Giovanni Marconi

Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients

FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2–2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.

Chromothripsis in acute myeloid leukemia: biological features and impact on survival

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study define incidence of chromothripsis in 395 newly-diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix®) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p=.002), ELN high risk (HR) (p<.001), lower white blood cell (WBC) count (p=.040), TP53 loss and/or mutations (p<.001) while FLT3 (p=.025) and NPM1 (p=.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p<.001) compared with HR patients (p=.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e. TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, 17. CBA. FISH showed that chromothripsis is associated with marker, derivative and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology.

Efficacy of Azacitidine in the treatment of adult patients aged 65 years or older with AML

ABSTRACT Introduction: Therapy for acute myeloid leukemia (AML) in elderly populations (>65 years) is still a challenge for scientists and hematologists worldwide, and represents an urgent medical need. Notably, the identification and the recognition of molecular and epigenetic mechanisms involved in the pathogenesis of such a heterogeneous disease, are providing new tools for a more successful and ‘targeted’ approach. Azacitidine is a hypomethylating agent (HMA) with relevant activity in patients affected by myelodysplastic syndrome (MDS) and AML with low blast cells percentage (>30%), in terms of reduction of transfusion dependence, and improvement of quality of life. Areas covered: This review summarizes the mechanism of action, safety profile and efficacy of azacitidine in the field of elderly AML populations, providing up-to-date references on this subset of high-risk patients. Expert opinion: HMAs are the first successful treatment for elderly patients with high-risk MDS and are effective for some AML subtypes. Translational studies based on gene expression profiling and molecular sequencing, would be able to identify, in the near future, patients with a favorable profile of response to these compounds suggesting new potential treatment combinations also.

Mesenchymal stromal cells from myelodysplastic and acute myeloid leukemia patients display in vitro reduced proliferative potential and similar capacity to support leukemia cell survival

BackgroundMesenchymal stromal cells (MSCs) are an essential element of the bone marrow (BM) microenvironment, playing a crucial function in regulating hematopoietic stem cell proliferation and differentiation. Recent findings have outlined a putative role for MSCs in hematological malignancy development. So far, conflicting results have been collected concerning MSC abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In particular, a considerable amount of evidence has been accumulated strongly supporting a permissive role of MSCs in malignancy evolution to MDS, while a potentially causative or promoting function performed by MSCs in AML has not yet been fully clarified. Here, we compared MSCs isolated from healthy, MDS, and AML subjects to investigate MSC alterations and to emphasize putative common and/or diverse features.MethodsWe isolated and expanded MSCs from AML patients (AML-MSCs) and MDS patients (MDS-MSCs), and we analyzed and compared their phenotypic and functional properties with respect to each other and versus healthy donor-derived MSCs (HD-MSCs).ResultsWe found that stable MSC cultures could be easily established from HD and MDS mononuclear BM-derived cells, while a substantial fraction (25%) of AML patients failed to yield MSCs. Nevertheless, isolated MDS-MSCs and AML-MSCs, as well as HD-MSCs, contained the basic features of MSCs. Indeed, they displayed similar surface marker expression and efficient capacity to differentiate versus osteogenic and adipogenic lineage in vitro. We also proved that MDS-MSCs and AML-MSCs, analyzed by fluorescence in-situ hybridization, did not harbor leukemic cell cytogenetic abnormalities. Moreover, MDS-MSCs and AML-MSCs were similar in terms of ability to sustain AML cell viability and immune-regulatory capacity. However, we were also able to detect some differences between AML-MSCs and MDS-MSCs. Indeed, we found that the frequency of rescued MSCs was lower in the AML group than in the HD and MDS groups, suggesting that a reduced number of MSC precursors could inhabit AML BM. Instead, MDS-MSCs showed the lowest proliferative capacity, reflecting some intrinsic and particular defect.ConclusionsOverall, our results elucidated that MDS-MSCs and AML-MSCs did not show macroscopic and/or tumor-related defects, but both displayed functional features potentially contributing to favor a leukemia-protective milieu.

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.

Tyrosine Kinase Inhibitors (TKI) in Relapsed/Refractory (RR) Patients with FLT3-ITD Positive Acute Myeloid Leukemia (AML) Confer Better Survival than Chemotherapy, Due to a Better Safety Profile

S200 will be efficacious in treating in vivo murine leukemias that are currently in development. The next phase of this project will aim to determine mechanisms of sensitivity and resistance to PARP inhibition in AML.

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

Abstract 3472: Separase overexpression defines a new subset of acute myeloma leukemia patients characterized by high CD34 and MYC levels

The endopeptidase Separase, encoded by the ESPL1 gene, plays a key role in faithful segregation of sister chromatids by cleaving the cohesin complex at the metaphase to anaphase transition. Its overexpression associates with aneuploidy and bad prognosis in solid tumors. Little is known in Acute Myeloid Leukemia (AML). We profiled the genomic landscape of 405 and 78 AML cases by SNP array (SNP 6.0 and Cytoscan HD, Affymetrix) and whole exome sequencing (100 bp, paired-end, Illumina), respectively. Bone marrow blasts from 61 patients were analyzed by gene expression profiling (HTA 2.0, Affymetrix). Separase expression was determined by Immunohistochemistry (1:600 antibody dilution Abnova, clone 6H6) in 44 AML and 4 control bone marrow specimens. One patient exhibited a nonsynonimous mutation in ESPL1 (1.3%), which was predicted to alter the protein function. Moreover, ESPL1 copy number gain was observed in 5/405 cases (1.2%): 2 hyperdiploid AML, one trisomy 12 and 2 cases with a short gain at 12q. Notably, protein level detection in one of the 12q-gain cases confirmed Separase overexpression. To determine the incidence of Separase overexpression, we performed Immunohistochemistry on additional 43 AML. Separase was overexpressed in 29/44 AML (66%, Separase-high), being comparable to control marrow biopsies in the remaining 15 samples (Separase-low). Sixty-two percent of Separase-high AML were aneuploid. However, no significative association was observed, as previously reported for mutations in the cohesin genes in AML. Separase overexpression correlated with increased patients’ age (median age 64 vs. 57 years, p=.01), 17-fold upregulation of CD34 (p=.004) and a trend towards reduced overall survival (6-years follow-up). Separase overexpression was not mutually esclusive with cohesin gene mutations, it co-occurred with NPM1 and FLT3 lesions and frequent mutations in genes involved in protein post-translational modification and ubiquitination (p=.04). Separase-low cases were enriched for mutations in RAS signaling pathway (NRAS, KRAS, NF1, RIT1, GRAP2, RALGDS; p=4.5x10-5) and in cell migration-related genes (LIMS2, S1PR1, PPIA, PLXNB1, FAT1). Separase-high cases also showed a defined transcriptomic profile, characterized by reduced expression of HOXA/B family genes, the DNA damage repair gene ATM, the p53 regulator MDM2 and forced expression of the cell cycle markers CDC20, AURKB, NUSAP1 and of MYC, independently of chromosome 8 gain. Taken together, our data suggest that genomic lesions targeting ESPL1 are a rare event in AML. However, Separase overexpression is a common feature and defines a new subset of AML cases with a distinct gene expression profile, which may benefit of innovative targeted therapies including CDC20 and bromodomain inhibitors. Supported by: ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Giorgia Simonetti, Antonella Padella, Simona Righi, Maria Chiara Fontana, Marco Manfrini, Cristina Papayannidis, Giovanni Marconi, Carmen Baldazzi, Marianna Garonzi, Alberto Ferrarini, Massimo Delledonne, Nicoletta Testoni, Elena Sabattini, Giovanni Martinelli. Separase overexpression defines a new subset of acute myeloma leukemia patients characterized by high CD34 and MYC levels [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 3472. doi:10.1158/1538-7445.AM2017-3472

Abstract 2451: Genomic wide microarray analysis identifies novel copy number alterations in adult acute myeloid leukemia

Introduction: Novel array-based technique as SNP microarray can detect losses or gains of chromosomic material, which could be predictive of response and can help define therapeutic strategies. The aim of this study is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis, by a SNP array-based genotyping approach. Materials and Methods: We performed SNP 6.0 or Cytoscan HD (Affymetrix) in 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 v8.0 (BioDiscovery) and R Core Team. Results: Copy Number Alterations (CNAs) were scattered across all chromosomes and all pts showed CNA events. SNP array analysis showed that several genes were 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%), while the 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 Conclusion: We have identified new CNAs and pathways involving novel potential leukemia-related genes. Our results suggest that the comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes implicated in deregulated pathways deserve further investigation in order to identify new candidate genes which could be relevant AML biomarkers. Acknowledgements: ELN,AIL,AIRC,progetto Regione-Universita 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY. Citation Format: Maria Chiara Fontana, Giovanni Marconi, Cristina Papayannidis, Eugenio Fonzi, Giorgia Simonetti, Antonella Padella, Anna Ferrari, Emanuela Ottaviani, Silvia Lo Monaco, Stefania Paolini, Simona Soverini, Giovanni Martinelli. Genomic wide microarray analysis identifies novel copy number alterations in adult acute myeloid 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 2451. doi:10.1158/1538-7445.AM2017-2451

Abstract 515: Alterations in phosphatidylinositol 3-phosphate (PI3P) pathway and cAMP pathway confirm poor prognosis and reduced overall survival (OS) in a series of 209 acute myeloid leukemia patients

Introduction PI3P is a molecule that regulate cell growth and mediates cell proliferation via PI3K/AKT/mTOR in response to various growth signals. Abnormal activation of genes in its pathway is associated to oncogenic activity and poor Overall Survival (OS). AMPK plays a role as a regulator of cellular energy homeostasis. Aims The aim of the this study is to define the role of PI3P pathways and AMPK pathway in AML. Methods In this work we analyzed 208 consecutive newly diagnosed non M3 AML patients, screened for TP53, FLT3, NMP1, IDH1, IDH2, and DNMT3A mutations. Remission status was assessed with bone marrow biopsy. We performed Microarray-based Comparative Genomic Hybridization with Affymetrix SNP array 6.0 or Cytoscan HD in all the patients; we performed Whole Exome Sequencing (WES)in 80/208 patients. Survival data were collected prospectively, with a median follow-up of 18 months. Survival analysis was performed with Kaplan Meyer method using log rank test. Univariate and multivariable regression and Cox Hazard Ratio(HR) model was performed. Correlation between variables was assessed with Fisher’s exact test. Results We selected genes in pathways basing on literature and GO data. Alterations in these pathways involved 103/209 patients (48%). We analyzed the gene in two different pathways. PI3K/AKT/mTOR pathway includes the following genes: pik3ca, cdkn1a, akt1, akt3, mtor and pten, pdk1,pik3r1 and irs1. The second one is AMPK pathway and it include: sesn, prkaa1, prkab1, prkag1, prkag3. Alterations in PI3K/AKT/mTOR pathway confer worst OS (p = .035) when compared with unaltered patient, but events in these pathways did not affect therapy response. Alterations in AMPK pathway confer worst OS (p Conclusions Our work investigates the role of PI3P and cAMP pathways in AML. Surprisingly, it showed that alterations in these pathways are associated with poor prognosis. Significantly, alterations in cAMP pathways were associated with therapy resistance. Acknowledgement: ELN, AIL, AIRC, PRIN, Progetto Regione-Universita 2010-12, FP7 NGS-PTL project, HARMONY Citation Format: Mariachiara Abbenante, Mariachiara Fontana, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Elena Tenti, Eugenia Franchini, Anna Ferrari, Sarah Parisi, Emanuela Ottaviani, Nicoletta Testoni, Viviana Guadagnuolo, Chiara Sartor, Silvia Lo Monaco, Cristina Papayannidis, Giovanni Martinelli. Alterations in phosphatidylinositol 3-phosphate (PI3P) pathway and cAMP pathway confirm poor prognosis and reduced overall survival (OS) in a series of 209 acute myeloid leukemia patients [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 515. doi:10.1158/1538-7445.AM2017-515