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Катедра по „Обща, експериментална и генетична психология, СУ „Св. Климент Охридски“ ** Катедра Психология, Университет на Нюкасъл, Австралия * Department of General, Experimental, and Genetic Psychology, Sofia University ** School of Psychology, University of Newcastle, Australia, Two empirical studies investigated the relation between different types of social groups and four core types of ingroup identification. It was hypothesized that particular types of group would be associated with particular types of ingroup identification. With minor discrepancies across samples, participants showed stronger social identification with social category groups, stronger communal identification with intimacy groups, and stronger interdependent identification with task groups. The results confirmed predictions and provided sufficient evidence to conclude that the manifestation of different types of ingroup identity varies as a function of the type of group that is most salient at the moment of identification.–––––––––––––––––––––––––––––––––––––––––––––––––––––– Две емпирични изследвания разкриват връзката между различни типове социални групи и четири основни видa вътрешногрупова идентификация. Предположено е, че конкретни типове групи ще бъдат свързани с конкретни видове вътрешногрупова иден- тификация. С малки несъответствия между извадките, участниците в изследванията показват по-силна социална идентификация със социална категория групи, по-силна общностна идентификация с интимни групи и по-силна взаимозависима идентифика- 120 ция с работни групи. Резултатите потвърждават предвижданията и предоставят доста- тъчно доказателства, за да се заключи, че проявата на различни видове групова иден- тификация варира като функция на типа на групата, която е най-актуална в момента на идентифициране.

Background: The activation of inositide signalling pathways, such as Akt/mTOR, has been demonstrated in high-risk MDS. Akt also binds PI-PLCgamma1, leading to a deregulation of stem cell proliferation, differentiation and apoptosis. These processes are critical in low-risk MDS, that usually show a marked apoptosis and a low proliferation rate, which can be rapidly reversed, thus leading to a worse clinical status. Introduction: Lenalidomide is currently used in the treatment of del(5q) low-risk MDS patients, where it may suppress the del(5q) clone and restore a normal erythropoiesis. Lenalidomide also shows anti-angiogenic activity and suppresses inflammatory cytokine release. The exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still unclear, even though it has been demonstrated that in Lenalidomide-sensitive del(5q) cell lines, Akt phosphorylation is inhibited. Purpose: We studied the effect of Lenalidomide on nuclear inositide signalling pathways in del(5q) low-risk MDS. Materials and Methods: We studied the expression of inositide signalling molecules in 6 patients diagnosed with del(5q) MDS (IPSS: Low or Int-1) who were given Lenalidomide. Given the limited number of cells, we quantified the expression of Akt and PI-PLCgamma1 in bone marrow total mononuclear cells. As for Akt phosphorylation, we analyzed its localization along with RPS14, in order to specifically detect the del(5q) clone. Moreover, by Real-Time PCR analyses, we assessed the expression of Beta-Globin, to evaluate the effect of the drug on erythropoiesis. Results: In our case series, 4 out of 6 del(5q) low-risk MDS patients responded to Lenalidomide and showed an activation of erythropoiesis, in that Beta-Globin levels increased, as compared with baseline. Moreover, these subjects also displayed an activation of PIPLCgamma1 and Akt. Interestingly, Akt resulted to be specifically phosphorylated in cells not showing the 5q deletion, hinting at a clonal activation of this pathway. The 2 non responder patients early discontinued Lenalidomide for adverse events, and for these patients neither a clinical assessment of Lenalidomide effect, nor a molecular analysis, were possible. Conclusions: Our data show Akt/PI-PLCgamma1 activation during Lenalidomide treatment, and confirm the activation of erythropoiesis in responder patients. In addition, our results indicate that Akt is specifically phosphorylated in the 5q+ clone. Therefore, it is conceivable that Lenalidomide strengthens the proliferation of the 5q+ clone, whilst the del(5q) clone undergoes an apoptotic process, allowing the restoration of the normal erythropoiesis. This is extremely important, not only for MDS pathogenesis, but also for the development of innovative targeted therapies.

Azacitidine (AZA) has proven effective in Myelodysplastic Syndromes (MDS), and the currently approved AZA regimen is 75 mg/sqm/die subcutaneously for 7 days every 28 days. Subsequently, other alternative and more convenient AZA dosing regimens have shown to be effective, in terms of hematologic responses (Lyons, 2009). From September 2004, in our Institution, 57 MDS patients (pts) (43 males), with a median age of 70 (37-84) yrs, were treated with AZA, following 4 different treatment regimens. Group 1 (10 pts), received the currently approved regimen (AZA 7). Group 2 (6 pts), received the AZA 7 regimen with valproic acid and all-trans-retinoic acid. Group 3 (29 pts) received the AZA 5-2-5 regimen: 50 mg/sqm/die SC for 10 days/28 days. Group 4 (12 pts) received the AZA 5 regimen: 75 mg/sqm/die SC for 5 days/28 days. Moreover, we quantified the degree of phosphoinositide-phospholipase C (PI-PLC) beta1 methylation and gene expression before and during AZA administration. At AZA onset, IPSS risk was: low: 3 pts; intermediate-1: 12 pts; intermediate-2: 33 pts; high: 9 pts. R-IPSS risk was: very low: 1 pt; low: 3 pts; intermediate: 7 pts; high: 10 pts; very high: 36 pts. WPSS risk was low: 3 pts; intermediate: 5 pts; high: 9 pts; very high: 40 pts. 9 pts had therapy-related MDS. ECOG-PS was poor (≥2) in 15 pts. Transfusion need was high (≥4 RBC units/8 weeks) in 34 pts. 6 pts presented circulating blasts. Following Itzykson’s AZA prognostic scoring system, the risk was low in 7 pts (12.3 %), intermediate in 48 pts (84.1 %), and high in 2 pts (3.6 %). The pts received a median of 8 cycles of AZA (range: 1-59). 50 pts were considered evaluable for response (at least 6 cycles): 34/50 pts (68%) showed a favourable response following IWG criteria (Cheson, 2006): complete remission (CR) in 7 pts (14% %), hematologic improvement (HI): 27 pts (54%). The median duration of response was 12 (range: 1-88) months. Group 1: 5 responders (62.5%) (1 CR and 4 HI); Group 2: 3 responders (50%) (3 HI); Group 3: 19 responders (73.1%) (5 CR, 14 HI); Group 4: 7 responders (70%) (2 CR, 5 HI). A significant toxicity (grade >2) was observed in 23 (40.4 %) pts. 37 pts died, 13 for AML, 9 for infection, 17 for other causes. Median OS from the start of AZA was 18 (range: 5-108) months. The detection of an increase in PIPLCbeta1 gene expression within the first three cycles of AZA therapy was significantly associated with a better clinical outcome and a longer hematologic response.

The present research investigated linguistic description as a moderator of biased evaluations of counterstereotypical individuals. Members of an online participant pool (N = 237) indicated their liking for stereotypical and counterstereotypical individuals who were described using adjectives or behaviors. There was a significant interaction between target typicality and linguistic description: People liked counterstereotypical individuals more than stereotypical individuals when target individuals were described using adjectives. In contrast, they showed no bias or a negative bias against counterstereotypical individuals who were described using behaviors. This interaction effect generalized across gender targets (men/women) and sexuality targets (gay/straight), and it was partially mediated by subjective processing fluency. Implications for the backlash effect and prejudice reduction are discussed.

Introduction. Azacitidine (AZA) has proven effective in myelodysplastic syndromes (MDS). The duration of haematological response is limited (median 13.6 months) (Fenaux, 2009), although some patients (pts) show a prolonged response. These data prompted us to retrospectively analyse our MDS pts treated with AZA, in order to enucleate longresponder pts (duration of response ≥20 months). METHODS. From September 2004, in our Institution, 52 MDS pts (40 males), median age: 70 (37-85) yrs, were treated with AZA, following 4 different treatment regimens: 9 pts received the AZA 7 regimen (AZA 7: 75 mg/sqm/die SC for 7 days/28 days); 6 pts received the combination of AZA 7 with valproic acid and all-trans-retinoic acid ; while 25 and 12 pts respectively received the alternative regimens AZA 5-2-5 and AZA 5 (Lyons, JCO 2009). 37 pts (71%) showed a IPSS high-risk MDS, while 15 pts (29%) with IPSS low-risk MDS received AZA because of refractoriness or ineligibility to erythropoietin, or secondary MDS. Moreover, as our group (Follo, 2009) demonstrated that phosphoinositide-phospholipase C (PIPLC) beta1 may represent a target for AZA, we quantified the degree of PI-PLCbeta1 methylation and gene expression before and during AZA administration. RESULTS. 9 pts (17.3%) showed a prolonged hematologic response (≥ 20 months). Pre-treatment clinical and haematologic features of long-responders: sex (M/F): 4/5; median age: 69 (52- 84); WHO: RCMD-RS: 1 pt; RAEB-1: 1 pt; RAEB-2: 7 pts; IPSS risk: low: 1 pt; int-1: 2 pts; int-2: 5 pts; high: 1 pt; IPSS cytogenetic risk: low: 7 pts; interm: 1 pt; high: 1 pt; ECOG: 0-1: 8 pts, ≥ 2: 1 pt; transfusion need (N° U)/8 weeks: < 4 : 4 pts; ≥ 4 : 5 pts; time from diagnosis (months): < 6 : 6 pts; ≥ 6 : 3 pts. Therapeutic regimen: AZA 7: 3 pts; AZA 5: 3 pts; AZA 5-2-5: 3 pts. Therapeutic response: median number of cycles: 19 (8-59); median time to 1st response: 3 (2-6) months; type of response: Complete Remission (CR): 3 pts; Hematologic Improvement (HI): 6 pts; cytogenetic remission: 1 pt; median duration of response: 30 (24-66) months; doubling of platelet count after 1st cycle: 4 pts; toxicity (grade > 2): 3 pts; 4 pts are still maintaining hematologic response, 3 pts are still alive but discontinued treatment because of disease progression, and 2 pts died (1 for AML and 1 for cachexy). Median survival (from the start of AZA): 38 (25-103) months. All the pts showed an increase in PI-PLCbeta1 expression, that was maintained along with the hematologic response. 15 pts (28.8%) showed a short-lived reponse (< 20 months), 5 pts (9.6%) show a shorter response but are still on treatment, 2 pts underwent allogeneic transplantation after 5 and 10 months. 7 pts (13.4%) are not evaluable for response (< 6 cycles), and 14 pts (26.9%) did not respond to AZA. Conclusions. Our data show that a limited but significant fraction of MDS pts show a long-lasting hematologic and molecular response to AZA.

The most recent clinical trials on adult acute lymphoid leukaemia (ALL) have shown complete remission and disease-free survival (DFS) rates of 80-85% and 30-40%, respectively (Annino, et al, Durrant, et al, Kantarjian, et al, Larson, et al, Ribera, et al, Rowe). Intensified consolidation, particularly with high-dose methotrexate and high-dose cytarabine, may be one of the reasons for the improved outcome in recent series (Bassan and Hoelzer, Hoelzer and Gokbuget, Kebriaei and Larson). In addition, risk-adapted and subtype-oriented therapy may have contributed to this better outcome. However, the long term outcome of adult patients is still dismal, with approximately one third of the cases only being cured. At present, therapeutic options include conventional chemotherapy (CHT), high dose therapy with autologous and, especially, allogeneic stem cells transplantation (SCT) and, for certain subsets, such as BCR-ABL1+ ALL, specific targeted therapy (Piccaluga, et al). Although SCT has been used in adult ALL for more than 20 years, its role remains controversial as demonstrated by conflicting results in various studies. Previous casecontrolled studies did not show that allogeneic SCT (alloSCT) provided any advantage over CHT (Horowitz, et al, Zhang, et al) while in some studies there was an advantage, but restricted to young adults (Oh, et al). The number of controlled published or ongoing trials is remarkably small and some of them did not include both standard-risk and high-risk patients. Thus, it is difficult to draw definitive conclusions from their results. In fact, while some authors did not report any differences between alloSCT and chemotherapy or autologous SCT (ASCT)(Gupta, et al, Labar, et al), others only found differences favouring allogeneic SCT in standard risk (Goldstone, et al) or high-risk ALL patients (Sebban, et al, Thiebaut, et al, Thomas, et al). In this chapter, the Authors reviewed data concerning alloSCT in adult ALL and discuss current controversial and possible perspectives.

Introduction. Inositide signalling pathways are involved in cell growth, differentiation and apoptosis and play a role in the progression of MDS towards AML. In particular, an altered expression of nuclear PI-PLCbeta1 and activated Akt can lead to a deregulation of cell cycle processes, therefore affecting the survival of primary MDS cells. Moreover, we postulated an inverse correlation between PI-PLCbeta1 expression and Akt activation in MDS. Indeed, these processes are critical especially in low-risk MDS, that usually show a marked apoptosis and a low proliferation rate, which can be rapidly reversed, thus leading to a worse clinical status. Lenalidomide is currently used in the treatment of del(5q) low-risk MDS patients, to compensate and counteract their ineffective erythropoiesis. In fact, this drug has anti-angiogenic activity, suppresses inflammatory cytokine release, induces the erythroid differentiation and enhances the EPO receptor signalling. The exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still unclear, even though it can target signalling pathways playing a role in the maintenance of the balance between apoptosis, proliferation and differentiation, such as PI3K/Akt. Methods. We studied 6 patients diagnosed with del(5q) Low-Risk MDS (IPSS: Low or Int-1) who were given Lenalidomide. We quantified the expression of several genes implicated in inositide signalling, such as PI-PLCbeta1 splicing variants and PIPLCgamma1, as well as Cyclin D3 and Beta-Globin, in order to assess the effect of Lenalidomide on erythropoiesis and cell cycle. Results. In our case series, 4 out of 6 del(5q) Low-Risk MDS patients responded to Lenalidomide and showed an activation of erythropoiesis, in that BetaGlobin levels increased. Moreover, these subjects also displayed an activation of PI-PLCgamma1, which is associated with PI3K/Akt activation. As for the other 2 cases, patients early discontinued Lenalidomide for adverse events, and for these patients a clinical assessment of Lenalidomide effect was not possible. Conclusions. Our data support the hypothesis of a role for PI-PLCgamma1 activation during Lenalidomide treatment, and confirm the activation of erythropoiesis in responder patients. In fact, Lenalidomide increased the expression of genes specifically associated with erythropoiesis, like Globin genes, in our responder patients. Our results also indicate that both PI-PLCbeta1 splicing variants, as well as Cyclin D3, are not significantly affected by Lenalidomide, whereas PIPLCgamma1 is specifically induced. Taken together, these results point to a specific activation of this pathway during the therapy and possibly pave the way to a larger investigation aiming to assess the role of these pathways in Lenalidomide response.

Introduction. Erytropoiesis-stimulating agents (ESAs) are effective in 20-30% of patients (pts) with Myelodysplastic Syndromes (MDS), but response rates are higher when pts are selected on the basis of clinical and hematologic parameters: baseline serum erythropoietin (EPO) level 4 units/8 weeks): 6 pts (17.6%); long interval from diagnosis to the start of EPO ( > 6 months) : 20 pts (58.8%). Starting weekly EPO dose: 80.000 U: 20 pts; 40.000 U: 14 pts. Outcome: median time to response: 8 (4-32) weeks; type of response: Complete Response (CR): 14 pts (44.1%); Haematologic Improvement (HI): 19 pts (55.1%); median duration of response: 47.5 (20-125) months; 15 pts (44.1%) were able to shift to a lower maintenance dose. Relapse occurred in 7 pts (20.6%) ( in 2 pts because of disease progression) after a median of 48 months. 24 pts are still alive, 5 pts died (none of them for AML), and 5 pts were lost at follow-up. Median survival (from the start of EPO): 51 (22-128) months. 13 pts show a shorter response but are still on treatment , 12 pts showed a short-lived reponse (< 20 months), and 10 pts did not respond to EPO. Conclusions. Our data show that, although in MDS the duration of response to EPO is limited, a substantial fraction of pts may show a long-lasting response, and that, unexpectedly, some of them may show unfavourable pre-treatment prognostic features (WPSS risk, high transfusion need).

Background: Deletions of IKZF1 gene, encoding for a regulator of lymphocyte differentiation, occur in more than 80% of adult patients (pts) with Ph+ ALL and significantly affect the prognosis. The deletions either involve the entire IKZF1 locus, resulting in loss of function, or delete an internal subset of exons, resulting in the expression of dominant negative isoforms. To better stratify Ph+ ALL pts according to IKZF1 status, we set up, validate and assess the routine applicability of a IKZF1 deletion screening strategy based on a multiplex-PCR. Patients and methods: We studied 87 adult BCR-ABL1+ ALL pts. For each type of common IKZF1 deletion (D4-7, D2-7, D4-8) an appropriate pair of primers will be designed using Primer 3 (http://frodo.wi.mit.edu/primer3/). Their ability to efficiently amplify the deletion was tested. PCR amplifications was performed using 50 ng genomic DNA as template for each reaction and a FastStart Taq DNA Polymerase (Roche Diagnostics). For each patient 3 amplicons were generated and sequenced (amplicon A for D4-7, amplicon B for D2-7 and amplicon C for D4-8). Moreover, a multiplex amplification strategy was assessed and a forth amplicon was generated and sequenced (forward primers were localized on intron 1 and 3, the reverse ones in the intron 7 and at the end of the gene, after the exon 8). The size of amplicons depends on the positions of the breakpoints in the IKZF1 gene and on the number of nucleotides added at the conjunction. It was in the range of 450-600 nucleotides. A positive control was used for each run. Results: On 87 pts previously analyzed by SNPs array, we identify IKZF1 deletions in 69/89 (78%) samples. Deletions were: 51 D4-7, 12 D2-7 and 4 D4-8; in 2 cases the deletion was extended to all gene, and 2 pts presented two breakpoints simultaneously. Multiplex PCR and subsequent sequencing were performed for most common deletions on 40 pts confirming previous results and indentifying the precise genomic positions of breakpoints and the nucleotides added at the conjunction. This rearrangement has been used to strictly monitor Minimal Residual Disease (MDR) during the follow-up and at relapse to confirm the clonal fidelity. Conclusions: We set-up a method rapid sensitive and with less amount of DNA sample, to screen Ph+ ALL pts at diagnosis and to monitor MRD during the treatment. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo, GIMEMA Onlus.

Loss of CDKN2A, encoding p16/INK4A and p14/ARF tumor suppressors, has been associated with poorer survival and higher leukemia-initiating- cell-frequency in xenograft models (Notta F et al. Nature 2011). Recently, using genome-wide single nucleotide polymorphisms arrays and gene candidate deep exon sequencing, we identified CDKN2A deletions in 29% (29/101) adult newly diagnosed Ph+ ALL patients and in 47% relapsed cases. Accordingly, here we aimed to investigate the functional consequences of genomic deletions and their prognostic value. By quantitative real-time PCR a significant difference in the expression of CDKN2A was observed among cases with heterozygous (p = 0.04) and homozygous (p = 0.01) loss compared to normal cases, suggesting that deletions lead to CDKN2A haploinsufficiency. In order to investigate their clinical implications, data were collected from 81 patients (median follow-up: 25.2 months- range 2.1-148.1; median age at diagnosis: 54 years- range, 18-76; CDKN2A loss in 29, 36%, cases). 72 patients (89%) were enrolled in the GIMEMA clinical trials (12 patients in GIMEMA LAL0201-B protocol, 13 in LAL2000 and 47 in LAL1205 protocols), while 9 patients (11%) were enrolled into institutional protocols. By univariate analysis a shorter overall survival (OS) and disease-free survival (DFS) were found in patients with CDKN2A deletion compared with those wild-type (OS: 27.7 v 38.2 months, respectively, p = 0.0206; DFS: 10.1 vs 56.1 months, respectively, p = 0.0010). Moreover, a higher cumulative incidence of relapse for patients with CDKN2A deletion (73.3 vs 38.1; p = 0.0014) was also recognized. The multivariate analysis confirmed the negative prognostic impact of CDKN2A deletion on DFS (p = 0051). These results show that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation, cell growth and tumor formation by the action of MDM2 and CDK4/6, attractive drugs to prevent disease recurrence could be represented by the inhibitors of MDM2 and CDK4/ CDK6. These drugs are currently under investigation in in vitro studies by our group. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program, Programma di Ricerca Regione – Università 2007 – 2009.

Introduction: Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed. Methods: 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions were genotyped in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) ALL and acute myeloid leukemia (AML) samples, and 183 healthy controls. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Results: A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with MAF 20% missing call rates, were instead excluded from the association analysis and potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus (exon 2) that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20 to 3.33; p= 7.1 x 10-3). Conclusions: Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Recently, the rs564398 has been demonstrated by Cunnington M.S. et al. (2010) to alter ANRIL expression leading in turn to a deregulation of CDKN2A/B genes. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program (PIO), PRIN 2008, Programma di Ricerca Regione – Università 2007 – 2009.

Background. Nelarabine (N) is approved for the treatment of T-ALL and T-LBL that have not responded to or has relapsed after treatment with at least 2 chemotherapy regimens. Aim. To evaluate safety profile and efficacy of N as savage therapy in 16 adult relapsed or refractory TALL or T-LBL. Methods. After obtaining an informed consent, 16 patients (median age 33 years, range 19-45, M/F= 13/3) affected by TALL (n=10) and T-LBL (n=6) received savage therapy with N (median cycle=1, range 1-3), administered at standard adult dosage (1500 mg/sqm on days 1, 3 and 5, every 21). Four patients were primary resistant to induction treatment, 7 patients were relapsed after two previous chemotherapy regimens (including allogeneic BMT in 4 cases and autologous SCT in 1 case); the remaining 6 patients had a molecular relapsed disease (MRD positive). Molecular characterization was performed, including NOTCH and WT-1 genes mutational status. GEP analysis, according to Ferrando A. stratification (Cancer Cell 2002), is still ongoing. Results. Currently, 12 out of 16 patients are evaluable, due to a too short follow up in the other 4 cases. Seven out of 12 patients obtained a complete remission (CR) (5 T-ALL 2 T-LBL);a partial remission (PR) was documented in 2 cases, with an overall response rate (ORR) of 75%. Median duration of CR was 10 weeks (range 2.8-54+). Among these, 2 out of 4 patients in molecular relapse reached a molecular CR and underwent an allogenic BMT (currently in CR after a median follow up of 12 months). Extra- hematological toxicity, not clearly related to the drug, occurred in 3 cases, determining, a complete and irreversible paraplegia, a condition of mental confusion, and a peripheral neuropathy, respectively. Conclusions. N showed a strong efficacy also in cases with low levels of residual disease, in addition to a good safety profile. Neurological toxicity needs to be strictly monitored. Acknowledgments. European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Background. Azacitidine (AZA), at a dosing schedule of 75 mg/m2/d subcutaneously for 7 days every 4 weeks, induces high hematologic response rates (60-80%) in patients with Myelodysplastic Syndromes (MDS), and prolongs overall survival in high-risk MDS patients (Silverman, 2002; Fenaux, 2009). However limited data are available concerning the efficacy and safety of AZA in low- risk MDS. Moreover, although continuation of AZA treatment is generally recommended in all responder patients, the optimal duration of therapy has not been clearly defined, especially for low-risk patients. Recently, a community-based study mainly involving low-risk MDS (Lyons, 2009), showed that the lower dose AZA 5 regimen (75 mg/m2/d subcutaneously for 5 days), which avoid week-end dosing, can induce therapeutic responses consistent with the currently approved schedule. Aims. These data prompted us to investigate the therapeutic effect of the AZA 5 regimen, administered as induction treatment for a total of 8 courses, in low-risk MDS patients with symptomatic anemia refractory to erythropoietin (EPO). Methods. From September 2008 to February 2010, 34 patients with low-risk MDS (IPSS score low or intermediate-1) were enrolled into the study. Age at diagnosis ranged between 56 and 84 years. Moreover, as our group (Follo, 2009) previously demonstrated that the inositide signalling pathways, in particular phosphoinositide-phospholipase C (PI-PLC) beta1, may represent a target for AZA, we quantified the degree of PI-PLCbeta1 methylation and gene expression before and during AZA administration. Results. According to the 2006 International Working Group criteria (Cheson, 2006), overall response rate (ORR) was 61% (14/23 evaluable patients), including Complete Remission (CR) (22%), and Hematologic Improvement (HI) (39%). Unexpectedly, in 3/14 responder patients we observed a long duration of response, still ongoing, after discontinuation of AZA. Patient 1, a 77 yr male, (WHO diagnosis: Refractory Anemia; IPSS risk: low; karyotype: normal) started AZA on September 2008. He showed a 1st response (HI, erythroid response), with a significant reduction of transfusions, after 3rd course of AZA, and became completely transfusion-independent from August 2009, 3 months after the completion of the 8th course (duration of response: 18 months). Patient 2, a 61 yr female, (WHO diagnosis: Refractory Anemia with Excess of Blasts-1; IPSS risk: intermediate-1; karyotype: normal) started AZA on October 2008, became transfusion-independent after 5th course, and achieved CR after 8th course (duration of response: 24 months). Patient 3, a 70 yr female, (WHO diagnosis: Refractory Cytopenia with Multilineage Dysplasia; IPSS risk: intermediate-1; karyotype: normal) started AZA on February 2009, and achieved HI (erythroid response) after 2nd course (duration of response: 22 months). All the patients showed an increase in PI-PLCbeta1 expression, in association with the hematologic response, and still maintain the molecular response. Summary/Conclusions. Our clinical and biological results support the feasibility and effectiveness of AZA treatment in low-risk MDS, especially in the case of EPO refractoriness. After achievement of hematologic response, discontinuation of AZA might be attempted, especially in lowrisk MDS, where the risk of leukemic evolution is lower. However, the effectiveness of AZA resumption, in case of relapse, is still unknown.

Lenalidomide (Len) has proven effectiveness in 70-80% of low-risk MDS cases with del(5q), resulting in transfusion-independence, and inducing a rise hemoglobin levels, suppression of the 5q clone and improvement of bone marrow morphologic features. In del(5q) MDS, Len might suppress the dysplastic clone, while in non-del(5q) it may promote effective erythropoiesis, via activation of EPO signalling, which in turn is associated with PI-PLCgamma1 pathway. However, the exact molecular mechanisms underlying the effect of Len in MDS cells are still unclear. Interestingly, Len targets the phosphatase PP2A, whose gene is located in the common deleted region and which usually targets Akt. Indeed, Akt-dependent pathways are critical in low-risk MDS, which display a marked apoptosis and a low proliferation rate. Recently, our group showed that inositide signalling pathways are involved in the MDS progression to AML. In particular, we demonstrated not only that MDS can show alterations on PI-PLCbeta1 and Akt pathways, but also that Akt is inversely correlated with PI-PLCbeta1, therefore affecting MDS cell survival and differentiation. Here, we report on a patient affected by MDS who was successfully treated with Len. The patient, a 58-year old female, was diagnosed with Refractory Anemia (IPSS: Low) and was given only supportive care before undergoing Len treatment. Shortly after the beginning of the therapy, in 2009, the patient showed a clinical favorable response to Len, and subsequently achieved complete hematologic and cytogenetic remission. To assess the molecular effects of Len on inositide signalling pathways, we analyzed the expression of critical genes involved in cell proliferation and differentiation, i.e. PI-PLCbeta1 and its downstream target Cyclin D3, as well as PI-PLCgamma1, which is linked with EPO signalling and Akt activation. That is why ongoing analyses are also trying to examine the effect of Len on Akt, and the correlation between Len and the expression of other genes specifically associated with erythropoiesis, like Globin genes. So far, our results indicate that both PI-PLCbeta1 and Cyclin D3 are not significantly affected by Len, whereas PI-PLCgamma1 is specifically induced. Consequently, these findings hint at a specific activation of PI-PLCgamma1 signalling following Len treatment, and possibly pave the way to further investigations aiming to better understand the role of these pathways in the mechanism of action of Len in del(5q) MDS.

Background: Azacitidine (AZA) has proven effective (response rate: 60–80%) in myelodysplastic syndromes (MDS). The currently approved AZA regimen is 75 mg/sqm/die subcutaneously (SC) or intravenously (IV) for 7 days every 28 days (Silverman, 2002; Fenaux, 2009). Introduction: Recently 3 different AZA dosing regimens, which avoid week-end dosing, have shown to induce therapeutic responses consistent with the currently approved schedule (Lyons, 2009). In particular, one of them, i.e. the AZA 5–2-5 regimen (50 mg/m2/d subcutaneously for 5 days, followed by 2 days no treatment, then 50 mg/m2/d for 5 days) allows the administration of a nearly equivalent monthly total dose of AZA. Moreover, some data suggest the possibility that prolonged exposure to lower doses of AZA may increase the response rate (Gore, 2006). However, the communitybased study of Lyons mainly involved lower-risk MDS patients (pts). Purpose: These data prompted us to investigate the therapeutic effect of the more convenient AZA 5–2-5 regimen in higher-risk MDS pts (i.e.: IPSS risk: high or intermediate-2). Material and Methods: From December 2007, in our Institution, 18 IPSS high-or-intermediate-2 risk MDS pts. (12 males), with a median age of 69 (37–81) yrs, were treated with the AZA 5–2-5 regimen. Moreover, as our group (Follo, 2009) previously demonstrated that the inositide signalling pathways, in particular phosphoinositidephospholipase C (PI-PLC) beta1, may represent a target for AZA, we quantified the degree of PI-PLCbeta1 methylation and gene expression before and during AZA administration in this group of pts, and in a control group of high-risk pts previously treated with the conventional AZA regimen. Results: The pts received a median number of 7.5 (1–15) AZA cycles. Among the 14 evaluable pts (i.e.: ≥6 cycles), 12 (85.7%) showed a favourable response, following IWG criteria (Cheson, 2006): 5 Complete Remission (CR), and 7 Hematologic Improvement (HI). First response occurred after a median of 3 (3–7) cycles. The remaining 2 pts maintained a stable disease (SD). 4 pts showed evolution into Acute Myeloid Leukemia (AML) (3 died), and 2 pts died for other causes (infection, heart failure). PI-PLCbeta1 methylation and gene expression appeared to be related to the therapeutic response, but not to the dose schedule. Conclusions: Our data seem to confirm, in a population of highrisk MDS, the effectiveness, in terms of hematologic and molecular response, of the more convenient AZA 5–2-5 regimen. Our results need to be supported by larger studies.

Background. Fludarabine plus Cytarabine and Idarubicine (FLAI) was proved to be an effective and well tollerated induction regimen for treatment of acute myeloid leukaemia (AML). The trial objective was to assess the efficacy of Etoposide when used in combination with FLAI schedule. Design and Methods. We retrospectively report clinical outcome results of 101 newly diagnosed and younger than 60 years AML patients (median age 46 years, range 18-60 years) treated in a Phase III clinical trial, with FLAIE induction chemotherapy, including Etoposide to the FLAI schedule. Induction consisted of Fludarabine 25mg/m2/day on days 1-5, Idarubicin 6 mg/m2/day on days 1, 3 and 5, Cytarabine 2 g/m2 infused in 4 h, daily on days1–5 and Etoposide 100 mg/m2/day on day 1-5. After induction, all the patients underwent consolidation with Cytarabine (2 g ⁄sqm i.v. infusion on days 1–5) and Idarubicin (12 mg⁄ sqm i.v. infusion on days 1, 3 and 5). All the patients shared the same strategy for intensification, that was allogenic or autologous stem cell transplantation. After consolidation, maintenance treatment with Cytarabine was given to patients who obtained a complete remission but who could not undergo allogenic or autologous stem cell transplantation. More than half of the patients had abnormal karyotypes. Molecular analysis at diagnosis for the more frequent abnormalities was performed. Duration of CR and overall survival was estimated according to the Kaplan-Meier method. The CR duration was dated from start of CR to first evidence of recurrence. Results. After informed consent was obtained, the patient received a single induction course of FLAIE; 73 pts obtained a CR (72.2%) and 8 pts a CRp (7.9%) for an overall response rate of 80.2%. Fifteen patients (14.9%) had resistant disease, and 5 (4.9%) died during induction. After a median follow-up of 33 months, 75 patients (76%) are in continuous CR. The median CR duration and OS were 45 and 55 months, respectively. 11 pts underwent ABMT and 44 a BMT. Relapses were more frequent in patients who were not submitted to allogenic stem cell transplantation. Of the 55 transplanted patients, 28 (51.9%; 1 with chromosome 7 abnormality), were alive in CR after a median follow up of 10 months (range, 2 to 45 months) after transplantation, 14 (25.9%) relapsed (median DFS 4 months), and 13 (24%) died in CR or CRp of transplant related complications. The most common grade 3 adverse events included gastro-intestinal toxicities(i.e. nausea, vomiting, mucositis and diarrhoea), liver dysfunction, and skin rash. Conclusion. The combination of etoposide to FLAI is safe and active. Further studies exploring different dosing and scheduling are warranted, particularly in patients with poor-risk AML. Acknowledgments. Work supported by European LeukemiaNet, FIRB 2008, AIRC, AIL, COFIN, University of Bologna and BolognAIL.

Background: Genetic heterogeneity in drug-metabolizing enzyme and transporter genes affects specific drug-related phenotypes in cancer. In order to investigate a relationship between genetic variation and response in AML, we comprehensively assessed the allele frequencies of 1936 genetic variations of 225 absorption, distribution, metabolism and excretion genes in 94 CD33-positive AML patients younger than 65 years, using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform. Patients and methods: All patients were enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy (ClinicalTrials.gov NCT00909168). Cytogenetics, multidrug-resistance phenotype, FLT3 and NPM mutation status, as well as WT1 quantitative expression analyses were performed at diagnosis. Furthermore, high-resolution single nucleotide polymorphism (SNP) array and DMET genotyping analyses (Affymetrix) were also performed. Results: Of the 94 patients, genotype results were evaluable for 91 cases. The median call rate was 99.48 (range, 96.32-100). Firstly, we tested the association among SNPs and response to the induction cycle (FLAI + GO) comparing the genotyping profile of 80 patients in complete (85%) and partial (3%) remission with that of patients (12%) with no response. A highly significant difference (p < 0.001) in the allele frequency of 2 variants, in complete linkage disequilibrium, in the alcohol dehydrogenase enzyme (ADH1A) was found. ADH1A metabolizes the conversion of ethanol to acetaldehyde which is thereafter converted to acetate by ALDH1, which is well known as stem cell marker. These variants were not associated with high risk AML, FLT3 and NPM1 mutations, but strongly influenced response to the induction phase also in a multivariate analysis. Secondly, SNPs were stratified according to liver toxicity and a significant difference in the allele frequency in CYP2E1, involved in the alcohol metabolism was found to be associated with a grade I/II liver toxicity. Conclusions: Genetic variations in ADH1A and CYP2E1 genes were for the first time identified and associated with response and toxicity in AML patients treated with a combination of GO and FLAI regimen. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007–2009.

Background: In acute myeloid leukemia (AML) the presence or absence of cytogenetic abnormalities allows the identification of favorable, intermediate and unfavorable subgroups. However, besides these specific subgroups, little it is known about the genetic variations influencing specific drug-related phenotypes. Aims: To perform an exploratory pharmacogenomic study that relates genetic variations in multidrug enzymes and transporters genes with the efficacy and toxicity to treatment. Patients and methods: We analyzed 94 CD33-positive AML patients younger than 65 previously untreated and enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy (eudract: 2007-005248-26; ClinicalTrials.gov NCT00909168). The induction regimen (GO-FLAI) included fludarabine (25 mg/sqm) and Ara-C (2 g/sqm) on days 1-5, idarubicin (10 mg/sqm) on days 1, 3, and 5 and GO (3 mg/sqm) on day 6. Hematopoietic stem cell transplant was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin. Cytogenetics, multidrug-resistance phenotype, FLT3 and NPM mutation status, as well as WT1 quantitative expression analyses were performed at diagnosis in all patients. Furthermore, high-resolution single nucleotide polymorphism (SNP) array analysis (Affymetrix, Inc. Santa Clara, CA, USA) was also performed. The allele frequencies of 1936 genetic variations of 225 absorption, distribution, metabolism and excretion were assessed using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform (Affymetrix). All statistical analyses were performed using the R package 2.11.1. Results: Of the 94 patients, genotype results were evaluable for 91 cases. The median call rate was 99.48 (range, 96.32-100). Three samples were run in duplicate and results with “passed call rate” were compared across all the polymorphic sites, showing a repeatability of 99.99%. In an initial screening procedure, we tested the association among SNPs and response to the induction cycle (FLAI + Gemtuzumab-Ozogamicin). Therefore, the genotyping profile of 80 patients in complete (85%) and partial (3%) remission was compared to that of patients (12%) with no response. We found a highly significant difference (p < 0.001) in the allele frequency of 2 variants, in complete linkage disequilibrium, in the alcohol dehydrogenase enzyme (ADH1A). These variants were not associated with high risk AML, FLT3 and NPM1 mutations, but strongly influenced response to the induction phase also in a multivariate analysis. Since genetic polymorphisms may influence the toxicity of chemotherapy drugs, we stratified SNPs according to liver toxicity and a significant difference in the allele frequency of a member of the cytochrome P450 family which is involved in the alcohol metabolism (CYP2E1) was found to be associated with a grade I/II liver toxicity. Conclusions: A pharmacogenomic panel made up of 1 gene (ADH1A) associated with clinical outcome and 1 gene (CYP2E1) associated with toxicity was for the first time identified in AML patients younger than 65 years treated with a combination of GO and FLAI regimen. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program, PRIN 2008, Programma di Ricerca Regione – Università 2007 – 2009.

Azacitidine (AZA) has proven effective (response rate: 60-80%) in myelodysplastic syndromes (MDS). The currently approved AZA regimen (AZA 7) is 75 mg/sqm/die subcutaneously (SC) for 7 days every 28 days. Recently a different AZA dosing schedule (AZA 5-2-5: 50 mg/m2/d subcutaneously for 5 days, followed by 2 days no treatment, then 50 mg/m2/d for 5 days), which avoids week-end dosing, has shown to induce therapeutic responses consistent with the currently approved schedule, in a population of mainly low risk pts (Lyons, 2009). These data prompted us to investigate the therapeutic effect of the AZA 5-2-5 regimen in high risk MDS pts (IPSS risk: high or intermediate-2). From September 2004, in our Institution, 28 high risk MDS pts. were treated with 2 different AZA dosing schedules. Group 1 (9 pts, 8 males, median age: 68, range 60-84 yrs) received the AZA 7 regimen, while group 2 (19 pts, 13 males, median age: 69, range 37-81 yrs) received the 5-2-5 AZA regimen. Moreover, as our group (Follo, 2009) demonstrated that phosphoinositide-phospholipase C (PI-PLC) beta1 may represent a target for AZA, we quantified the degree of PI-PLCbeta1 methylation and gene expression before and during AZA administration in both groups. Pts of group 1 received a median number of 12 (1-59) AZA cycles. Among the 8 evaluable pts (i.e.: at least 6 cycles) 5 (62.5%) showed a favourable response, following IWG criteria (Cheson, 2006): 1 Complete Remission (CR), 1 Partial Remission (PR) and 3 Hematologic Improvement (HI). 3 pts died because of evolution into Acute Myeloid Leukemia (AML), and 4 pts for other causes. One pts, still alive and under imatinib therapy, developed Ph1+ Chronic Myeloid Leukemia (CML) after 59 courses of AZA. Mean follow-up of group 1 pts: 31 (13-79) months. Pts of group 2 received a median number of 8 (1-17) AZA cycles. Among the 15 evaluable pts, 13 (86.6%) showed a favourable response: 5 CR (33.3%) and 8 HI. 3 pts died because of evolution into AML, and 2 for other causes. 2 pts underwent allogeneic stem cell transplantation, both after achieving CR. 12 pts are alive, 9 of them still under AZA treatment. Mean follow-up of group 2 pts: 14 (1-35) months. PI-PLCbeta1 methylation and gene expression appeared to be related to the therapeutic response, but not to the dose schedule. Our results, although larger studies are required, seem to confirm the effectiveness of the more convenient AZA 5-2-5 regimen, even in high risk MDS.

Background: Inter-individual variations in genes encoding drug metabolizing enzymes and transporters have been demonstrated to influence the response to therapy. However so far, how these genetic variations interact to produce specific drug related phenotypes in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) has not yet been investigated. Aim: In order to investigate potential genetic structure and related pharmacogenetic profiles, around 2000 variants in more than 200 genes involved in drug absorption, distribution, metabolism and elimination were genotyped and studied with a population genetics approach in 45 Ph+ ALL patients. Methods: The Drug Metabolizing Enzymes and Transporters (DMET™, Affymetrix) platform, covering more than 90% of the most biologically relevant drug absorption, distribution, metabolism and excretion (ADME) markers was used for successfully genotyping 1931 variants in Ph+ ALL patients treated with the tyrosine kinase inhibitor Dasatinib. A model-based clustering method for inferring population structure using genotype data was applied by means of the Structure software assuming a model in which there are K populations - each one of them being characterized by a set of allele frequencies at each locus - to which individuals are probabilistically assigned according to their genotypes. Distribution of the genetic variance observed among the identified leukemia sub-groups was investigated with a locus by locus Analysis of the Molecular Variance (AMOVA) by means of the Arlequin 3.01 package, exploiting information on genotypes allelic content and frequencies. Results: Three different sub-groups (G1, G2, G3), made up of 2, 12 and 31 patients respectively, were identified in the examined ALL sample, according to their different patterns of allele frequency. A statistical support for this finding was provided by AMOVA results which pointed out a substantial level of genetic differentiation among G1 and the other two sub-groups (Fst = 0.099, p0.3) and significant Fst values; whereas a total of 50 loci, located on the NAT2, VKORC1, CYP4F2, CYP2B6, UGT2B7 and CYP2D6 genes, showed moderate to high (>0.08) significant Fst values in the G2/G3 comparison. Conclusions: Differences of allele frequencies observed among the identified ALL sub-groups prove that an evident genetic structure is detectable in our sample by genotyping loci involved in drug metabolism. Supported by: Fondazione GIMEMA Onlus, 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.

Introduction. T-ALL represents 15% of childhood and 25% of adult ALL. Despite a cure rate of almost 80% in children and adolescents, adult ALL remains a difficult disease to cure, due to a high risk of relapse. Effective treatment of relapsed acute T-ALL is limited with a low CR rate, a high treatment-related mortality, and a very low prolonged disease-free survival. Nelarabine is a pro-drug of ara-G, approved by the FDA for the treatment of T-ALL and T-LBL that have not responded to or has relapsed after treatment with at least 2 chemotherapy regimens. Similar to other nucleoside analogues, Nelarabine acts by inhibiting DNA synthesis and inducing apoptosis in malignant cells. Our aim was to evaluate safety profile and efficacy of Nelarabine treatment as savage therapy in 9 adult relapsed or refractory T-ALL or T-LBL. Methods. After obtaining an informed consent, ten patients (median age 31 years, range 19-37, M/F=9/0) affected by T-ALL (N=6) and T-LBL (N=4) received a savage therapy with Nelarabine (median cycle=1, range 1-3). Nelarabine was administered at standard adult dosage (1500 mg2 on days 1, 3 and 5, every 21). Eight patients were relapsed after two previous chemtotherapy regimens, including allogeneic bone marrow transplantation; the remaining two patients were primary resistant to standard induction treatment. Results. Five out of ten patients obtained a complete morphological remission (4 T-ALL patients and 1 T-LBL patient), whereas a partial remission was documented in three cases, with an overall response rate of 80%. Median duration of complete response was 6 weeks (range 3-6 weeks). Nelarabine was well tolerated, and no significant adverse events were registered. Extra- hematological neurological toxicity, not clearly related to the drug, occurred in two cases, determining, in one patient a complete and irreversible paraplegia, and in the second one a condition of mental confusion (grade III), which resolved after few days. Conclusions. In our experience Nelarabine was successfully administered in such a high risk patients population. The drug showed a relevant efficacy and a good safety profile. Acknowledgments. This work was supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione - Università 2007-2009.

Introduction. Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed. Methods. 23 SNPs spanning the MTAP, CDKN2A/Band CDKN2BAS loci, as well as relative intergenic regions were genotyped in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples, and 183 healthy controls. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Results. A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with MAF 20% missing call rates, were instead excluded from the association analysis and potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20 to 3.33; P=7.1¥103). Conclusions. Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione - Università 2007-2009.

BACKGROUND: Ph+ ALL is observed in about 20-30% of adult ALL and is associated with a very poor prognosis and early relapse. Tyrosine kinase inhibitors have improved overall treatment results, with a rapid response and a complete remission (CR) rate ranging 90%. Nevertheless most patients experienced hematological relapse in a short time, also after hematopoietic stem cell transplantation. Molecular analysis based on quantitative assays (i.e. quantitative polymerase chain reaction, qPCR) provides detection of residual leukemic cells measuring BCR-ABL1 transcript level and becomes necessary in the monitoring of minimal residual disease to confirm molecular CR and to detect early relapse. AIM: To investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies in Ph+ ALL patients who obtained molecular remission as assessed by conventional qPCR. METHODS AND PATIENTS: The 12.765 Digital array (Fluidigm) is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Samples are portioned prior to qPCR into the single chambers of the panel; as fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Following amplification, digital raw data are processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per chamber using the Poisson probabilistic distribution. At the time of writing, we analyzed 22 Ph+ ALL samples (11 positive for the P190 BCR-ABL1 isoform and 11 for the P210 ) who were in complete (87%) or major (13%) molecular response (BCR-ABL1/ABL ratio ≤ 0.001 or < 0.1, respectively) as assessed by conventional qPCR; RNA integrity was evaluated using the control gene ABL. RESULTS: First, we assessed the sensitivity and reproducibility of the assay using six serial dilutions of plasmids (Ipsogen) expressing known copy number of BCR-ABL1 P190 transcript (10000; 1000; 100; 50; 10; 1 copies). A 2 µl volume of input cDNA was loaded and two panel for each dilution were used. Analysis parameter chosen for digital raw data processing were automated set threshold of 0.65 and target Ct range 20-40. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates (p= 0.0014, Paired t TEST analysis). We then analyzed duplicates of Ph+ ALL samples with a positive control for each chip: digital array resulted positive in 58% of complete molecular response samples, with 5.5 as median number of copies detected (range 0.5-11). CONCLUSIONS: The Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript with greater accuracy than conventional qPCR, as demonstrated for samples in molecular remission, and could provide an accurate monitoring method for Ph+ ALL CR patients. Further studies to confirm these results are actually ongoing. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Introduction: Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for its development. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and may be used to better classify the disease by dissection of genomic subsets. Patients and Methods: 105 adult patients with acute myeloid leukemia were analyzed. These cases included all French-American-British subtypes, miscellaneous cytogenetic abnormalities and normal karyotype subgroups. Among these, the M3 subtype included 28 patients, representing the 33% of the whole study population. Genomic DNA was isolated from blast cells and applied to Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) following the manufacturer’s instructions. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: A wide spectrum of different copy number alterations (CNAs) were identified in all cases and no significant difference in the average number of alterations was detected among different leukemia cytogenetic subgroups except for the complex subgroup, which had an average of 55 CNA/patient. In APL cases an average of 8 CNAs per case (range, 1-24) was found. The macroscopic alterations were rare, confirmed conventional cytogenetics and involved trisomy of chromosome 8 in 3 cases, loss of chromosome 6, loss of chromosome 20 and deletions on chromosome 9 and 7. Microscopic CNAs (< 1.5 Mbps) involved every chromosome at least once and predominantly chromosomes 1, 2, 9, 15 and 17. For each alteration we interrogated a collated library of copy-number variants (CNVs, Database of Genomics Variants and USCS Genome Browser) to assure that these regions were not known as CNVs and therefore to decrease the noise of raw copy number data. Genetic gains were more common than losses and their median size was 300 kb (range 0.2- 1.4 Mb). The majority of lesions were not recurrent, being identified in only a single patient. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes, in genes involved in activation of transcription (loss of LMX1 on 1q23.3, loss of MLXIPL and BCL7 on 7q11.23), regulation of cell cycle (gain of PVT1 and MYC on 8q24) and cell adhesion (gain of NCAM1 on 11q23). In order to identify potential pathogenetic alterations, all microscopic CNAs were compared with the list of genes from the Cancer genome project (http://www.sanger.ac.uk/genetics/CPG/Census) finding out that six alterations involved a known cancer-related gene. Most of these genes encode tyrosine kinase proteins (ERBB4) or transcription factors (ETV1, ETV6, ERG). Copy neutral loss of heterozygosity events affected 1p34.2-1p32.3, 10p11.2 (MLLT10), 11p11.2 (WT1, CDKN1C, HRAS). Finally, patients with more than 10 CNAs were found to be associated with a worse prognosis. Conclusions: These data demonstrate that different cooperating events may be involved in the generation of APL. Furthermore, these novel findings may be used to stratify patients according to genomic changes. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants.

Introduction. The BCR-ABL1+ ALL is the most frequent and prognostically unfavorable subtype of ALL in adults. 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. The selected cases had previously been profiled by high-resolution SNP and gene expression arrays and candidate gene re-sequencing. 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 generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, most of which successfully mapped to the reference sequence of the human genome. With the exclusion of the T315I BCR-ABL mutation, 7 novel missense mutations were detected after applying stringent criteria to reduce the SNV discovery false positive rate: 4 were exclusively found in the primary ALL sample and affected genes involved in metabolic processes (DPEP1, ZC3H12D, TMEM46) or transport (MVP); 3 relapse-related mutations affected genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21). Differences in mutational patterns suggest that the leukemia clone from which relapsed cells have been developed was not the predominant one at diagnosis and that relapse specific variants were mutations probably acquired during progression. Moreover, 4,334 and 3,651 primary and relapse isoforms with at least one AS event were identified. An average of 1.5 and 1.3 AS per isoform was estimated. Finally, a detailed gene expression profile was obtained indicating that more than 60% of annotated human genes were transcribed in leukemia cells in both diagnosis and relapse phases. Approximately 23% of genes were up-regulated at relapse compared to diagnosis, and most of them affected cell cycle progression (AURORA A, SURVIVIN, PLK1, CDK1, Cyclin A, Cyclin B), suggesting that the loss of cell cycle control may play a role in disease progression. Conversely, only 9% of active genes in both samples were down-regulated at relapse compared to diagnosis. 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 demonstrating that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations. Supported by AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna,FIRB 2006, PRIN 2008, European LeukemiaNet, GIMEMA ONLUS, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione - Università 2007-2009.

Background. This locus 9p21, containing the p16/CDKN2A (cyclindependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15/CDKN2B, is a major target in the pathogenesis of a number of human tumors. Patients and methods. In order to assess whether and how it is inactivated in adult BCR-ABL1-positive ALL, we studied 112 adult patients: 78 (70%) were de novo ALL, 15 (13%) were unpaired relapsed cases and 19 (17%) were paired relapsed cases. Their median age was 53 years (range: 18-76) and their median blast percentage was 90% (range, 18-99). Affymetrix single nucleotide polymorphism (SNP) arrays were used to identify at a high resolution copy number changes on 9p21. Mutation screening of all exons by cloning and subsequent sequencing were also performed. Results. SNP array analysis revealed CDKN2A/ARF and CDKN2B genomic alterations in 33% and 24% of diagnosed patients, respectively. In 70% of cases, deletions were limited to CDKN2A/CDKN2B genes, whereas in 30% they also affected neighbour genes and/or the entire chromosome 9. In order to assess whether CDKN2A loss is responsible for progression, 34 patients were analyzed at the time of relapse and a significant increase in the detection rate of CDKN2A/ARF loss (53%) compared to diagnosis (P=0.04) was found. In contrast, CDKN2B deletions were found to be not significantly different between diagnosis and relapse (41% vs. 24%, P=0.07). The mutation screening of all exons showed that the 9p21 locus is rarely affected by point mutations, since we only identified the D146N and the R128 in the exon 2 of CDKN2A/ARF and the P83 silent mutation in the exon 2 of CDKN2B gene. These mutations were mutually exclusive and were found in only single cases. Conclusions. Loss of the tumor suppressor gene CDKN2A/ARF by genomic deletions is a frequent event in adult Ph+ ALL and it is involved in disease progression. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, PRIN 2008, Project of integratad program (PIO), Programma di Ricerca Regione, Università 200-2009.