Valentina Robustelli

Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Abstract 2723: The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia

Different Checkpoint kinase inhibitors (Chk-i) have been developed to increase the cytotoxic effect of genotoxic agents inhibiting the key elements of the DNA damage response (DDR) pathways. Our group has already showed the efficacy of this class of compounds in single agent in different in vitro/ex vivo/in vivo studies for the treatment of acute lymphoblastic leukemia (ALL). The aim of the study was to evaluate the efficacy of a Chk1/Chk2 inhibitor in combination with the topoisomerase II inhibitor doxorubicin for the treatment of ALL. Firstly we evaluate the efficacy of doxorubicin on human B (NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathways. Cells were treated with doxorubicin (0.25-2.5 uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. The induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was also evaluated in all the cell lines. Due to the inhibitory effect of the compound on the topoisomerase II enzyme and due to the activation of the cell cycle checkpoint, cells were arrested in G2/M phase. Then the effectiveness of the Chk-i as a chemo-sensitizer agent was evaluated. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk-i (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a synergistic effect in term of reduction of the cell viability and induction of apoptosis. The effect of the combination was also analyzed using western blot looking for specific marker of activation of the DDR pathway showing the same synergistic effect. Moreover the effect of the combination on cell cycle profile was evaluated using a double staining Pi/Anti-phospho-Histone H3 ser10 (marker of mitosis). Cell lines were pre-treated for 18 hours with doxorubicin and then with the Chk-i for different time points (1, 2, 3, 6 and 9 hours). The treatment with Chk-i removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells positive for the mitotic marker p-HH3 (ser10) and increasing the percentage of cells in sub-G1 phase. In our opinion the combination between the Chk1 inhibitor,LY2606368, and the topoisomerase II inhibitor, doxorubicin, could be a promising strategy for the treatment of B/T-ALL. Supported by ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni Martinelli. The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2723.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia

BackgroundDespite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study.MethodsThe effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts.ResultsWe showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation.ConclusionsOur data suggest that WEE1 plays a role in ALL blast’s survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

Abstract 294: Override the doxorubicin-induced G2/M checkpoint using cell-cycle checkpoint inhibitors on acute lymphoblastic leukemia

The topoisomerase 2 inhibitor, doxorubicin, has been showed by different groups to induce cell cycle arrest in various kind of tumor cells. Specifically doxorubicin-treated cells activate the G2/M cell cycle checkpoint as a consequence of the induction of DNA damages. During the last years many studies have been showed the efficacy of different cell cycle checkpoint inhibitors in single agent or in combination with various DNA damaging agents. These studies showed that the inhibition of key proteins of the cell cycle, like Chk1 and Wee1, deeply sensitize tumor cells to the treatment with genotoxic agent. On these bases, the aim of the study was to evaluate the efficacy of a selective Chk1/Chk2 inhibitor and a Wee1 inhibitor in combination with doxorubicin for the treatment of acute lymphoblastic leukemia. Firstly we evaluate the effect of doxorubicin treatment on a panel of human B and T ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DNA damage response. For this reason the cells were treated with doxorubicin (0.25, 0.5 and 1uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. Then the induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was evaluated in all the cell lines. In line with the literature the treatment with doxorubicin arrested the cells in G2/M phase. Then the effect of the combinations between doxorubicin and the two checkpoint kinase inhibitor was assessed in all the cell lines. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk1/Chk2 inhibitor (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a additively effect in term of reduction of the cell viability and induction of apoptosis. Different cell lines were pre-treated for 18 hours with doxorubicin and then with Chk1/Chk2 inhibitor for different time points. Interestingly the inhibition of both Chk1/Chk2 proteins removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells in sub-G1 phase. Similar results were seen combining a Wee1 inhibitor with doxorubicin on several ALL cell lines. In our opinion the combination between the cell cycle checkpoint inhibitors and doxorubicin could be a promising strategy for the treatment of B/T-ALL. Supported by ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Anna Ferrari, Valentina Robustelli, Cristina Papayannidis, Maria Chiara Abbenante, Antonella Padella, Giovanni Marconi, Sandro Grilli, Giovanni Martinelli. Override the doxorubicin-induced G2/M checkpoint using cell-cycle checkpoint inhibitors on acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 294. doi:10.1158/1538-7445.AM2017-294

Novel fusion transcripts identified by RNAseq cooperate with somatic mutations in the pathogenesis of acute myeloid leukemia

Acute Myeloid Leukemia (AML) is a highly heterogeneous disease and somatic mutations and fusion genes have a crucial diagnostic, prognostic and therapeutic role in AML. However, the leukemogenic potential of the fusions, their cooperation with somatic mutations and their prognostic role are still unknown. The aim of the study was to identify novel rare gene fusions having a causative role in leukemogenesis and to identify cooperative somatic mutations as potential targets for personalized therapies in AML cases with rare and poorly described chromosomal translocations. We performed RNAseq on bone marrow samples from 5 AML patients (#59810, #20, #84, #21 and #32). The presence of gene fusions was assessed with deFuse and Chimerascan. Putative fusion genes were prioritized using Pegasus and Oncofuse to select biologically relevant fusions. WES was performed on samples #59810, #20 and #21 and variants were detected using the software Mutect and GATK. The CBFβ-MYH11 chimera was identified in sample #84, with inv(16), thus confirming the reliability of our analysis. Sample #59810 carried the fusion transcript ZEB2-BCL11B (Driver Score, DS = 0.7), an in-frame fusion and a rare event in AML associated with t(2;14). The fusion transcript showed 3 splicing isoforms and ZEB2 and BCL11B transcripts were upregulated in patient9s blasts, compared with 53 AML samples with no chromosomal aberrations in the 14q32 region. The WT1-CNOT2 chimera was also detected, which is a novel out-of-frame fusion (DS = 0.008) related to t(11;12) translocation. WES analysis showed SNVs in TET2 and BCOR genes. In sample #20 we identified two different fusion transcripts: CPD-PXT1 (DS = 0.07), which was the reciprocal fusion product of t(6;17) translocation, and SAV1-GYPB, which was cryptic at cytogenetic analysis (DS = 0.8). Interestingly, the latter fusion involved a tumour suppressor gene. Moreover, mutations in BCOR and NRAS were detected by WES. RNAseq on sample #21 identified a novel fusion event between chromosomes 19 and 7, involving the genes OAZ and MAFK (DS = 0.9). The 3’ partner gene is a transcription factor, a functional class recurrently altered in AML. This patient showed SNVs in CBL and SMC1A genes. Finally, no chimeras were confirmed in sample #32 having a t(12;18) translocation. Our data suggest that fusion events are frequent in AML and a number of them cannot be detected by current cytogenetic analyses. Gene fusions cooperate with somatic mutations in the genomic landscape driving AML pathogenesis and its heterogeneity. Moreover, the results indicate that different approaches, including WES, RNAseq bioinformatic and statistic tools need to be integrated in order to identify potential targets for personalized therapies. Acknowledgements: ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Anna Ferrari, Giulia Paciello, Elisa Zago, Carmen Baldazzi, Viviana Guadagnuolo, Cristina Papayannidis, Valentina Robustelli, Enrica Imbrogno, Nicoletta Testoni, Massimo Delledonne, Ilaria Iacobucci, Tiziana Clelia Storlazzi, Elisa Ficarra, Pier-Luigi Lollini, Giovanni Martinelli. Novel fusion transcripts identified by RNAseq cooperate with somatic mutations in the pathogenesis of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 113.

Abstract B03: Very poor outcome and chemoresistance of acute myeloid leukemia patients with TP53 mutations: Correlation with complex karyotype and clinical outcome.

Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated. Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of 172/886 AML pts treated at the Seragnoli Institute of Bologna between 2002 and 2013. Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation Deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Number™ v7.5 (BioDiscovery). Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc). Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype. Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious. Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively). Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (OS) (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Anna Ferrari, Stefania Paolini, Carmen Baldazzi, Chiara Sartor, Maria Chiara Abbenante, Sarah Parisi, Francesca Volpato, Ilaria Iacobucci, Antonella Padella, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Claudia Venturi, Giorgia Simonetti, Elisa Zuffa, Eugenia Franchini, Emanuela Ottaviani, Nicoletta Testoni, Giovanni Martinelli. Very poor outcome and chemoresistance of acute myeloid leukemia patients with TP53 mutations: Correlation with complex karyotype and clinical outcome. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B03.

Abstract 4906: TP53 mutations are mutually exclusive with FLT3 and NPM mutations in AML patients and are strongly associated with complex karyotype and poor outcome

AML is a heterogeneous disease with a variety of structural and numerical chromosomal and genetic alterations that provided important prognostic information capable to guide therapy and predict outcome. The reported TP53 mutation rate in AML is low (2.1%). By contrast, the incidence of FLT3 disruption is frequent (20-30%) and is an independent predictor of unfavourable outcome of acquired clinical resistance to FLT3 inhibitors. TP53 mutations in AML with a complex karyotype (CK) is higher (69-78%), but few data are available for association between the most common AML associated lesions such as FLT3, NPM1, DNMT3A, IDH2 and TP53 mutation or CK. Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the molecular abnormalities, the correlation with known molecular alterations (FLT3, NPM, etc.) and the prognostic role TP53 mutations in adult AML pts. Patients and Methods: 886 AML patients were analysed for cytogenetic and for a panel of genetic alterations (FLT3, NPM, WT1, DNMT3A, IDH1-2 etc). Of these, 200 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq 2000 platform (38/200) and were correlated with cytogenetic analysis. Results: 55 pts (27.5%) showed 3 or more chromosome abnormalities (CK-AML), 83 (41.5%) presented one or two cytogenetic abnormalities (other-AML) and 43 pts (21.5%) have normal karyotype (nK). In 19 cases the karyotype was not available. Sanger sequencing analysis detected TP53 mutations on 29 patients with 36 different types of mutations (32 deleterious point mutations; 4 deletions); seven pts (4%) have 2 mutations. Mostly (23/29) of the TP53 mutated pts (79.3%) had CK while only 6/29 (21%) mutated pts have “no CK”. Overall, between pts with CK, TP53 frequency is 41.8% (P>0,0001). 120 pts were analysed for concomitant presence of TP53 mutations and FLT3/NPM1 disruption/mutation: revealing a significant relation between pts with FLT3-ITD or NPM1 and TP53 wild-type (p = 0.043 and 0.022 respectively). As for clinical outcome alterations of TP53 were significantly associated with poor outcome (OS and EFS p WES analysis done in 38 pts (33 TP53 wt and 5 pts TP53 mutated) revealed no genes exclusively mutated in the 5 TP53 mutated pts. Conclusions: Our data demonstrated that TP53 mutations occur in 14.5% of AML with a higher frequency in the subgroup of CK-AML (p Citation Format: Anna Ferrari, Cristina Papayannidis, Elisa Zuffa, Carmen Baldazzi, Antonella Padella, Eugenia Franchini, Ilaria Iacobucci, Stefania Paolini, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Claudia Venturi, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Francesca Volpato, Federica Cattina, Giorgia Simonetti, Maria Chiara Fontana, Maria Teresa Bochicchio, Federica Frabetti, Elisa Lani, Katia Mancuso, Beatrice Zannetti, Simona Luatti, Emanuela Ottaviani, Nicoletta Testoni, Giovanni Martinelli. TP53 mutations are mutually exclusive with FLT3 and NPM mutations in AML patients and are strongly associated with complex karyotype and poor outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4906. doi:10.1158/1538-7445.AM2015-4906

Abstract CT312: Ponatinib is well tolerated and active in patients with relapsed/refractory philadelphia positive leukemias: The Bologna experience

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Ponatinib, a third generation BCR-ABL inhibitor, has shown relevant activity against native and mutant forms of BCR-ABL, including the T315I mutant. The aim of this compassionate protocol was to confirm the efficacy and the safety of the compound in patients with advanced Ph+ ALL and CML. Design and Methods: Ponatinib was obtained through a compassionate use named patient program, approved by ARIAD Pharmaceuticals and by the Bologna Ethical Committee. After informed consent was signed, 17 patients (M/F=8/9) have been treated (45 mg orally, once daily) between February 2012 and July 2013, including 14 Ph+ ALL and 3 blast phases of CML (11 p190, 5 p210, 1 p230). The median age of the patients was 64 years (range 23 -77). The median time from diagnosis was 2 years (range 0.2-6). Five patients (29%) had previously received an allogenic stem cell transplantation. All the patients were resistant or intolerant to previous TKIs (11 patients received >=2 TKIs). Median Hb, PLTs and WBC values were 10,1 g/dl (range 8.3-13.9), 100000/mmc (range 14000-325000) and 4400/mmc (range 1700-17000), respectively. In 6 out of 17 patients (35%), additional cytogenetic alterations were revealed. Mutational analysis showed the presence of T315I mutation (8 pts), G250E (1 pt), T315I and Y253H (1 pt), T315I and Y253A (1 pt), V299L (1 pt). Results: The median treatment duration was 153 days (range 42-594+). With a median follow up of 8 months (range 2-21+), a maHR was obtained in 12/17 patients (71%). Among these, 6 were T315I mutated. After one month of treatment, a reduction of BCR ABL fusion transcript to undetectable level was observed in 3/17 patients (18%), which increased to 29% (5/17) after two additional cycles. The progression free survival (PFS) at 12 months was 35% (median 57 days), without differences between patients with T315I mutations and patients harboring other mutations. Furthermore, we observed that, in responder patients, mutations disappeared in all except one case. In contrast, in resistant patients, a specular mutational profile before and after treatment was detected. Currently, 6/17 patients are still on study (35%). Six patients died due to progression disease. In five cases, the response allowed the patients to proceed to a savage allogenic stem cell transplantation. The drug was well tolerated. No SAEs were detected and no thrombotic arterial/venous events occurred. Conclusion: In our experience, the activity of Ponatinib in advanced Ph+ leukemias was confirmed.No treatment-related deaths occurred. The understanding of molecular mechanisms responsible for resistance or lack of response to the drug will be necessary in order to identify patients who could take advantage of this treatment. Acknowledgments: Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Caterina De Benedittis, Simona Soverini, Ilaria Iacobucci, Maria Chiara Abbenante, Chiara Sartor, Maria Teresa Bochicchio, Anna Ferrari, Claudia Venturi, Valentina Robustelli, Andrea Ghelli Luserna di Rora, Viviana Guadagnuolo, Emanuela Ottaviani, Nicoletta Testoni, Carmen Baldazzi, Simona Luatti, Sarah Parisi, Stefania Paolini, Alberto Conficoni, Federica Frabetti, Elisa Lani, Silvia Piccari, Paolo Di Bartolomeo, Roberto Di Lorenzo, Renato Fanin, Giuseppe Cimino, Fabio Ciceri, Giovanni Martinelli. Ponatinib is well tolerated and active in patients with relapsed/refractory philadelphia positive leukemias: The Bologna experience. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT312. doi:10.1158/1538-7445.AM2014-CT312

Abstract 570: Tp53 mutation screening in adult acute myeloid leukemia (AML) patients shows a strong association with complex karyotype and poor outcome

Introduction: AML is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome. The TP53 gene is the most frequently mutated gene in human tumors. The reported TP53 mutation rate in AML is low (2.1%). In contrast, the incidence of TP53 mutations in AML with a complex aberrant karyotype (CK-AML) is higher (69-78%). Aims: to investigate the frequency and the prognostic role of TP53 mutations in adult AML patients (pts) focusing the screening on subgroups of pts with chromosome abnormalities. Patients and Methods: 106 adult AML pts with FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype (nK-AML, 14/106 pts) were examined. Forty-four pts (41.1%) showed 3 or more chromosome abnormalities (CK-AML), 42 (39.6%) presented one or two cytogenetic abnormalities (other-AML) and in 6 cases the karyotype was not available. Genomic DNA and/or cDNA were isolated from mononuclear AML blast cells. TP53 mutation screening was performed on all 106 AML pts, in particular in 42 from exon (ex) 2 to 11, in 48 from ex 4 to 11 and in 16 pts from ex 2 to 8. Analysis was focused on coding sequences (RefSeq GRCh37/hg19 NG_017013.2). Results: By PCR and subsequent Sanger sequencing, mutations of TP53 were detected in 23 pts (21.1%). Seven pts revealed 2 mutations. 83% of all mutated pts had CK (19/23) by contrast the frequency of mutations was very low in “no CK-AML” pts (6.5%). Overall, mutated patients included 19/44 with CK-AML (43.2%); 1/6 (16.7%) with nK and 3/42 (7.1%) with other-AML. 26 TP53 point mutations [19 missense, 1 silent, 5 intron and 1 3’ untranslated region (UTR)] and 4 TP53 deletions were found. Twenty-six out of 30 mutations/deletions (86.6%) were located in the DNA binding domain, 2 in the carboxyl-terminal tetramerization and regulatory domains and 2 in the transcriptional activation domain. All mutations in coding regions were classified by the IARC database (http://p53.iarc.fr/TP53GeneVariations.aspx) as deleterious. Of note, alterations of TP53 were significantly associated with poor outcomes in terms of overall survival and disease free-survival (P Supported by: EuropeanLeukemiaNet, AIL, AIRC, PRIN 2010-2011, Fondazione del Monte di Bologna e Ravenna, European Union Seventh Framework Programme [FP7/2007-2013]. Citation Format: Anna Ferrari, Ilaria Iacobucci, Cristina Papayannidis, Carmen Baldazzi, Chiara Sartor, Emanuela Ottaviani, Nicoletta Testoni, Valentina Robustelli, Margherita Perricone, Claudia Venturi, Maria Chiara Abbenante, Viviana Guadagnuolo, Antonella Padella, Federica Cattina, Giorgia Simonetti, Domenico Russo, Giovanni Martinelli. Tp53 mutation screening in adult acute myeloid leukemia (AML) patients shows a strong association with complex karyotype and poor outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2014-570