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Papers by Anna Ferrari

Biomedicines

In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved e... more In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML ...

International Journal of Molecular Sciences

Adult rhabdomyosarcoma (RMS) represents an uncommon entity with an incidence of less than 3% of a... more Adult rhabdomyosarcoma (RMS) represents an uncommon entity with an incidence of less than 3% of all soft tissue sarcomas (STS). Consequently, the natural history and the clinical management of this disease are infrequently reported. In order to fill this gap, we investigated the molecular biology of an adult RMS case series. The expression of epithelial mesenchymal transition-related gene and chemoresistance-related gene panels were evaluated. Moreover, taking advantage of our STS translational model combining patient-derived primary culture and 3D-scaffold, the pharmacological profile of an adult head and neck sclerosing RMS was assessed. Furthermore, NGS, microsatellite instability, and in silico analyses were carried out. RT-PCR identified the upregulation of CDH1, SLUG, MMP9, RAB22a, S100P, and LAPTM4b, representing promising biomarkers for this disease. Pharmacological profiling showed the highest sensitivity with anthracycline-based regimen in both 2D and 3D culture systems. N...

Cell Biology and Toxicology

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and ... more Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the ...

International Journal of Molecular Sciences

Adult acute lymphoblastic leukemia (ALL) with BCR-ABL1 rearrangement (Philadelphia chromosome, Ph... more Adult acute lymphoblastic leukemia (ALL) with BCR-ABL1 rearrangement (Philadelphia chromosome, Ph) is a hematological aggressive disease with a fatal outcome in more than 50% of cases. Tyrosine kinase inhibitors (TKIs) targeting the activity of BCR-ABL1 protein have improved the prognosis; however, relapses are frequent because of acquired somatic mutations in the BCR-ABL1 kinase domain causing resistance to first, second and third generation TKIs. Axitinib has shown in vitro and ex vivo activity in blocking ABL1; however, clinical trials exploring its efficacy in ALL are missing. Here, we presented a 77-year-old male with a diagnosis of Ph positive ALL resistant to ponatinib and carrying a rare threonine to leucine (T315L) mutation on BCR-ABL1 gene. The patient was treated with axitinib at 5 mg/twice daily as salvage therapy showing an immediate although transient benefit with an overall survival of 9.3 months. Further dose-finding and randomized clinical trials are required to ass...

Journal of Clinical Oncology

11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predict... more 11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predictive of response and can help define therapeutic strategies. Our aim is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis by a SNP array-based genotyping approach. Methods: We performed SNP 6.0/Cytoscan HD (Affymetrix) on 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number (BioDiscovery) and R Core Team. Results: We found several genes preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p < ....

Cancers

Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of t... more Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. Methods: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. Results: This study reports, for the first time, the efficacy of the concomi...

Blood

2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the C... more 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roch...

Experimental and Molecular Therapeutics

Cancer research, Jan 12, 2017

Philadelphia Chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is currently treated with... more Philadelphia Chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is currently treated with BCR-ABL1 tyrosine kinase inhibitors (TKI) in combination with chemotherapy. However, most patients develop resistance to TKI through BCR-ABL1-dependent and -independent mechanisms. Newly developed TKI can target Ph+ ALL cells with BCR-ABL1-dependent resistance; however, overcoming BCR-ABL1-independent mechanisms of resistance remains challenging because transcription factors (TF), which are difficult to inhibit, are often involved. We show here that: i) the growth of Ph+ ALL cell lines and primary cells is highly dependent on MYB-mediated transcriptional upregulation of CDK6, cyclin D3, and BCL2 and ii) restoring their expression in MYB-silenced Ph+ ALL cells rescues their impaired proliferation and survival. Levels of MYB and CDK6 were highly correlated in adult Ph+ ALL (p=0.00008). Moreover, Ph+ ALL cells exhibited a specific requirement for CDK6 but not CDK4 expression, most likely b...

Leukemia & Lymphoma

To date, the lack of chemo-naive cell models limited exploratory studies to define novel therapie... more To date, the lack of chemo-naive cell models limited exploratory studies to define novel therapies for Burkitt lymphoma (BL). To obtain a useful tool for biological and translational studies for this tumor, we established the RALE051 cell line from the malignant ascitic fluid cells of a patient at initial diagnosis, not previously exposed to any treatment. The cell line was characterized by karyotyping, fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) array, immunohistochemistry, and RNA-Seq, revealing the persistence of biological and molecular features observed in the primary ascitic fluid, such as the cell morphology and immunophenotype, the occurrence of a t(8;22) translocation deregulating MYC, knock-out ID3 mutations in a compound homozygous state, and a mutated TP53. Moreover, additional alterations characterizing the ex vivo transformation included the emergence of three novel fusion transcripts and a nonsense mutation affecting TP53. The establishment of this cell line would be beneficial for future biological and therapeutic studies of BL.

Oncotarget, Jan 11, 2016

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed f... more 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 resu...

Journal of hematology & oncology, Jan 5, 2015

Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored t... more Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. 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. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent N-ethyl-N-nitrosourea. Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment...

Cancer Research, 2015

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: In... more Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: In Acute Myeloid Leukemia (AML) there is a strong need to develop new diagnostic and therapeutic options: to identify the genes mostly predictive of treatment response, we use Single Nucleotide Polymorphism (SNP) Arrays and Whole Exome Sequencing (WES) in AML patients with heterogeneous karyotypes and different subgroups. Materials and Methods: SNP arrays (CytoScan HD Array, Affymetrix Inc.) was positively done in 58 AML samples and all of them were analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.) and Nexus Copy Number™ v7.5 software (BioDiscovery), while Next Generation Sequencing (NGS)-WES HiSeq 2000 (Illumina) was done in 35 AML patients. Results: We treated 58 AML patients (pts) with a median age of 52 years, of which 9 were insensitive pts and 49 sensitive to therapies, all obtaining complete clinical remission. Copy Number Alterations (CNAs) were detected in all patients affecting all the chromosomes, in particular our cohort of pts present a percentage of CNA, divided as follow: 58% of LOH, 15% of gain, 16% of loss, 3% of high copy gain and 4% of homozygous copy loss. We found that several cancer genes were preferentially amplified: IGH@, KIT, IGL@, TSC1, NOTCH2, SETD2, EZH2, while in high copy gain we found TSC1, PTEN, RB1, IKZF1, ZRSR2, IGH@, NF1, MYC, KRAS. Then several genes were preferentially deleted: CRLF2, ATRX, JAK2, BCOR, PHF6, GATA1, KDM6A and in homozygous copy loss: JAK2, CRLF2, RB1, PDGFRA, RUNX1. Moreover we genes in LOH: DDX5, MTCP1, HOOK3, ZRSR2, GATA1, KDM6A, BCOR, NF1, BRAF, ATRX. We focused on two losses of JAK2: the first deletion, detected in 18/58 (31%) pts, goes from 5030 to 5038 Kbp (7,43 Kbps) including intron 4-5; the second minimal common region of loss, detected in 5/58 (8,6%) pts, goes from 5083 to 5098 Kpb (15 Kbps) including intron 19-20 and exons 20, 21 and 22, suggesting a defective transduction, in fact we showed that the overall survival rate is better for the group of pts which present a deletion of JAK2 rather than the group with an amplification of this gene (p-value < 0,01). We have also found three other genes which are preferentially lost: SIRPB1, ADAM3A and STAG2 with a percentage of 50%, 43% and 69% respectively. By NGS-WES we analyzed 35 AML samples at diagnosis and we searched for point mutations, insertion/deletion or other abnormalities, involved in biomarkers of response to treatment. We found mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR. Conclusion: By SNP arrays we have identified Copy Number Alterations involving important cancer genes AML and we showed that a new deletion in JAK2 may have a role in overall survival rate. Future prospective will be to correlate the cancer genes alteration and mutations with the prognosis of AML, in order to identify new biomarkers relevant for the disease. Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Universita 2010-12 (L. Bolondi), FP7NGS-PTL project Citation Format: Viviana Guadagnuolo, Maria Chiara Fontana, Antonella Padella, Ilaria Iacobucci, Cristina Papayannidis, Giorgia Simonetti, Anna Ferrari, Giovanni Marconi, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Francesca Volpato, Chiara Sartor, Emanuela Ottaviani, Massimo Delledonne, Michele Cavo, Guido Biasco, Giovanni Martinelli. SNP array reveals a new deletion of JAK2 in AML patients. [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 4848. doi:10.1158/1538-7445.AM2015-4848

Cancer Research, 2015

AML is a heterogeneous disease with a variety of structural and numerical chromosomal and genetic... more 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

Cancer Research, 2014

Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequ... more Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs. The recent WHO classification (2008) includes an indolent form (ISM), an aggressive form (ASM) and a leukemic subvariant (MCL). The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 22 patients (male/female=11/11) affected by SM have been referred to our Institution. Among these, 12 (55%) presented with systemic symptoms associated with signs of organ involvement (skeletal lesions, ascites, liver function impairment or bon marrow disfunction), thus identifying an ASM. Therefore, since a first line therapy (IFNalfa, Imatinib and 2CdA in 56%, 11% and 33% of the patients, respectively) and supportive care with histamine receptor antagonists weren’t followed by a significant benefit, a personalized use of PKC412 was asked and obtained for 9 out of 12 ASM patients. Thus, from March 2011 9 (M/F =3/9) patients with ASM have been treated with PKC412, administered orally, at the dosage of 100 mg twice daily, without rest periods. The median age was 60 years (range 39-75); the median time from diagnosis was 6 months (range 2-53). Median serum tryptase level was 100 mcg/L(range 19.3-1160). C-kit mutation D816V was present in 8 out of 9 patients. Cytogenetic analysis was normal in all the patients. Results. Seven out of nine patients were evaluable for response. The median duration of therapy was 517 days (range 327-970+). According to European Criteria, a Major response was observed in one patient, and a partial response in 6 patients. Overall, the drug was well tolerated, and no serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. Conclusions. In a small cohort of ASM patient, the prolonged therapy with PKC412 is safe and effective, mainly on symptoms improvement and haematological profile. Nevertheless, the persistence of the D816V c-kit mutation, despite significant responses, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. UDS approaches are needed in order to clarify this issue. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Simona Soverini, Caterina De Benedittis, Maria Chiara Abbenante, Chiara Sartor, Ilaria Iacobucci, Carmen Baldazzi, Emanuela Ottaviani, Anna Ferrari, Viviana Guadagnuolo, Alberto Conficoni, Strefania Paolini, Sarah Parisi, Federica Frabetti, Silvia Piccari, Sandro Grilli, Elisa Lani, Giovanni Martinelli. PKC412 (Midostaurin) is safe and highly effective in systemic mastocytosis patients: Follow up of a single-center Italian compassionate use. [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 746. doi:10.1158/1538-7445.AM2014-746

Cancer Research, 2014

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Ponatinib, a th... more 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

Cancer Research, 2013

Background. Precursor T cell LBL/ALL occurs most frequently in late childhood, adolescence, and y... more Background. Precursor T cell LBL/ALL occurs most frequently in late childhood, adolescence, and young adulthood, with a 2:1 male predominance; it comprises 15 and 25 percent of childhood and adult ALL, respectively, and 2 percent of adult non-Hodgkin lymphoma. Nelarabine is an anticancer prodrug of arabinofuranosylguanine (ara-G); it inhibits DNA synthesis and leads to high molecular weight DNA fragmentation and cell death. Nelarabine has showed relevant efficacy in phase II clinical trials, both in pediatric and in adult LBL/ALLpopulations. Design and Methods. We report clinical outcome results of 9 newly diagnosed and younger than 60 years T-ALL patients (median age 29 years, range 22-45 years, 3/6 M/F, 8 T-ALL, 1 T-LBL) treated according to pediatric-like adapted schedule. Cytogentics data and molecular biologic features will be provided on site. Induction cycle included Vincristine, daunoblastine, L-asparaginase and Prednisone. After induction, all the patients received consolidation therapy with cyclophosphamyde, L-asparaginase, Cytarabine and 6-Mercaptopurine. Subsequently all the patients receveid Nelarabine 1500 mg/sqm (days 1-3-5 every 21) for two cycles. All the patients shared the same strategy for intensification, which consisted in allogenic stem cell transplantation, if available, or additional courses of consolidation chemotherapy. Durations of complete remission (CR) and overall survival (OS) were estimated according to the Kaplan-Meier method. The CR duration was dated from start of CR to first evidence of disease recurrence. Results. After a single induction course, 9/9 patients obtained a CR (100%). Eight patients underwent an allogenic bone marrow transplantation. After a median follow-up of 24 months, 7/9 patients (78%) are alive in CR. The median CR duration and OS were 13.4 and 24.4 months, respectively. Neurological toxicity of grade 3 has not been reported. We did not observe grade 3-4 haematological toxicity. Conclusion. Nelarabine is a promising drug, which induces a remarkable complete remission rate at the expense of a very low and manageable toxicity. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, University of Bologna and BolognAIL. Citation Format: Chiara Sartor, Mariachiara Abbenante, Cristina Papayannidis, Ilaria Iacobucci, Stefania Paolini, Emanuela Ottaviani, Annalisa Lonetti, Sarah Parisi, Anna Ferrari, Viviana Guadagnuolo, Sandro Grilli, Nicoletta Testoni, Michele Cavo, Giovanni Martinelli. Nelarabine front-line therapy for adult T-lymphoblastic leukemia/lymphoma (T-LBL/ALL): preliminary results of a single center experience. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1179. doi:10.1158/1538-7445.AM2013-1179

Cancer Research, 2010

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

Background: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic ... more Background: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult BALL patients (106 BCR-ABL1-positive and 38 BALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling.

Biomedicines

In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved e... more In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML ...

International Journal of Molecular Sciences

Adult rhabdomyosarcoma (RMS) represents an uncommon entity with an incidence of less than 3% of a... more Adult rhabdomyosarcoma (RMS) represents an uncommon entity with an incidence of less than 3% of all soft tissue sarcomas (STS). Consequently, the natural history and the clinical management of this disease are infrequently reported. In order to fill this gap, we investigated the molecular biology of an adult RMS case series. The expression of epithelial mesenchymal transition-related gene and chemoresistance-related gene panels were evaluated. Moreover, taking advantage of our STS translational model combining patient-derived primary culture and 3D-scaffold, the pharmacological profile of an adult head and neck sclerosing RMS was assessed. Furthermore, NGS, microsatellite instability, and in silico analyses were carried out. RT-PCR identified the upregulation of CDH1, SLUG, MMP9, RAB22a, S100P, and LAPTM4b, representing promising biomarkers for this disease. Pharmacological profiling showed the highest sensitivity with anthracycline-based regimen in both 2D and 3D culture systems. N...

Cell Biology and Toxicology

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and ... more Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the ...

International Journal of Molecular Sciences

Adult acute lymphoblastic leukemia (ALL) with BCR-ABL1 rearrangement (Philadelphia chromosome, Ph... more Adult acute lymphoblastic leukemia (ALL) with BCR-ABL1 rearrangement (Philadelphia chromosome, Ph) is a hematological aggressive disease with a fatal outcome in more than 50% of cases. Tyrosine kinase inhibitors (TKIs) targeting the activity of BCR-ABL1 protein have improved the prognosis; however, relapses are frequent because of acquired somatic mutations in the BCR-ABL1 kinase domain causing resistance to first, second and third generation TKIs. Axitinib has shown in vitro and ex vivo activity in blocking ABL1; however, clinical trials exploring its efficacy in ALL are missing. Here, we presented a 77-year-old male with a diagnosis of Ph positive ALL resistant to ponatinib and carrying a rare threonine to leucine (T315L) mutation on BCR-ABL1 gene. The patient was treated with axitinib at 5 mg/twice daily as salvage therapy showing an immediate although transient benefit with an overall survival of 9.3 months. Further dose-finding and randomized clinical trials are required to ass...

Journal of Clinical Oncology

11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predict... more 11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predictive of response and can help define therapeutic strategies. Our aim is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis by a SNP array-based genotyping approach. Methods: We performed SNP 6.0/Cytoscan HD (Affymetrix) on 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number (BioDiscovery) and R Core Team. Results: We found several genes preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p < ....

Cancers

Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of t... more Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. Methods: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. Results: This study reports, for the first time, the efficacy of the concomi...

Blood

2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the C... more 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roch...

Experimental and Molecular Therapeutics

Cancer research, Jan 12, 2017

Philadelphia Chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is currently treated with... more Philadelphia Chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is currently treated with BCR-ABL1 tyrosine kinase inhibitors (TKI) in combination with chemotherapy. However, most patients develop resistance to TKI through BCR-ABL1-dependent and -independent mechanisms. Newly developed TKI can target Ph+ ALL cells with BCR-ABL1-dependent resistance; however, overcoming BCR-ABL1-independent mechanisms of resistance remains challenging because transcription factors (TF), which are difficult to inhibit, are often involved. We show here that: i) the growth of Ph+ ALL cell lines and primary cells is highly dependent on MYB-mediated transcriptional upregulation of CDK6, cyclin D3, and BCL2 and ii) restoring their expression in MYB-silenced Ph+ ALL cells rescues their impaired proliferation and survival. Levels of MYB and CDK6 were highly correlated in adult Ph+ ALL (p=0.00008). Moreover, Ph+ ALL cells exhibited a specific requirement for CDK6 but not CDK4 expression, most likely b...

Leukemia & Lymphoma

To date, the lack of chemo-naive cell models limited exploratory studies to define novel therapie... more To date, the lack of chemo-naive cell models limited exploratory studies to define novel therapies for Burkitt lymphoma (BL). To obtain a useful tool for biological and translational studies for this tumor, we established the RALE051 cell line from the malignant ascitic fluid cells of a patient at initial diagnosis, not previously exposed to any treatment. The cell line was characterized by karyotyping, fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) array, immunohistochemistry, and RNA-Seq, revealing the persistence of biological and molecular features observed in the primary ascitic fluid, such as the cell morphology and immunophenotype, the occurrence of a t(8;22) translocation deregulating MYC, knock-out ID3 mutations in a compound homozygous state, and a mutated TP53. Moreover, additional alterations characterizing the ex vivo transformation included the emergence of three novel fusion transcripts and a nonsense mutation affecting TP53. The establishment of this cell line would be beneficial for future biological and therapeutic studies of BL.

Oncotarget, Jan 11, 2016

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed f... more 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 resu...

Journal of hematology & oncology, Jan 5, 2015

Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored t... more Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. 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. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent N-ethyl-N-nitrosourea. Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment...

Cancer Research, 2015

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: In... more Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: In Acute Myeloid Leukemia (AML) there is a strong need to develop new diagnostic and therapeutic options: to identify the genes mostly predictive of treatment response, we use Single Nucleotide Polymorphism (SNP) Arrays and Whole Exome Sequencing (WES) in AML patients with heterogeneous karyotypes and different subgroups. Materials and Methods: SNP arrays (CytoScan HD Array, Affymetrix Inc.) was positively done in 58 AML samples and all of them were analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.) and Nexus Copy Number™ v7.5 software (BioDiscovery), while Next Generation Sequencing (NGS)-WES HiSeq 2000 (Illumina) was done in 35 AML patients. Results: We treated 58 AML patients (pts) with a median age of 52 years, of which 9 were insensitive pts and 49 sensitive to therapies, all obtaining complete clinical remission. Copy Number Alterations (CNAs) were detected in all patients affecting all the chromosomes, in particular our cohort of pts present a percentage of CNA, divided as follow: 58% of LOH, 15% of gain, 16% of loss, 3% of high copy gain and 4% of homozygous copy loss. We found that several cancer genes were preferentially amplified: IGH@, KIT, IGL@, TSC1, NOTCH2, SETD2, EZH2, while in high copy gain we found TSC1, PTEN, RB1, IKZF1, ZRSR2, IGH@, NF1, MYC, KRAS. Then several genes were preferentially deleted: CRLF2, ATRX, JAK2, BCOR, PHF6, GATA1, KDM6A and in homozygous copy loss: JAK2, CRLF2, RB1, PDGFRA, RUNX1. Moreover we genes in LOH: DDX5, MTCP1, HOOK3, ZRSR2, GATA1, KDM6A, BCOR, NF1, BRAF, ATRX. We focused on two losses of JAK2: the first deletion, detected in 18/58 (31%) pts, goes from 5030 to 5038 Kbp (7,43 Kbps) including intron 4-5; the second minimal common region of loss, detected in 5/58 (8,6%) pts, goes from 5083 to 5098 Kpb (15 Kbps) including intron 19-20 and exons 20, 21 and 22, suggesting a defective transduction, in fact we showed that the overall survival rate is better for the group of pts which present a deletion of JAK2 rather than the group with an amplification of this gene (p-value < 0,01). We have also found three other genes which are preferentially lost: SIRPB1, ADAM3A and STAG2 with a percentage of 50%, 43% and 69% respectively. By NGS-WES we analyzed 35 AML samples at diagnosis and we searched for point mutations, insertion/deletion or other abnormalities, involved in biomarkers of response to treatment. We found mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR. Conclusion: By SNP arrays we have identified Copy Number Alterations involving important cancer genes AML and we showed that a new deletion in JAK2 may have a role in overall survival rate. Future prospective will be to correlate the cancer genes alteration and mutations with the prognosis of AML, in order to identify new biomarkers relevant for the disease. Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Universita 2010-12 (L. Bolondi), FP7NGS-PTL project Citation Format: Viviana Guadagnuolo, Maria Chiara Fontana, Antonella Padella, Ilaria Iacobucci, Cristina Papayannidis, Giorgia Simonetti, Anna Ferrari, Giovanni Marconi, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Francesca Volpato, Chiara Sartor, Emanuela Ottaviani, Massimo Delledonne, Michele Cavo, Guido Biasco, Giovanni Martinelli. SNP array reveals a new deletion of JAK2 in AML patients. [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 4848. doi:10.1158/1538-7445.AM2015-4848

Cancer Research, 2015

AML is a heterogeneous disease with a variety of structural and numerical chromosomal and genetic... more 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

Cancer Research, 2014

Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequ... more Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs. The recent WHO classification (2008) includes an indolent form (ISM), an aggressive form (ASM) and a leukemic subvariant (MCL). The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 22 patients (male/female=11/11) affected by SM have been referred to our Institution. Among these, 12 (55%) presented with systemic symptoms associated with signs of organ involvement (skeletal lesions, ascites, liver function impairment or bon marrow disfunction), thus identifying an ASM. Therefore, since a first line therapy (IFNalfa, Imatinib and 2CdA in 56%, 11% and 33% of the patients, respectively) and supportive care with histamine receptor antagonists weren’t followed by a significant benefit, a personalized use of PKC412 was asked and obtained for 9 out of 12 ASM patients. Thus, from March 2011 9 (M/F =3/9) patients with ASM have been treated with PKC412, administered orally, at the dosage of 100 mg twice daily, without rest periods. The median age was 60 years (range 39-75); the median time from diagnosis was 6 months (range 2-53). Median serum tryptase level was 100 mcg/L(range 19.3-1160). C-kit mutation D816V was present in 8 out of 9 patients. Cytogenetic analysis was normal in all the patients. Results. Seven out of nine patients were evaluable for response. The median duration of therapy was 517 days (range 327-970+). According to European Criteria, a Major response was observed in one patient, and a partial response in 6 patients. Overall, the drug was well tolerated, and no serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. Conclusions. In a small cohort of ASM patient, the prolonged therapy with PKC412 is safe and effective, mainly on symptoms improvement and haematological profile. Nevertheless, the persistence of the D816V c-kit mutation, despite significant responses, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. UDS approaches are needed in order to clarify this issue. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Simona Soverini, Caterina De Benedittis, Maria Chiara Abbenante, Chiara Sartor, Ilaria Iacobucci, Carmen Baldazzi, Emanuela Ottaviani, Anna Ferrari, Viviana Guadagnuolo, Alberto Conficoni, Strefania Paolini, Sarah Parisi, Federica Frabetti, Silvia Piccari, Sandro Grilli, Elisa Lani, Giovanni Martinelli. PKC412 (Midostaurin) is safe and highly effective in systemic mastocytosis patients: Follow up of a single-center Italian compassionate use. [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 746. doi:10.1158/1538-7445.AM2014-746

Cancer Research, 2014

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Ponatinib, a th... more 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

Cancer Research, 2013

Background. Precursor T cell LBL/ALL occurs most frequently in late childhood, adolescence, and y... more Background. Precursor T cell LBL/ALL occurs most frequently in late childhood, adolescence, and young adulthood, with a 2:1 male predominance; it comprises 15 and 25 percent of childhood and adult ALL, respectively, and 2 percent of adult non-Hodgkin lymphoma. Nelarabine is an anticancer prodrug of arabinofuranosylguanine (ara-G); it inhibits DNA synthesis and leads to high molecular weight DNA fragmentation and cell death. Nelarabine has showed relevant efficacy in phase II clinical trials, both in pediatric and in adult LBL/ALLpopulations. Design and Methods. We report clinical outcome results of 9 newly diagnosed and younger than 60 years T-ALL patients (median age 29 years, range 22-45 years, 3/6 M/F, 8 T-ALL, 1 T-LBL) treated according to pediatric-like adapted schedule. Cytogentics data and molecular biologic features will be provided on site. Induction cycle included Vincristine, daunoblastine, L-asparaginase and Prednisone. After induction, all the patients received consolidation therapy with cyclophosphamyde, L-asparaginase, Cytarabine and 6-Mercaptopurine. Subsequently all the patients receveid Nelarabine 1500 mg/sqm (days 1-3-5 every 21) for two cycles. All the patients shared the same strategy for intensification, which consisted in allogenic stem cell transplantation, if available, or additional courses of consolidation chemotherapy. Durations of complete remission (CR) and overall survival (OS) were estimated according to the Kaplan-Meier method. The CR duration was dated from start of CR to first evidence of disease recurrence. Results. After a single induction course, 9/9 patients obtained a CR (100%). Eight patients underwent an allogenic bone marrow transplantation. After a median follow-up of 24 months, 7/9 patients (78%) are alive in CR. The median CR duration and OS were 13.4 and 24.4 months, respectively. Neurological toxicity of grade 3 has not been reported. We did not observe grade 3-4 haematological toxicity. Conclusion. Nelarabine is a promising drug, which induces a remarkable complete remission rate at the expense of a very low and manageable toxicity. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, University of Bologna and BolognAIL. Citation Format: Chiara Sartor, Mariachiara Abbenante, Cristina Papayannidis, Ilaria Iacobucci, Stefania Paolini, Emanuela Ottaviani, Annalisa Lonetti, Sarah Parisi, Anna Ferrari, Viviana Guadagnuolo, Sandro Grilli, Nicoletta Testoni, Michele Cavo, Giovanni Martinelli. Nelarabine front-line therapy for adult T-lymphoblastic leukemia/lymphoma (T-LBL/ALL): preliminary results of a single center experience. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1179. doi:10.1158/1538-7445.AM2013-1179

Cancer Research, 2010

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

Background: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic ... more Background: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult BALL patients (106 BCR-ABL1-positive and 38 BALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling.