Francesca Volpato

Complex karyotype, older age, and reduced first-line dose intensity determine poor survival in core binding factor acute myeloid leukemia patients with long-term follow-up

Approximately 40% of patients affected by core binding factor (CBF) acute myeloid leukemia (AML) ultimately die from the disease. Few prognostic markers have been identified. We reviewed 192 patients with CBF AML, treated with curative intent (age, 15–79 years) in 11 Italian institutions. Overall, 10‐year overall survival (OS), disease‐free survival (DFS), and event‐free survival were 63.9%, 54.8%, and 49.9%, respectively; patients with the t(8;21) and inv(16) chromosomal rearrangements exhibited significant differences at diagnosis. Despite similar high complete remission (CR) rate, patients with inv(16) experienced superior DFS and a high chance of achieving a second CR, often leading to prolonged OS also after relapse. We found that a complex karyotype (i.e., ≥4 cytogenetic anomalies) affected survival, even if only in univariate analysis; the KIT D816 mutation predicted worse prognosis, but only in patients with the t(8;21) rearrangement, whereas FLT3 mutations had no prognostic impact. We then observed increasingly better survival with more intense first‐line therapy, in some high‐risk patients including autologous or allogeneic hematopoietic stem cell transplantation. In multivariate analysis, age, severe thrombocytopenia, elevated lactate dehydrogenase levels, and failure to achieve CR after induction independently predicted longer OS, whereas complex karyotype predicted shorter OS only in univariate analysis. The achievement of minimal residual disease negativity predicted better OS and DFS. Long‐term survival was observed also in a minority of elderly patients who received intensive consolidation. All considered, we identified among CBF AML patients a subgroup with poorer prognosis who might benefit from more intense first‐line treatment. Am. J. Hematol. 90:515–523, 2015.

Posterior Reversible Encephalopathy Syndrome in a B-Cell Acute Lymphoblastic Leukemia Young Adult Patient Treated with a Pediatric-Like Chemotherapeutic Schedule

We report here the case of a young adult affected by pre B-cell acute lymphoblastic leukemia (ALL), who developed, during a pediatric-like chemotherapy consolidation schedule with high dosage of Methotrexate, a severe neurological toxicity. Clinical presentation and neuroimaging data were diagnostic for posterior reversible encephalopathy syndrome (PRES). A complete resolution was quickly obtained with medical blood pressure control and anticonvulsants administration. To the best of our knowledge, this is the first case of PRES described in the adult ALL setting. Currently, the clinical management of this aggressive disease is moving towards a pediatric-like approach also in adult patients, due to the better outcome reached with intensive chemotherapeutic regimens in children population. However, therapy-related toxicities have to be taken into account, since their onset may adversely affect patients’ clinical outcome.

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 4848: SNP array reveals a new deletion of JAK2 in AML patients

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

Abstract 4835: A new biomarker of response to 5-azacitidine therapy in MDS and AML patients: SIRPB1

Introduction: Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are a group of diseases of the elderly that initiates in a hematopoietic stem cell and are characterized by clonal hematopoiesis and uncertain prognosis, mostly due to cytogenetic background. In both diseases, 5-azacitidine (5-Aza) has been successful, inducing prolonged survival and delayed AML evolution. Aim: To identify the genes mostly predictive of treatment response, we use high-throughput genomic analysis (SNP arrays and/or NGS-RNA-seq and/or NGS-WES) in azacitidine-sensitive and resistant MDS/AML patients. Materials and Methods: NGS-WES or RNA seq HiSeq 2000 (Illumina) was positively done in 35/214 AML samples (16%). SNPs arrays (CytoScan HD Array, Affymetrix Inc.) was done in 125/214 AML samples (58%) and 18/32 MDS samples (56%) at diagnosis, then analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.), Nexus Copy Number™ v7.5 (BioDiscovery) and GeneGo MetaCore™ software. Results: We treated 246 adult patients (pts) with MDS or AML: 214 pts were AML and 32 were MDS with a median age of 59 and 70 years, respectively. Forty-five pts were treated with 5-Aza (32 MDS / 13 AML), while 201 AML were treated with conventional chemotherapy. Forty-five MDS/AML pts were treated with at least one complete cycle of 5-Aza (75 mg/sqm/daily). SNP arrays was done in 22/45 (49%), 13 pts were defined “insensitive/resistant”. Macroscopic CNAs affecting a complete chromosome or its arms were detected in 5 of 22 pts (23%), while classical cytogenetic was able to detect only two cases of trisomy 8 (9%), suggesting superiority of SNPs array for CNAs identifications. Chromosomic aberrations disease-related are more statistically frequent on pts “insensitive” versus pts. “sensitive” (64% vs. 35%) (p≤0.01). Moreover we found that from the median of chromosomic alterations lenghts (in kbp) the group of “insensitive” MDS/AML patients to 5-Aza therapy present more losses than “sensitive” ones. By Nexus Copy Number software, we identify 137 genes highly differentially gain (SIRPB1 and KIT with p ≤ 0.05) or loss (SIRPB1, LCE1C, BCAS1, EXD3 with p ≤ 0.05) or LOH between “insensitive” versus “sensitive” to 5-Aza (p ≤ 0.05). Among these genes, we focused on SIRPB1 (cytoband 20p13, 56Kbps), since it was loss on 14/22 (64%) “insensitive” pts (p = 0,023) and gain on 7/22 (32%) “sensitive” ones (p = 0,0324), respectively. SIRPB1 common deletion region length is 27 kbps and the common amplified region length is 30 kbps. By NGS-WES we analyzed 35/214 (16%) AML samples at diagnosis. We found mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR and again in SIRPB1 an heterozygous missense variant (rs45545343; p. H/D). Conclusions: We conclude that SIRPB1 is a promising marker of response to 5-Aza treatment in myelodysplastic syndromes and in acute myeloid leukemia. Citation Format: Viviana Guadagnuolo, Cristina Papayannidis, Ilaria Iacobucci, Giorgia Simonetti, Antonella Padella, Stefania Paolini, Mariachiara Abbenante, Sarah Parisi, Francesca Volpato, Chiara Sartor, Maria Chiara Fontana, Massimo Delledonne, Michele Malagola, Carla Fili, Domenico Russo, Sandro Grilli, Michele Cavo, Giovanni Martinelli. A new biomarker of response to 5-azacitidine therapy in MDS and AML patients: SIRPB1. [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 4835. doi:10.1158/1538-7445.AM2015-4835

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 2243: Gene expression signature of aneuploidy in acute myeloid leukemia

Acute Myeloid Leukemia (AML) is a heterogeneous malignancy characterized by the expansion of myeloid precursor cells with limited or abnormal differentiation capacity. A relatively common event in AML is represented by chromosome gain or loss. Numerical chromosome abnormalities, which define the aneuploid condition, have a detrimental effect in primary non-malignant cells, since they dramatically reduce cellular fitness. However evidence suggests that they have a causative role in tumorigenesis and that they are well tolerated in transformed cells belonging to the myeloid lineage. Aim of the study is to elucidate the pathogenic mechanisms that sustain and contribute to aneuploidy in AML. We have performed gene expression profile (GEP) analysis of bone marrow cells from 42 AML patients, including 19 aneuploid cases and 23 cases with normal karyotype. All samples contained more than 80% blast cells. The aneuploid cohort included AML cases carrying one (or more) monosomy, trisomy or a monosomal karyotype. Our analysis covered more than 245,000 and 40,000 coding and non-coding transcripts, respectively (the latter comprising microRNAs), and a significant number of exon-exon junctions, which allow the analysis of multiple splicing isoforms. Quality controls confirmed that the data show comparable signal values. The gene expression signature of aneuploid samples have been compared to normal karyotype ones. We have identified a set of coding and non-coding transcripts which are differentially expressed between the two groups (p≤0.05, including more than 20 genes with a fold difference ≥2) and defined a gene signature that allows the discrimination between aneuploid and euploid samples in our dataset. The analysis of an increased number of cases will confirm the results and allow the sub-stratification of aneuploid samples according to their GEP. Our data will be further validated by comparing them with published GEP datasets and the gene signature will be characterized by pathway analysis. This study provides novel insights into the molecular mechanism that sustain aneuploidy in AML. The biological validation of genes which are commonly and specifically deregulated in aneuploid AML patients will guide the design of future therapeutic strategies targeting key players in the disease. Acknowledgements: European LeukemiaNet, AIRC, AIL, Prin 2010-2011, FP7 NGS-PTL project. Citation Format: Giorgia Simonetti, Antonella Padella, Viviana Guadagnuolo, Cristina Papayannidis, Francesca Volpato, Emanuela Ottaviani, Serena Formica, Annalisa Astolfi, Ilaria Iacobucci, Giovanni Capranico, Daniel Remondini, Giovanni Martinelli. Gene expression signature of aneuploidy in acute myeloid leukemia. [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 2243. doi:10.1158/1538-7445.AM2014-2243