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Background: Fusions (Fs) are major molecular biological abnormalities in acute leukemias (ALs), and all well-known Fs in leukemias are founder variations are routinely used as molecular markers for the diagnosis, classification, risk stratification, and targeted therapy but there is a considerable part of ALs that are not screened for other known/unknown transcripts. For example in B-Other B Acute Lymphoblastic Leukemia (B-ALL) [Ph-/-/-; negative for t(9;22); t(1;19); t(4;11); 61% of adult B-ALL], many chimeric genes have been identified leading to a refined classification of B-ALL and to, in some cases, tailored therapies. At this point, a RNA-seq approach is needed but challenging for many aspects, among them a not standardized and very heterogeneous F data analysis. Aims: We developed and validated our integrated pipeline in order to assess targetable biomarkers and to better classify patients (pts). Methods: we performed 1385 gene RNAseq (Illumina) of 224 adult AL samples (112 Ph-/-/-, 41 Ph+, 6 t(1;19), 16 T-LBL/T-ALL and 49 AML). Starting from Ph-/-/-, we developed a combined 4 tool analysis that is further implemented with a filtering strategy with a specific ALs fusion literature filter (Patent PCTEP2021-065692 and 749 ALL Fs database copyright) (Fig.1A). We validate our strategy using: RT-PCR, FISH SNP Arrays; MLPA and total RNA-seq. Results: From 3022 candidate Fs, we retained 160 of them (5.3%; excluding WHO canonical Fs) not otherwise detected, in 120 pts with a high Fs rate in T-LBL/T-ALL, Ph-/-/- and Ph+ (62.5%, 62.2% and 61% respectively) denoting that ALs are not deeply characterized (Fig 1B). The lower rate was found in AML (26.5%), but we were able to identify new ETV6 rearrangement (r) and a FISH cryptic F in a 46, XY pt (TBL1XR1-MECOM). We validated 98 Fs that have been already reported in the literature and 43 novel F transcripts. We obtained a validation rate of 81%. The majority of fused samples (65.2%) had only one detectable F while a smaller group was characterized by multiple Fs. Many of these Fs were previously described in B/T-ALL and AML (e.g. KMT2A-MLLT1, ABL1/2-RCSD1, IGH-MYC, NUP214-SET, ETV6-MECOM). In our bigger sub-cohort (Ph-/-/-), 44 Fs out of 109 (40.3%) were never been reported in Ph- ALL cases. In 15 Ph-like pts, we identified and validate 11 new transcripts. Ph-/-/- F detection help to sub-classify our fused pts in Ph-/-/- subgroups (ZNF384r-11.8%; Ph-like-19.1%; DUX4r- 4.4%; HLFr, MLLr and BCL2/MYC in 2.9%; MEF2Dr-1.5%). Conclusions: we identified pivotal transcripts in all ALs and an unexpected high rate of secondary Fs in adult ALs subgroups (52.4%) that are not characterized with conventional diagnostic methods. The use of an NGS approach and a powerful pipeline permit us to detect Fs useful for a better classification, prognostic identification (e.g. TBL1XR1-MECOM) and in some cases to find targetable Fs (e.g. ABL1-2/RCSD1, NUMA1-CSF1R, ZMYM2-FLT3). Supported by: L3P2505. Citation Format: Anna Ferrari, Silvia Vitali, Eugenio Fonzi, Andrea Ghelli Luserna Di Rora, Chiara Domizio, Cristina Papayannidis, Maria Teresa Bochicchio, Giovanni Marconi, Daniele Dall'Olio, Michela Tebaldi, Michela Rondoni, Barbara Giannini, Fabio Giglio, Crescenza Pasciolla, Monica Fumagalli, Sara Galimberti, Gastone Castellani, Daniel Remondini, Giorgia Simonetti, Giovanni Martinelli. Fusion landscape in acute leukemias: A submerged world of not routinely characterized transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6163.

Currently, the role of autophagy in acute lymphoblastic leukemia (ALL) is not fully understood however it has been linked to drug resistance. Several autophagy modulators have proven their efficacy in single agents or as chemo-sensitizer in ALL. Recently, the 3rd generation BCR-ABL1 inhibitor, ponatinib, has been showed to promote autophagy in chronic myeloid leukemia (CML) cells and the combination between ponatinib and hydroxychloroquine (HCQ) has proven its efficacy in neuroblastoma cells. Here, we tested the efficacy of combining different autophagy modulators (ponatinib, HCQ and rapamycin) against ALL cell lines and primary leukemic ALL cells. Firstly, we established the IC50 values of a panel of Philadelphia (Ph)-positive/negative B-/T-ALL cell lines (n=6) treated with ponatinib, HCQ and rapamycin in single agent. Then, using sub-toxic concentrations, we evaluated the efficacy of the combinations HCQ+Ponatinib and HCQ+Rapamycin in the reduction of the cell viability after 24, 48 and 72 hours of simultaneous treatment. Combination index analyses showed a synergic time and dosage-dependent reduction of the cell viability in all the cell lines treated with HCQ+Ponatinib. The combination HCQ+Rapamycin resulted synergic only in Ph-negative ALL cell lines. These results were confirmed by the significant induction of apoptosis (Annexin V and/or Caspase-3 staining) in the samples treated with HCQ+Ponatinib. The two combinations also significantly reduced the proliferation capacity of ALL cell lines in comparison with controls and single treatments. Moreover, the efficacy of the two combinations was confirmed on primary leukemic blasts isolated form adult ALL patients(n=6). Indeed, a significant induction of apoptosis was seen in samples treated with the two combinations in comparison with single treatments. To deeper understand the mechanism of action of the combinations we evaluated LC3A/B expression by flow cytometry. Interestingly, the expression of LC3A/B was modified heterogeneously among the different ALL cell lines and did not correlate with the response in term of cell viability or induction of apoptosis. Light microscopy analyses showed a significant increment in the number and diameter of autophagy vesicles in both ALL cell lines and primary leukemic ALL cells treated with HCQ+Ponatinib in comparison with single treatments. Interstingly, no significant effect on autophagy vesicles number or diameter was seen in the samples treated with HCQ+Rapamycin in comparison with single treatments. In conclusion, our findings show that the identified combinations effectively inhibit cell viability and induce apoptosis in Ph-positive/negative B-/T-ALL cells, suggesting the fundamental role of autophagy regulation in ALL survival. Citation Format: Andrea Ghelli Luserna Di Rora, Eugenio Fonzi, Lorenzo Ledda, Roberta Napolitano, Anna Ferrari, Antonella Padella, Martina Ghetti, Maria Teresa Bochicchio, Mouna Jandoubi, Cristina Mazzotti, Matteo Paganelli, Claudio Cerchione, Giovanni Marconi, Gerardo Musuraca, Giovanni Martinelli, Giorgia Simonetti. Targeting autophagy in acute lymphoblastic leukemia: Synergism between ponatinib, hydroxychloroquine and rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3001.

Background: The genetically heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL group that doesn’t have the most recurrent adult rearrangements (t(9;22); t(1;19); t(4;11)) is collectively referred to as “triple negative” (Ph-/-/-). CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Understand the genomic background of all Ph-/-/- ALL and subsequently clustering Ph-/-/- considering CRLF2 overexpression event, in order to define new biomarkers in these subgroups. Pts and Methods: Ph-/-/- pts were sequenced by WES (44pts/93 samples) and 92 B-Other pts were analyzed with NGS target seq (NTS) to validate the 8 most mutated genes (Fig1A). GEP were performed on 55 Ph-/-/-, 29 B-ALL Ph+ and on 7 donors. We cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in a top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (Pancancer and/or RNASeq; dMLPA-MRC-Holland; SNP Array; 454 Junior and PCR). Results: WES analysis identified some recurrent mutated genes (NRAS, PAX5, KRAS, PTPN11, EP400, JAK2, TP53, CREBBP) previously reported to be involved in B-ALL, confirming the pivotal roles of these gene in ALL. For the first time we described a little known gene PKHD1L1 as highly mutated (7.2%). TP53 was the most mutated gene (Fig1A) and that between these gene is the only one associated to a worst OS (p=0.004). Combining our new Ph-/-/- GEP clustering, WES, NTS, Fusion and CNA results we identify a defined 2-clusters-subdivision (Gr1 and Gr2). The Gr2 (14.1% of all B-ALL) is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) co-overexpression. The Gr2 GEP is similar to Ph+ one (Fig1C). Gr1 represents 46.9% of all B-ALL. Fusion, CNA and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mut or del), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. We also validated not previously described fusions. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upreg in the group1 and CDK6 in the Gr2. Preliminary data seems to confirm an higher effect on cell viability of a TKI on Gr2 primary cell pt (vs Gr1 pt). Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B. This new subclassification identifies new potential therapeutic targets with available drugs (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; TKIs already effective on Ph+ and Ph-like) to test. Citation Format: Anna Ferrari, Silvia Vitali, Valentina Robustelli, Andrea Ghelli Luserna di Rorà, Eugenio Fonzi, Simona Righi, Carmen Baldazzi, Michela Tebaldi, Samanta Salvi, Cristina Papayannidis, Giovanni Marconi, Mariachiara Fontana, Enrica Imbrogno, Antonella Padella, Giorgia Simonetti, Alessandra Santoro, Jesus María Hernández-Rivas, Maria Teresa Bochicchio, Fabiana Mammoli, Benedetta Giannini, Nicoletta Testoni, Daniele Calistri, Massimiliano Bonafè, Gastone Castellani, Elena Sabattini, Daniel Remondini, Giovanni Martinelli. “3c-up” a new adult Philadelphia negative acute lymphoblastic leukemia subgroup: Novel molecular markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2140.

Introduction: PPM1D (wild-type p53-inducible protein phosphatase WIP1) is a member of the PP2C family serine/threonine phosphatase involved in negative regulation of cell stress response pathways, leading to suppression of p53 after stress. To restore p53 function through MDM2 inhibition (Nutlin-3a) is a promising approach in AML. Promising results of the combination of MDM2 inhibitors and WIP1 inhibitor (WIP1i), obtained in many preclinical studies of solid tumors, paved the way for their application in AML. We investigated whether the inhibition of WIP1 (GSK2830371) could sensitize AML cell lines and primary cells to Nutlin-3a (Nut-3a) in order to obtain a novel therapeutic strategy for AML patients, based on restoration of p53 activity. Methods: In vitro viability (by WST-1 reagent) and Annexin-V/PI apoptosis assay were performed on a panel of TP53-wt AML cells (MOLM-13, MV-4-11 and OCI-AML3), on TP53-mutated NOMO-1 AML cells and on primary AML samples. Gene expression profile (GEP) and Western Blot (WB) analyses were performed on MV-4-11 and NOMO-1 after 16h. Results: Combined inhibition of increasing dosage of Nut-3a (0.5 to 5 uM) and WIP1i (5 to 20 uM) synergistically reduces TP53-wt AML cells viability, while NOMO-1 resulted to be insensitive to the combination. The combination index analyses showed a synergistically effect of the combination of both compounds on TP53-wt AML cells. Annexin V/PI staining showed that WIP1i sensitizes TP53-wt AML cell lines and primary samples to Nut-3a-induced apoptosis, when compared with single treatment. No effect was seen in NOMO-1. GEP demonstrated that MV-4-11 cells exhibits a major response to drug combination with an upregulation of cell cycle control genes, a downregulation of DNA repair machinery genes, upregulation of MDM2 and of TP53-downstream genes (eg.CDKN1A), confirming the activation of p53 pathway. NOMO-1 cells showed upregulation of resiliency-mechanism and confirmed insensitivity to both treatment. WB analysis confirmed GEP data showing an increased expression of p53 and p21 in wt-TP53 cell line after 16h of combined-treatment. Conclusions: We identified a novel synergistic drug combination between Nut-3a and WIP1i that induces apoptosis in AML cell lines and primary samples. GEP and WB of TP53-wt MV-4-11 and TP53-mutated NOMO-1 cells showed mechanisms underlying drug sensitivity and resistance giving novel insights on potential markers of response and novel drugable targets. In vivo studies are needed to confirm these preclinical data. Supported by: AIRC, FP7-NGS-PTL, Fondazione del Monte, HARMONY. Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giovanni Marconi, Martina Pazzaglia, Matteo Bocconcelli, Antonella Padella, Simona Soverini, Ilaria Iacobucci, Cristina Papayannidis, Anna Ferrari, Maria Teresa Bochicchio, Enrica Imbrogno, Michele Cavo, Andrea Ghelli Luserna di Rora, Giorgia Simonetti, Giovanni Martinelli. Pharmacological inhibition of WIP1 by GSK2830371 sensitizes AML cells to MDM2 inhibitor Nutlin-3a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2964.

Genomic and functional alterations of enzymatic activity drive cancer metabolic reprogramming along with mutations of tumor suppressors and oncogenes. IDH1/2 lesions represent a paradigmatic example in acute myeloid leukemia (AML). The study aimed to stratify AML patients based on their metabolic landscape and to map potential connections between their metabolic and genomic profile. The genomic landscape of 166 AML patients was obtained by whole exome sequencing. Variants were called by MuTect and Varscan2. Six additional cases were analyzed by targeted sequencing (SOPHiA GENETICS). Metabolites were quantified by mass spectrometry of bone marrow cells (35 CD34+ and 15 CD33+ AML from the above-mentioned cohort) and compared with CD34+ cord blood and CD33+ healthy blood cells (n=21 each, Metabolon) by Welch's t-test. In AML, 17% of somatic variants targeted metabolism-related genes: 38% were enzymes (according to Recon2), including 3% of electron transport chain genes encoded by mitochondrial DNA (COX1-3, ND1-6), 3% were AML-related genes with a known involvement in cell metabolism (e.g. IDH1/2, MYC, KRAS) and 59% were metabolic regulators, defined by gene ontology annotation. Ninety-one% of patients carried at least one mutation in a metabolism-related gene, with 43% of variants rated as damaging. The most represented pathways were lipid, carbohydrate, nucleotide (AK9, H6PD), amino acids (IDO2) and glucose metabolism (PKM, HK3). Principal component analysis of metabolic data showed a distinct profile between AML and healthy cells, with a predictive accuracy of 86% and 95% for CD34+ and CD33+ cells, respectively. Conversely, few differences were observed between CD34+ and CD33+ AML. Unsupervised hierarchical clustering clearly defined 3 AML clusters (C1-3). Moreover, 3 subgroups could be identified in C3 without ambiguous assignments. C1 was enriched for NPM1-mutated (mut) cases (83%, 33%, 27% in C1, C2 and C3, respectively, p=0.03). NPM1-mutated AML were distinguished by a 12-metabolites signature. They showed increased levels of spermidine, cytidine 2′ or 3′-monophosphate (P), thymidine 3′-monoP, uridine-2',3'-cyclic monoP and decrease of inosine 5'-monoP, suggesting altered polyamine, pyrimidine and purine metabolism. Moreover, NPM1-mut AML had reduced levels of intermediates involved in acyl carnitine, lysophospholipid, phosphatidylethanolamine and sphingolipid metabolism. Overall, mutations of metabolism-related genes are common in AML. We defined a metabolic-based classification of AML and identified a new metabolic signature based on 12 metabolites that distinguish NPM1-mut AML from wild-type cases and healthy CD34+/CD33+ cells. Major alterations in the nucleotide and polyamine pathways suggest novel potential therapeutic approaches. Supported by: EHA research fellowship award, AIRC, FP7-NGS-PTL, Fondazione del Monte. Citation Format: Giorgia Simonetti, Antonella Padella, Eugenio Fonzi, Martina Pazzaglia, Margherita Perricone, Maria Chiara Fontana, Samantha Bruno, Maria Teresa Bochicchio, Eugenia Franchini, Jacopo Nanni, Giovanni Marconi, Italo F. do Valle, Rossella De Tommaso, Anna Ferrari, Enrica Imbrogno, Claudio Cerchione, Cristina Papayannidis, Emanuela Ottaviani, Daniel Remondini, Giovanni Martinelli. Metabolic profiling defines a new characterization of acute myeloid leukemia and identifies NPM1-mutated cases as a distinct subgroup [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5279.

Introduction. Intensive induction chemotherapy in non-M3 young Acute Myeloid Leukemia (AML) patients is represented by the association of an antracycline and Cytarabine. Some treatment regimens including fludarabine or the addition of Gemtuzumab Ozogamicin (GO) as a third or fourth drug, proved to give a benefit in terms of CR rates. Aims of the study. In a group of 49 patients treated with intensive chemotherapy, we evaluated chromosomal abnormalities with SNP 6.0 and Cytoscan HD (Affymetrix) in order to improve conventional cytogenetic analysis and discover novel chromosomic aberrations related to clinical data and therapy response. Patients and Methods. From 2001 to 2014, 489 patients were treated in our Institution. Among those, in 49 newly diagnosed AML patients (median age 54 (range 19-71)), SNP microarray based-genotyping were performed and then analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team. According to karyotype, FLT3 and NPM1 mutational status, 55.9% of the patients were considered at High Risk (HR) and 4.1% at low risk (LR). Ten patients had secondary AML. Patients were treated with induction schemes including MyFLAI, MyAIE, FLAI, FLAN, FLAG, 3+7 or DAE. Results. The CR rate after induction was 87.8% (43/49 patients). Deaths during induction (DDI), occurring in the first 50 days from 1st line therapy, were 1/49. The median OS was 135 months, the 5-years OS in our patients was 55,1%. Patients treated with GO showed a non-statistical trend toward a better OS than patients treated with other regimens (median OS not reached vs 133 months, respectively). We explored the alterations found by SNP array in our patients searching for novel markers of therapy resistance. We found a median of 192,5 total copy number aberrations (range 72- 1071): a median of 145,5 total copy number aberrations in responding patients group (RPG), and a median of 361 total copy number aberrations (p = ns) in non-responding patients group (NRPG). We compared the frequency of detected aberrations in RPG and in NRPG with Fisher's exact test. We found that PIK3CA, Gain chr3:178,927,088-178,929,550 (p = 0,0016), SMAD4, Gain chr18:48,573,154-48,573,255 (p = 0,0166) and several other gene's loci (CASC18, TCF12, UTY, GRB10, ZFY) are significant aberrations in NRPG compared with RPG. Conclusions. We identified a number of genes with significant aberrations in NRPG, particularly PIK3CA, a protein-coding gene involved in cell proliferation and metabolic pathway with interaction with HRAS/KRAS and EGF, and SMAD4, a transcription factor activated by TGF-beta. Those 2 genes were found overexpressed in other solid tumors. We suppose that those genes may be involved in a hyper-proliferative pathway that underlies a mechanism of chemo-resistance. Acknowledgments Work supported by ELN, AIL, AIRC, Progetto Regione-Universit⁁ 2010-12 (L.Bolondi), FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Maria Chiara Fontana, Giovanni Marconi, Viviana Guadagnuolo, Giorgia Simonetti, Antonella Padella, Simona Soverini, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Silvia Lo Monaco, Marco Manfrini, Elisa Zuffa, Eugenia Franchini, Claudia Venturi, Maria Teresa Bochicchio, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Giovanni Martinelli. Specific chromosomic alterations confer therapy resistance in a cohort of 49 patients with newly diagnosed acute myeloid leukemia treated with intensive chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 368.

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

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 Rorà, 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

Background & Aims - In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants, routinely assessed by Sanger sequencing (SS). We took advantage of ultra-deep sequencing (UDS) in order to: 1) resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs; 2) investigate their clonal structure and evolution in relation to time and treatment. Methods - We retrospectively performed a longitudinal analysis of 111 samples from 32 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS. UDS of the BCR-ABL KD was done using Roche 454 technology (lower detection limit, 0.1%). Seqnext software was used for alignment and variant identification; Jalview and Figtree softwares were used for haplotype and phylogenetic reconstruction. Results - UDS showed that SS often misclassifies or underestimates BCR-ABL KD mutation status. In more than half of the cases, UDS revealed that up to five ‘minor’ mutations with 1-20% abundance were present, either in samples scored as wild-type by SS or in samples already bearing one or more dominant mutations. The high degree of complexity was even more evident when the clonal relationships of multiple mutations were reconstructed and the relative abundance of all mutant subclones coexisting at each timepoint was assessed. This revealed that identical mutations may be acquired in parallel by independent subclones (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). Longitudinal quantitative follow-up showed that the landscape of all competing mutant subclones is highly dynamic, and that dominant subclones may be replaced as quickly as within one month in case of selective pressure change. Earlier identification of emerging TKI-resistant mutants was made possible by UDS. Conclusions - 1) sequential changes in the selective pressure exerted by TKIs may result in a heterogeneous mosaic of subclones harbouring different mutations or mutation combinations 2) The ‘ecosystem’ of mutant subclones is highly dynamic: acquisition of additional mutations dictates quick shrinkage or expansion of subclones over time 3) Inherent sensitivity to a specific TKI is necessary but not sufficient to determine the ‘fitness’ of a mutant subclone: competition with coexisting subclones also concurs to shape its fate 4) Reasoning on the basis of mutations detectable by SS may not always be sufficient to predict responsiveness to a TKI. Supported by Fondazione CARISBO, PRIN, IGA MZCR NT11555. Citation Format: Simona Soverini, Caterina De Benedittis, Katerina Machova Polakova, Adela Brouckova, Fausto Castagnetti, Cristina Papayannidis, Gabriele Gugliotta, Francesca Palandri, Hana Klamova, Ilaria Iacobucci, Claudia Venturi, Federica Cattina, Paola Bresciani, Valeria Coluccio, Marzia Salvucci, Mario Tiribelli, Gianni Binotto, Tamara Intermesoli, Mario Luppi, Maria Teresa Bochicchio, Emanuela Ottaviani, Domenico Russo, Rosti Gianantonio, Michele Baccarani, Giovanni Martinelli. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain in Philadelphia-positive leukemias. [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 4632. doi:10.1158/1538-7445.AM2013-4632