Abstract: Tyrosine kinase inhibitors have improved survival for patients with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL). However, prognosis for old or unfit patients remains poor. In the INCB84344-201 (formerly GIMEMA LAL 1811) prospective, multicenter, phase 2 trial, we tested the efficacy and safety of ponatinib plus prednisone in newly diagnosed patients with Ph+ ALL ≥60 years, or unfit for intensive chemotherapy and stem cell transplantation. Forty-four patients received oral ponatinib 45 mg/d for 48 weeks (core phase), with prednisone tapered to 60 mg/m2/d from days-14-29. Prophylactic intrathecal chemotherapy was administered monthly. Median age was 66.5 years (range, 26-85). The primary endpoint (complete hematologic response [CHR] at 24 weeks) was reached in 38/44 patients (86.4%); complete molecular response (CMR) in 18/44 patients (40.9%) at 24 weeks. 61.4% of patients completed the core phase. As of 24 April 2020, median event-free survival was 14.31 months (95% CI 9.30-22.31). Median overall survival and duration of CHR were not reached; median duration of CMR was 11.6 months. Most common treatment-emergent adverse events (TEAEs) were rash (36.4%), asthenia (22.7%), alanine transaminase increase (15.9%), erythema (15.9%), and γ-glutamyltransferase increase (15.9%). Cardiac and vascular TEAEs occurred in 29.5% (grade ≥3, 18.2%) and 27.3% (grade ≥3, 15.9%), respectively. Dose reductions, interruptions, and discontinuations due to TEAEs occurred in 43.2%, 43.2%, and 27.3% of patients, respectively; 5 patients had fatal TEAEs. Ponatinib and prednisone showed efficacy in unfit patients with Ph+ ALL; however, a lower ponatinib dose may be more appropriate in this population. This trial was registered at www.clinicaltrials.gov as #NCT01641107.

Abstract: The causes of the aggressive nature of BCR-ABL1–positive adult acute lymphoblastic leukemia (ALL) are unknown. To identify, at the submicroscopic level, oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed an investigation of genomic copy number alterations using single nucleotide polymorphism array, genomic polymerase chain reaction, and sequencing of candidate genes. Patients and Methods Eighty-three patients with de novo adult Philadelphia chromosome (Ph) –positive ALL were enrolled onto institutional (n = 17) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Working Party delle Leucemia Acute (n = 66) clinical trials. Treatments included tyrosine kinase inhibitor (TKI) alone, conventional chemotherapy, or a combination of TKI and chemotherapy. Results A 7p12 deletion of IKZF1 (Ikaros) was identified in 52 (63%) of 83 patients. The pattern of deletion varied among different patients, but the two most common deletion types were loss of exons 4 to 7 in 31 (37%) of 83 patients and loss of exons 2 to 7 in 17 (20%) of 83 patients. Disease-free survival (DFS) was shorter in patients with IKZF1 deletion versus patients with IKZF1 wild type (10 v 32 months, respectively; P = .02). Furthermore, a significantly shorter cumulative incidence of relapse was recorded in patients with IKZF1 deletion versus patients with IKZF1 wild type (10.1 v 56.1 months, respectively; P = .001). Multivariate analysis confirmed the negative prognostic impact of IKZF1 deletion on DFS (P = .04). Conclusion We conclude that IKZF1 deletions are likely to be a genomic alteration that significantly affects the prognosis of Ph-positive ALL in adults.

Abstract: The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1–positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Δ4-7) and by removal of exons 2 through 7 (Δ2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Δ4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Δ2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Abstract: Background: We have explored if the administration of two TKIs, Nilotinib (NIL) and Imatinib (IM) can improve the results without increasing the toxicity in the elderly Ph+ Acute Lymphoblastic Leukemia (ALL) patients. We investigate the type and number of BCR-ABL kinase domain mutations developing during and after the study. Methods: We have designed a study (ClinicalTrials.gov. NCT01025505) in which patients more than 60 years old or unfit for intensive chemotherapy and SCT where treated with two TKIs, NIL 400 mg twice daily, and IM 300 mg twice daily, alternating for 6 weeks for a minimum of 24 weeks (study core) and indefinitely in case of response. The 6-weeks rotation schedule was respected, irrespectively of temporary discontinuations. The primary end-point was the rate of Disease Free Survival (DFS) at 24 weeks (4 courses of treatment); the secondary end points included the evaluation of CHR, CCgR and CMR rates. Results: 39 patients have been enrolled in 15 Italian hematologic Centers (median age 66 years, range 28-84). Among these, 8 patients were unfit for standard chemotherapy or SCT (median age 50 years, range 28-59). 27 patients were p190, 5 were p210 and 7 were p190/p210. After 6 weeks of treatment, 36 patients were evaluable for response: 34 were in CHR (94%) and 2 in PHR (6%). 23 patients have already completed the study core (24 weeks), 87% were in CHR and 17 are currently continuing therapy in the protocol extension phase. Thus, the OS at 1 year is 79%, and 64% at 2 years. Overall, 1 patient was primarily resistant and 13 patients have relapsed, with a median time to relapse of 7.6 months (range 0.8-16.1 months), for a DFS of 51.3% at 12 months. Conclusions: In this small cohort of Ph+ ALL elderly/unfit patients, the rates of relapse and progression were not likely to be different from the rates observed with Imatinib alone. Acknowledgements: ELN, AIL, AIRC, PRIN, FP7 NGS-PTL project. Citation Format: Chiara Sartor, Cristina Papayannidis, Alfonso Piciocchi, Antonella Vitale, Ilaria Iacobucci, Simona Soverini, Pier Luigi Lollini, Francesco Di Raimondo, Stefania Paolini, Massimiliano Bonifacio, Angelo Michele Carella, Mario Cazzola, Antonio Cuneo, Pietro Leoni, Mario Luppi, Enrica Morra, Giorgina Specchia, Loredana Elia, Robin Foà, Michele Baccarani, Giovanni Martinelli. Sequential use of first and second generation TKIs are effective on prolonged overall survival in elderly population affected by Ph+ acute lymphoblastic leukemia: the GIMEMA experience. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2015-5491

Abstract: Background: Although the pathogenesis of BCR-ABL1+ ALL is mainly related to the expression of BCR-ABL1, additional genetic lesions are supposed to be involved in its development and progression. Aim: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. Methods: Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results: RNA-seq analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, respectively, detecting transcripts from 62% and 64% of human annotated genes. Low RPKM estimates (0.01 & lt;RPKM & lt; 10) were computed for the great majority of genes (78% at diagnosis and 73% at relapse), whereas a moderate expression (10 & lt;RPKM & lt; 100) was observed for 20% − 24% of active genes and only 2% − 3% of transcripts had high RPKM values (100 & lt;RPKM & lt; 8000). Moreover, 4,334 and 3,651 primary ALL and relapse isoforms with at least one AS event were identified. An average of 1.5 and 1.3 AS per isoform was estimated. The well-known alternatively spliced IKZF1 gene was also detected. Finally, 2,011 and 2,103 SNVs were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. As potential ALL-related mutations, 124 and 114 not annotated SNVs were found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 71 resulted private variants. The analysis was focused on the coding sequences of annotated genes, finding 12 non-synonymous changes: 1 affecting the PLXNB2 gene on both samples, 6 affecting genes involved in metabolic processes (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) and transport (MVP) at diagnosis and 3 affecting genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21) at relapse. Furthermore, the T315I mutation in the Bcr-Abl kinase domain was also identified. Conclusions: Discovery of novel missense mutations, as well as exhaustive alternative splicing and gene expression profiles were achieved for the first time for a BCR-ABL1+ positive ALL patient demonstrating that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations. Supported: AIL, AIRC, FIRB, European LeukemiaNet. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 651.

Abstract: Abstract 3136 Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the median blast percentage was 90% (range, 18–99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract: Background. The incorporation of tyrosine kinase inhibitors (TKIs) in treatment schemes of Ph+ ALL has remarkably improved survival. In adult patients with Ph+ ALL, ponatinib in combination with chemotherapy showed a 3-year event-free survival rate of 69%, a 3-year overall survival (OS) of 83%, and a higher rate of response when compared with dasatinib plus chemotherapy. However, in unfit or elderly ALL patients, TKIs combined with chemotherapy are associations with higher toxicity. Therefore, we examined the efficacy and safety of steroids plus ponatinib alone for the treatment of elderly or unfit patients with Ph+ ALL in a multi-center Phase II prospective clinical Italian trial, GIMEMA LAL1811 (EudraCT number 2012-002761-35). Methods. From March 2014 to December 2016, we enrolled 44 patients with untreated Ph+ ALL, ≥ 60 years or unfit (i.e. for intensive chemotherapy and stem cell transplantation). Two out of 44 patients were not elegible for the study. Patients received oral administration of 45 mg/day of ponatinib for 8 consecutive courses of 6 weeks (w). Steroids were administered from day -14 to day 29 during course 1. Intrathecal therapy with methotrexate, cytarabine and dexametasone was performed every 28 days for central nervous system (CNS) disease prophylaxis. In patients with CNS disease at diagnosis, intrathecal therapy was administered twice a week until complete remission. Dose reduction of ponatinib was allowed for adverse events. Patient samples were obtained at diagnosis and at every course, BCR-ABL mutational analisys and BCR-ABL/ABL ratio by quantitative real time PCR was performed. Complete molecular response (CMR) was defined as BCR-ABL/ABL ratio below 0.01 or undetectable, and with a sensitivity of at least 30,000 molecules of ABL. Results. Forty-two patients were eligible for the study. Median age was 68 years (range 27-85). Nine out of 42 patients were & lt;60 years and were considered unfit. Twenty-six out of 42 patients had the p190 fusion transcript, 4/42 had p210, 12/42 had p190/p210. Steroid pretreatment was administered to 39; 14/39 patients had a reduction in circulating blasts of 75% or more before starting ponatinib. Primary endpoint (Complete hematological response (CHR) at 24w in 75% of patients) was prematurely reached. CHR was obtained in 40/42 patients (95,2%) after course 1 (6w). Thirty-eight out of 42 patients (90,5%) were in CHR after 8 courses (24w); 2 patients stopped treatment after 6w for disease relapse (1) and for excessive toxicity (1). Two patients dropped out after 12w for medical decision. A CMR was detected in 11/24 patients at 24w (45.8%; 14/38 patients not evaluable). Considering a CMR test sensitivity of at least 10,000 ABL molecules and testing peripheral blood whenever a bone marrow was not obtained, 20/33 patients (60.6% 5/38 patients not evaluable) were in CMR at 24w (figure 1). The median follow-up of the enrolled patients was 11.4 months (range 6-34.5). Overall survival (OS) at 6 months and 1 year was 97.6% (C.I 95%: 93.1%-100.0%) and 87.5% (C.I. 95%: 76.5%-99,9%) respectively (figure2). At week 24, 15/42 patients still received 45 mg of ponatinib daily, only 4/42 patients permanently withdrew study drug. During the study, 75 adverse events (AE) were reported; 36 of the 75 AEs were considered related to ponatinib. Twenty-six of the 75 AEs were considered serious (SAE); 13/26 SAEs were considered related to ponatinib. A death was suspected to be related to ponatinib. We performed BCR-ABL mutational analysis in 22 patients at diagnosis, and 15 patients at 24w. T315L (abundance 100%) was detected in a patient relapsed during ponatinib therapy. We could not identify the emergency of other mutations. Conclusions. Ponatinib and steroid show a high efficacy in newly diagnosed unfit/elderly Ph+ ALL patients. Toxicities were manageable and cardiovascular AEs were limited. In the small cohort of patients relapsed in the study, relapse mechanisms were unclear; only one patient had evidence of mutations that caused resistance to ponatinib. The fast and deep reduction of the disease burden in the majority of patients, the ability of ponatinib to prevent the emergence of clones harboring BCR-ABL mutations, and the synthetic lethality with steroids on the BCR-ABL, FLT3, HCK, CDK6, MCL1 pathway could explain the therapeutic effectiveness. Acknowledgments. GIMEMA, ELN, AIL, AIRC, Regione-Università 2010-12, FP7 NGS-PTL, HARMONY, Fondazione del Monte BO e RA. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy. Bocchia: Novartis: Other: Travel grant; Celgene: Other: Travel grant; Roche: Other: Travel grant; Jansen: Other: Travel grant. Cuneo: Abbvie: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Gilead: Honoraria, Other: Advisory Board; Roche: Honoraria, Other: Advisory Board. Bonifacio: Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees. Falini: Roche: Research Funding. Galieni: Takeda: Other: Advisory Board; Abbvie: Other: Advisory Board. Foà: Sandoz: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Baccarani: Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte ARIAD: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.

Abstract: Abstract 3074 Poster Board III-11 Background BCR-ABL1-positve Acute Lymphoblastic Leukemia (ALL) is the most common ALL subtype in adults and is associated with poor prognosis. The pathogenesis of this leukemia is related to the expression of the BCR-ABL1 fusion transcript, but additional recurrent genetic lesions are suspected to be involved in its development and progression. Aim A Next-Generation Sequencing Technology was used to sequence the whole transcriptome of leukemia cells from a BCR-ABL1-positive ALL patient at diagnosis and at relapse following tyrosine kinase inhibitor (TKI) therapy with the aim to detect acquired mutations cooperating with BCR-ABL1 in leukemia manifestation and drug-resistance. Methods Poly(A) RNA was extracted from leukemia cells and used to prepare double-stranded cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained 36 base-pair (bp) sequence reads were mapped to the reference sequence of the human genome (UCSC hg18, NCBI build 36.1) to identify single nucleotide variants (SNVs) and to estimate reads density corresponding to RNA from each known exon, canonical splice event or new candidate gene. This approach allowed us to define a detailed Digital Gene Expression (DGE) profile. Reads that showed no match to the reference genome were subsequently mapped to a dataset of all possible splice junctions created by in silico pairwise combination of the exons of all annotated genes (UCSC knownGene file) to identify alternative splicing (AS) events. Results Whole Transcriptome Shotgun Sequencing (RNA-seq) analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples respectively, achieving approximately 90% diploid coverage and detecting transcripts from 62% and 64% of human annotated genes. The great majority of these active genes (78% at diagnosis and 73% at relapse) showed very low expression levels, with a number of reads per kilobase of exon model per million mapped reads (RPKM value) from 0.01 to 10, whereas 20% and 24% showed moderate expression levels (RPKM 10-100), as well as only 2% and 3% resulted highly expressed (RPKM 100-8000). Moreover, 6,390 and 4,671 AS events were also identified within 4,334 diagnosis and 3,651 relapse annotated transcripts, with the already described ALL-related Ik6 Ikaros isoform observed in both samples. Finally, 2,011 and 2,103 single nucleotide variants (SNVs) were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. Of greater interest as potential ALL-related mutations, 124 and 115 non annotated SNVs were also found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 72 resulted diagnosis and relapse private variants. In particular, the analysis was focused to the coding sequences of annotated genes, finding that non-synonymous changes were one out of the 19 shared between the two samples and affected a transmembrane receptor gene (PLXNB2). Six out of the 12 diagnosis private variants, affecting genes involved in metabolic process (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) or transport (MVP) and 5 out of the 30 relapse private variants, affecting genes involved in cell cycle regulation (ABL1, CDC2L1), catalytic activity (CTSZ, CXorf21) or with unknown function (FAM116B). Most of these diagnosis and relapse non-synonymous private mutations resulted highly expressed, showing frequencies of mutated unique reads higher than 50%. According to this pattern, diagnosis private mutations may be carried by primary leukemic clones that did not develop again at relapse, whereas relapse private mutations have greater probability to be variants acquired during the disease progression. Interestingly, the T315I point mutation in the Abl kinase domain, that confers resistance to many TKIs, was found at relapse but not at diagnosis. Conclusions An accurate expression profile was obtained for the leukemia cells of the examined ALL patient, as well as the discovery of several new non-synonymous mutations affecting genes from different pathways and for which no correlation was previously found with ALL pathogenesis. These findings demonstrate that RNA-Seq represents a suitable and cost-efficient approach for identifying new genes potentially involved in ALL development and progression. Acknowledgments AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo 60% grants, European LeukemiaNet. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 12 Background: Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, so far, the occurrence of PAX5 alterations and their clinical correlation has not been investigated in adults with BCR-ABL1-positive ALL. Aim: To characterize the rearrangements on 9p involving PAX5 and their clinical significance in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: By SNP array analysis we identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was hemizygous. Four patterns of PAX5 deletion were observed: 1) focal deletion involving only the PAX5 gene in one case (3%); 2) deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); 3) broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); 4) deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in either PAX5 haploinsufficiency or expression of PAX5 alleles with impaired DNA-binding or transcriptional activation activity. In one patient the deletion of PAX5 involved only a subset of exons (exons 2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used, demonstrating that genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Furthermore, both normal and deleted PAX5 subgroups were investigated for point mutations by sequencing analysis but we did not found nucleotide substitutions, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. However, when we investigated the association of PAX5 deletions with clinical outcome, no association with PAX5 status was detected (overall survival: p=0.3294; disease free-survival,DFS: p=0.9249 and cumulative incidence of relapse, CIR: p=0.945), suggesting that PAX5 deletions are not associated with outcome. Twenty-three patients (26%) had deletion of both PAX5 and IKZF1, encoding for the transcription factor Ikaros. We assessed the contribution of both PAX5 and IKZF1 alterations on clinical outcome finding out that the occurrence of both normal PAX5 and IKZF1 loss is associated with a shorter DFS (p=0.04) and higher CIR cumulative incidence of relapse in comparison to patients with both normal IKZF1 and PAX5 (p = 0.009). These results were confirmed by multivariate analysis. Conclusion: PAX5 deletions are frequent events in adult BCR-ABL1 positive ALL and are often associated with IKZF1 loss. The genomic status of both PAX5 and IKZF1 can be useful to identify at diagnosis groups of patients with worse prognosis. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures: No relevant conflicts of interest to declare.

Abstract: In Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts) treated with tyrosine kinase inhibitors (TKIs), responses are generally short-lived and relapse is most often accompanied by selection of point mutations in the Bcr-Abl kinase domain (KD). In order to assess whether mutations are already present at the time of diagnosis, we analyzed RNA samples from 13 pts with newly diagnosed Ph+ ALL enrolled on the GIMEMA LAL1205 protocol – designed to assess activity, safety and tolerability of dasatinib monotherapy as first-line treatment for adult Ph+ ALL. The pts investigated (males/ females: 6/7; median age: 56 years, range 30–73) included 6 pts who subsequently relapsed with dasatinib-resistant mutations, 2 pts who relapsed without evidence of mutations as assessed by direct sequencing and 5 pts in persistent remission. Screening for low level mutations was performed by cloning the entire Bcr-Abl KD (amino acid 206 through 524) in a bacterial vector and sequencing 150 to 200 independent clones for each pt. All pts, irrespective of their subsequent response to dasatinib, had evidence of aberrant KD sequences due to point mutations (13/13 pts), small insertions (2/13 pts) or small deletions (1/13 pts). Two to five point mutations were detectable for each pt. In some cases, multiple mutations could be found to co-exist in the same cloned fragment. A total of 45 different point mutations (including 18 silent mutations, 4 nonsense mutations and 23 missense mutations) were observed. Base substitutions were scattered all over the KD and there were no mutation hotspots. Thirty-nine out of 45 (87%) mutations were transitions: G 〉 A (n=14), A 〉 G (n=10), C 〉 T (n=9), T 〉 C (n=6). Such a high prevalence of transitions (which normally occur 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is predominant. Activation-induced cytidine deaminase (AID) has been recently shown to be aberrantly expressed in a proportion of Ph+ ALLs and has been implicated in the development of at least some Bcr-Abl mutations; correlation with AID expression will be presented. Mutations were detected in no more than three independent clones, suggesting that they were present at low levels. Mutations detected in less than two clones were considered only if independently validated by a specifically designed ASO-PCR assay run on the original cDNA sample. The majority of point mutations detected have never been reported in association with TKI resistance. However, three pts were found to harbor known imatinib- or dasatinib-resistant mutations, including two T315I mutations that were later on selected and led to relapse. This supports the theory that mutations randomly arise as a consequence of a high genetic instability and very few of them will confer a growth advantage under the selective pressure of TKIs. Six samples from newly diagnosed chronic phase CML (CP-CML) pts were screened for comparison with the same cloning approach, including 2 pts who later progressed with evidence of KD mutations after 3 and 6 months from the start of imatinib, respectively, and 4 pts who achieved a stable molecular response on imatinib. Both pts who experienced early relapse on imatinib had low level mutations detectable at the time of diagnosis, whereas none of the clones from CP-CML imatinib responders had evidence of KD mutations. The results herein presented support the concept that Ph+ ALL cells have a particularly high genomic instability that allows early escape from TKI inhibitor therapy. Only a fraction of CML cases share this feature. Our data highlight the importance of understanding the mechanism(s) responsible for this ‘mutator’ phenotype, as well as how to interfere therapeutically with it.