (n=4), and TLX3 rearrangements (n=2) including a case with a ABL1/NUP214 fusion and a focal biallelic PTPN2 deletion. Cryptic deletions in 4q31.3/FBXW7 and in 9q34 resulting in a SET/NUP214 fusion and duplication of MYB were observed in a single case. In B-cell ALL, recurring “cryptic” deletions were of IKZF1 (n=6), TOX (n=3), PAX5 (n= 5) CDKN2A (mono- or bi-allelic) (n=10), ETV6 (n=6), BTG1 (n=4), C20orf94 (n=3), EBF1 (n=2), TP53 (n=2), and miR650 (n=2). Single CNAs observed in B-cell ALL included: CDKN2C, LCK, BTLA, MECOM, TBL1XR1, AFF1, LEF1, HEF, RAG1, ATF7IP, JAK2, PTEN, ID4, CASC3, COMMD1, and the drug receptor gene NRCC1. Gains observed in residual or relapsed B-cell ALL were MYC, MLL, miR657, and at 13q31.3, which includes GPC5 and multiple miRNAs (latter also seen in three cases of T-cell), and PPP2R5A in a t(4;11) pt. Imbalances in TRG (7p14), TRB (7q34), and TRA/TRD (14q11.2) were noted in T-cell pts whereas IGH, IGK and/or IGL were clearly seen in B-cell pts. Three translocations, t(9;22), t(4;11) and t(12;21), were also detected by microarray using linear amplification followed by aCGH. These findings demonstrated that aCGH can improve the cytogenetic diagnosis of ALL, contribute to risk stratification, and provide genetic markers for MRD testing. Moreover, these results provide a rationale for the integration of targeted microarray technology in the clinical evaluation of ALL. Disclosures: Slovak: PerkinElmer: Employment. Hunger:bristol myers squibb: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees. Shaffer:PerkinElmer: Employment; American College of Medical Genetics Foundation: Membership on an entity's Board of Directors or advisory committees. Ballif:PerkinElmer: Employment. Schultz:PerkinElmer: Employment.