ALBERT

Description: 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.

Description: Human systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cells accumulation in various tissues. It usually occurs as a sporadic disease that is often persistent or progressive in adults. Clinical course is variable. The most aggressive forms have a rapid course and required treatments to reduce the neoplastic burden and to slacken the progression. Unfortunately no therapy have been demonstrated efficacy, mostly in the treatment of aggressive systemic mastocytosis (ASM). SM has been associated with constitutive activating c-kit somatic mutations, the most frequent of whom is the D816V mutation. Kinase inhibitors blocking constitutive c-kit activation, such as imatinib, have been used in SM, but they had no effect on D816V mutant kit. Here, we report on six patients with Systemic mastocytosis and with detectable D816V mutation treated with dasatinib 70 mg BID as in leukemia treatment. According to the WHO Classification of Systemic mastocytosis, we describe clinical characteristics of six cases of ASM with different organ damage treated with dasatinib for various period, and we made evaluation of response according to Valent criteria (Leuk Research, 2003). All patients excepting Patient 3 reached the dose of 140 mg daily. Patient 4 are on treatment after 10 months, without any signs or symptoms of disease progression. Patient 2 is still on treatment with low dose of dasatinib (20 mg daily) but he shows cytopenia and worsening clinical condition, he does not tolerate high dose of dasatinib for gastrointestinal toxicity and astenia. Patient 1 stopped therapy after 10 months of therapy and she relapsed with ascite and died after 1 month for progressive disease. Patient 3 and Patient 5 interrupted dasatinib for worsening clinical condition after 40 days and three months respectively. Patient 6 stopped dasatinib after five months for progression to acute leukemia. After dasatinib suspension, Patient 3 died for complication of leg fracture and Patient 5 died for progression of disease. We observed major response in three patients for the prompt resolution of sign of organ damage recorded during dasatinib treatment, and we also obteined a measurable decrease of the burden of neoplastic MCs by means of tryptase level monitoring in all but one patients. Toxicity of 140 mg daily in patients with advanced ASM is notable. All patients had to suspended therapy due to extra-haematological toxicity. The most commonly toxicity involved gastroenteric tract, with diarrohea and abdominal pain, hypotension and pleural effusion. Symptoms resolved or return to mild grade with dose reduction, but in frail patients therapy is not well tolerate. After interruption of therapy progression was recorded.

Description: Resistance to imatinib in Philadelphia-positive (Ph+) leukemia patients is often associated with selection of point mutations in the Bcr-Abl kinase domain (KD). Dasatinib and nilotinib are second-generation tyrosine kinase inhibitors (TKIs) with different binding modes with respect to imatinib, that have been shown to confer in vitro and in vivo activity against many Bcr-Abl mutated forms. However, both dasatinib and nilotinib have been shown to retain some ‘Achilles heels’, and they include both imatinib-resistant mutations (e.g., T315I) and some novel, inhibitor-specific ones. Selection of either type of KD mutations has frequently been observed in patients (pts) who relapse after an initial response to dasatinib or nilotinib and represents one of the major hurdles on the road to successful treatment of imatinib-resistant pts. We have monitored Abl KD mutation status in a total of 121 pts who received dasatinib (n= 78) or nilotinib (n=43) as 2nd TKI after imatinib failure since February 2005. Fifty-eight (48%) pts had chronic phase (CP) chronic myelogenous leukemia (CML), 63 pts (52%) had accelerated phase (AP) or blast crisis (BC) CML or Ph+ acute lymphoblastic leukemia (ALL). Median age was 55 years (range, 18–76); median time from diagnosis was 49 months (range, 4–181); median time on imatinib was 32 months (range, 4–66). Median follow-up of all pts who received a 2nd TKI is 7 months (range, 1–38). Median follow-up of pts who are still on 2nd TKI treatment is 32 months (range, 28–38). Relapses after an initial response have so far been observed in 46/121 pts. Thirty-eight out of these 46 pts had AP/BC CML or Ph+ ALL at the time 2nd TKI was started. Forty-one out of 121 (34%) pts have experienced relapse after an initial response during the first 12 months of 2nd TKI treatment (median time to relapse, 6,5 months; range 4–12 months), while only five of the 45 (11%) pts who were still on 2nd TKI treatment after 〉12 months have relapsed (at 13, 15, 18, 20 and 33 months, respectively). Interestingly, none of these 5 pts had never achieved more than a minor cytogenetic response (CgR), and 4/5 pts were receiving a reduced TKI dose because of toxicity. In 36/46 (78%) cases, relapse was associated with newly acquired Abl KD mutations. In particular 26/30 (87%) pts who relapsed on dasatinib and 10/16 (63%) pts who relapsed on nilotinib had evidence of a newly acquired KD mutation presumably responsible for treatment failure. Newly acquired mutations in pts who relapsed on dasatinib as 2nd TKI were T315I (n= 12 pts) F317L (n= 8 pts) T315A (n=3 pts); V299L (n=3 pts); F317I (n=2 pts); 2 pts had multiple mutations. Newly acquired mutations in pts who relapsed on nilotinib as 2nd TKI were E255K (n=3); E255V (n=2); Y253H (n=2); T315I (n=1); F359V (n=1); F359C (n=1). Sixteen pts (but none of those harboring the T315I) switched to dasatinib or nilotinib or high-dose imatinib as 3rd TKI and this rescued hematologic or even cytogenetic responses in a proportion of cases. Our observations suggest that: newly acquired mutations leading to relapse in Ph+ leukemia pts receiving dasatinib or nilotinib as 2nd TKI usually arise rapidly; the likelihood of mutation selection consistently decreases over time, and seems mainly confined to advanced phase pts and to pts with no or minor CgR; almost all (87%) cases who developed resistance to dasatinib had newly acquired KD mutations - suggesting that the higher potency with respect to imatinib can overcome Bcr-Abl gene amplification and that Src kinase inhibition may turn off Bcr- Abl-independent resistance mechanisms; a lower incidence (63%) of newly acquired KD mutations was observed in pts who developed resistance to nilotinib; with the exception of T315I, there is little if no overlap between dasatinib and nilotinib-resistant mutants, which may allow to regain responses by switching TKIs.

Description: Among all cytogenetic subtypes of ALL, the BCR-ABL1 fusion gene, which is causative of chronic myeloid leukemia (CML), defines the subgroup of ALL with the worst prognosis. The reasons of the aggressive nature of BCR-ABL1-positive ALL have not yet been completely understood. To identify, at the submicroscopic level, oncogenic lesions that cooperate with BCR-ABL1 to induce ALL and blastic transformation of CML, we used an integrated molecular approach including high resolution genomic analysis of copy number alterations by single nucleotide polymorphism (SNP) arrays (500K, Affymetrix Inc., Santa Clara, CA, USA), genomic amplification, cloning, deep sequencing, gene expression profiling (GEP) and analysis of epigenetic changes to study 97 de novo BCR-ABL1- positive ALL adult patients (pts). High level amplification and homozygous deletions were identified in all pts, with deletions outnumbering amplification almost 3:1. The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros identified in 59/97 pts (61%). Ikaros is required for the earliest stages of lymphoid lineage commitment and acts as tumor suppressor in mice. The IKZF1 deletions were predominantly mono-allelic (64% vs 36%) and were limited to the gene in all cases, identifying IKZF1 as the genetic target. Real-time quantitative PCR and FISH analysis confirmed SNP results. We characterized and mapped all breakpoints to recognize that two major deletions occur in BCR-ABL1-positive ALL: type A characterized by loss of exons 4–7 (37/97, 38%) and due to breakpoints occurring in the region spanning from 50380371 to 50380388 and from 50431123 to 50431167 in introns 3 and 7, respectively; type B characterized by removal of exons 2–7 (18/97, 19%) and due to a variable pattern of breakpoints in introns 1 (from 50317112 to 50317936) and 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 both deletions correlated with the expression of a dominant-negative isoform Ik6 with cytoplasmic localization and oncogenic activity, and of an aberrant transcript containing only exons 1 and 8, respectively. Interestingly, 2 pts with a homozygosis deletion showed simultaneously both type A and B deletions. In other 2 pts, the deletion involved the whole IKZF1 gene. The IKZF1 alteration was not a frequent event across different leukemias, since it was identified also at the progression of CML to lymphoid blast crisis (66%), but never in myeloid blast crisis CML (n=10), chronic phase CML (n=30) and acute myeloid leukemia (n=28). Known DNA sequences and structural features were mapped along the breakpoint cluster regions including heptamer recombination signal sequences recognized by the RAG enzymes during V(D)J recombination and activation-induced cytidine deaminase (AID) consensus motifs (DGYW/WRCH), suggesting that IKZF1 deletions could arise from aberrant RAG and/or AID-mediated recombination. GEP analysis of pts with IKZF1 deletion vs wild-type pts identified a unique signature characterized by a down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3), demonstrating that genomic IKZF1 alterations have a strong impact on trascriptoma and contribute to a block of B-cell differentiation. For 83 out of 97 BCR-ABL1 ALL pts correlation between molecular data and clinical outcome was available. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). In conclusion, deletion of IKZF1 resulting in either haploinsuffciency and in the expression of the dominant negative isoforms is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome.

Description: 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.

Description: Abstract 2027 Poster Board II-4 Background. The prognosis of Ph+ adult ALL patients treated with standard therapies, including multi-agent chemotherapy, Imatinib, and allogeneic stem cell transplantation, is still dismal, due to a high risk of relapse. Dasatinib and Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a BCR-ABL P210. Nineteen patients received Dasatinib, 2 patients Nilotinib and the remaining 4 patients were treated with third generation TKIs. Fourteen patients (56%) were in first relapse, and 7 (28%), 3 (12%) and 1 (4%) were in second, third and fourth relapse, respectively. A mutational analysis was performed in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also a cytogenetic response (CyR) and 6 patients a molecular response (MolR). With a median follow up of 10.8 months (range 2-29 months), median duration of HR, CyR and MolR were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs. Since TKIs are different and have different efficacy, pharmacokinetic, pharmacodynamic and toxicity profiles, and since are all effective, an alternative experimental option for the treatment of Ph+ ALL could be a combination or a rotation of two or more than two TKIs. Moreover, the addition of new promising targeted molecules, such as Aurora kinase or T315I inhibitors may contribute to overcome the problem of resistance to TKIs. Acknowledgments. Work supported by European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: No relevant conflicts of interest to declare.

Description: Nucleophosmin (NPM) is a multifunctional phosphoprotein that acts as nucleocytoplasmic shuttling protein, with tumour suppressor and oncogenic functions. Recently, NPM mutations have been found in a subset of adults with de novo acute myeloid leukemia (AML). These mutations occur in the last coding exon (exon 12), causing a frameshift and the formation of novel C-termini. The abnormal mutated NPM protein shows aberrant cytoplasmic localization and is frequently associated with FLT3 mutation. These observations provide basis for studies of the pathogenesis in AML. We did sequential analysis on patient samples during the clinical course to investigate the stability and pathogenetic role of NPM mutation in AML and the association with FLT3 mutations. The NPM mutations were determined by D-HPLC analysis; samples exhibiting an abnormal D-HPLC profile were confirmed by direct sequencing. We investigated 28 patients of de novo AML. Eleven samples were sequenced since they showed an heteroduplex D-HPLC profile. Type A mutation (960_963dupTCTG) was the commonest observed change, occurring in 9/11 samples, followed by type B mutation (960_963insCATG) in 1 case and type D mutation (960_963insCCTG) in 1 case. Furthermore, we observed that 5/11 patients harboring NPM mutation presented also mutant FLT3 at diagnosis. Analyzing NPM mutations during progression of disease, we observed that NPM mutation disappeared at complete remission and the same mutation reappeared at relapse. No differences were found in wild type NPM. Instead, we found a modification of FLT3 status associated to evolution of disease in 7/28 patients: 2 patients lost the mutation at relapse, 4 patients acquired the mutation at relapse and 1 patient modified the mutation from D835 to ITD (Table 1). Together these results suggest that NPM mutations and not FLT3 mutations may have utility as a potential marker for monitoring minimal residual disease. Studies on the biological effects of NPM mutations will contribute to disclose the role of NPM mutations in the pathogenesis of AML and their interactions with other genetic alterations such as FLT3.

Description: Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphia-positive leukemias. To assess how Bcr-Abl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for T315I. Patients already harboring mutations had higher likelihood of relapse associated with development of further mutations compared with patients who did not harbor mutations (23 of 51 vs 8 of 44, respectively, for patients who relapsed on second TKI; 13 of 20 vs 1 of 6, respectively, for patients who relapsed on third TKI).

Description: Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph+ acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non–DNA-binding Ik6 isoform was detected in 49% of Ph+ ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph+ ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph+ ALL patients.

Description: Abstract 4147 Background Standard induction chemotherapy for Acute Myeloid Leukaemia (AML) is represented by a combination of cytarabine and antracyclin with or without the addition of a third drug (e.g. etoposide), with a Complete Remission (CR) rate of about 60-65%. Aim The main goal of adding further drugs to induction therapy is to increase the CR rate in order to extend the opportunity of intensification therapies (e.g. autologous or allogeneic stem cell transplantation). Design and methods Eighty-four consecutive AML patients younger than 65 yrs were treated with a four-drug induction regimen Fludarabine (25 mg/sqm), Ara-C (2 g/sqm), Etoposide 100 mg/sqm on days 1-5, Idarubicin (6 mg/sqm) on days 1, 3, and 5, between 2002 and 2008 in two Italian Hematological Centres. The median age of this series of patients was 45 years (range 18-65), 39 were males and 44 were females. According to karyotype at diagnosis, white blood count (WBC 〉 30.000/mmc) and secondary disease, 58/84 patients (69%) were considered at high risk Patients were evaluated for response rate and treatment-related adverse events. Stem cell transplantation was performed according to the risk stratification and donor availability. Results After induction with FLAIE, CR occurred in 62 of 84 patients (74%). There were two death during induction (DDI 2 %). Six out of 22 resistant patients (7%) achieved CR after the second course of therapy, that was HD-Ara-c (2 g/sqm) on days 1-5 + idarubicine (12 mg/sqm) on days 1-3. Three patients were lost to follow up after induction, while in CR. The toxicity of FLAIE was acceptable, with grade III/IV hematological and extra-hematological adverse events comparable with standard induction therapy. Fifty-five patients of the 81 evaluable (68%) underwent transplant procedures while in first CR (22/81, 27 %, auto-SCT and 33/81, 41 %, allo-SCT). Five patients received allo-SCT in second CR, after early relapse. Three patients received allo-SCT with active disease and died because of disease progression. Sixteen patients (20%) received only chemotherapy-based consolidation because of either resistant disease or lack of donors or unsuccessful peripheral blood harvestt. After a median follow-up of 46 months (range 1-81), 40 of 81 evaluable patients (49%) are alive (40/40 in CR). Relapse rate is 32% at 4 yrs. These results suggest that a four drugs induction chemotherapy is feasible in young AML patients and can effectively permit intensification chemotherapy in at least 2/3 of the patients. A larger number of patients, in the context of a randomized trial may better define its efficacy with respect to standard 2 – 3 drugs induction therapy. Disclosures: Baccarani: Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau.