164 PROGNOSTIC IMPACT OF P53 PROTEIN EXPRESSION IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES: A SINGLE CENTRE EXPERIENCE

Poster Presentations – 13th International Symposium on Myelodyspastic Syndromes / Leukemia Research 39 S1 (2015) S1–S166

S83

162 THE PRESENCE OF SUBCLONES WITHIN AN ABNORMAL KARYOTYPE IS ASSOCIATED WITH AN INFERIOR PROGNOSIS IN MDS PATIENTS J. Neukirchen1, B. Hildebrandt2, A.C. Nolting1, R. Haas1, U. Germing1 1 Department of Hematology Oncology and Clinical Immunology, Heinrich-Heine-Universität, Düsseldorf, Germany; 2Department of Human Genetics, Heinrich-Heine-Universität, Düsseldorf, Germany

163 MUTATIONAL PROFILING IN CHRONIC MYELOMONOCYTIC LEUKEMIA COMPARED TO PRIMARY MYELOFIBROSIS F. PS Santos1, R. Puga1, R. Helman1, W. Pereira1, T. Datoguia1, B. Lisboa1, M. Miyaji1, E. da Mata1, S. Nakashima1, I. Bello1, M. Diniz1, G. Perini1, B. Gusmao1, E. Velloso1, N. Hamerschlak1, P. Campregher1 1 Hematology/Oncology, Hospital Israelita Albert Einstein, Sao Paulo, Brazil

Introduction: Several chromosomal abnormalities have been shown to be important for the assessment of prognosis in patients with MDS and AML. Thus, the IPSS-R includes the karyotype as an important factor for prognostication. In AML, it has recently been shown that the occurrence of subclones is also associated with a worse prognosis. Therefore, we were interested in the outcome of MDS patients with subclones and correlated the chromosomal findings with survival. Methods: We screened the Duesseldorf MDS registry for patients with cytogenetic alterations according to the International System for Human Cytogenetic Nomenclature (ISCN) and categorized the karyotypes according to the prognostic subgroups of the IPSS-R. We then analyzed the survival rates of the patients presenting with subclones and compared them with those patients with respective karyotypes according to the IPSS-R risk groups without subclones. Patients that received induction chemotherapy or allogeneic stem cell transplantation were excluded from the analysis. A p-value <0.05 was considered as statistically significant. Results: 125 out of 595 patients (370 male, 225 female patients) with an abnormal karyotype presented with subclones (20.8%). Especially in patients with an advanced MDS subclones were detectable (60.4% of patients with subclones were classified as IPSS-R high or very high, table 1). Looking at the survival, we found a significantly superior survival without subclones (62 vs. 26 months, p=0.003) categorized within the intermediate cytogenetic risk group according to the IPSS-R. In those patients with good risk karyotypes, the survival in the cohort without subclones was with 64 months in comparison to 40 months in the cohort with subclones also favorable, but not significantly different. Patients with a more unfavorable karyotype (poor risk) showed an even worse survival of 26 vs. 15 months. Of note, due to a small amount of patients (16 and 4, respectively), the results were not statistically significant, too. Conclusions: In MDS patients, clonal evolution with development of subclones within an already altered karyotype is associated with an inferior prognosis as seen in AML patients. The influence on survival was evident especially in the cohort with an intermediate risk group. Therefore, existing subclones can serve as an additional risk factor for disease progression and may deliver some more information according to prognosis and treatment decision making.

Introduction: Chronic myelomonocytic leukemia (CMML) and primary myelofibrosis (PMF) are chronic myeloid neoplasms that can present with similar clinical features, including the presence of cytopenias, splenomegaly, leukocytosis with monocytosis, and bone marrow fibrosis. We hypothesized that molecular features could help to better differentiate these two entities. Objective: To compare the mutational profiling and activated pathways in patients with CMML and PMF Patients and methods: We selected 9 patients with CMML of the proliferative subtype and 45 patients with PMF. Paired DNA (sorted CD66b-granulocytes/skin biopsy) was subjected to whole exome sequencing on an Illumina HiSeq 2000 platform using Agilent SureSelect kit. Tumor coverage was 150x and germline coverage was 60x. Somatic variants calls were generated by combining the output of Somatic Sniper, Mutect and Pindel. The combined output of these 3 tools was further filtered by in-house criteria in order to reduce false-positive calls. All known oncogenic driver mutations were validated through Sanger sequencing. Chi-square/ Fischer exact test and Wilcoxon ranksum test were used for comparison of categorical and continuous variables, respectively. Q-values were calculated by the Benjamini-Hochberg method. All statistical calculations were done in Stata, v11.0. Results: We identified a total of 2,429 mutations in all patients. Most mutations (94.9%) were single nucleotide variants (SNVs) while the remaining were insertions and deletions (5.1%). The median mutational rate in CMML was 0.46 mutations/Mb (95% CI 0.280.61), and there was no difference compared to the mutational rate observed in PMF (0.36 mutations/Mb; 95% CI 0.32-0.57; p=0.58). After adjusting for multiple comparisons, the following molecular abnormalities had a differential frequency among patients with CMML compared to patients with MF: IDH1 mutations (11% vs. 0%; q-value=0.08), RUNX1 mutations (44% vs. 0%, q-value=0.004) and mutations activating the JAK-STAT pathway (22% vs. 93%; q-value=0.004). There was a trend for patients harboring both TET2 and SRSF2 mutations to have a diagnosis of CMML (33% vs. 4%, p=0.006), but this did not reach statistical significance after correcting for multiple comparisons (q-value=0.24). Conclusion: The presence of mutations of the myeloid transcription factor RUNX1 and absence of mutations activating the JAKSTAT pathway are the best molecular predictors of the diagnosis of CMML compared to a diagnosis of PMF. The combined presence of both TET2 and SRSF2 mutations also tend to be more frequent in patients with CMML. Sequencing a larger number of patients will help to determine the best molecular predictors of the diagnosis of these two entities.

Table 1. Distribution of patients with subclones according to the IPSS-R IPSS-R very low

n=106 0

low

22 (20.7%)

intermediate

20 (18.9%)

high

27 (25.5%)

very high

37 (34.9%)

164 PROGNOSTIC IMPACT OF P53 PROTEIN EXPRESSION IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES: A SINGLE CENTRE EXPERIENCE A. Ricco1, A. Napoli2, P. Carluccio1, A.V. Russo Rossi1, B. Daraia1, E. Maiorano2, G. Specchia1 1 Emergency and transplantation, Hematology, Bari, Italy; 2 Emergency and transplantation, Pathology section, Bari, Italy Introduction: AML following MDS has a very poor outcome, so accurate prediction is essential, especially in patients who

S84

Poster Presentations – 13th International Symposium on Myelodyspastic Syndromes / Leukemia Research 39 S1 (2015) S1–S166

could potentially be cured by stem cell transplantation. A strong association between p53 protein expression, the TP53 mutation and an adverse outcome has been reported in various hematologic malignancies including MDS. Strong nuclear staining of the p53 protein by immunohistochemistry (IHC) has been used as a surrogate marker for TP53 gene mutations in hematologic and other malignancies; a strong correlation of p53 nuclear expression with TP53 mutation has also been demonstrated. Patients and Methods: To assess the role of p53 expression in MDS patients as a new prognostic tool, as well as its feasibility in routine clinical practice, we analyzed the prevalence of p53 expression in a cohort of 40 consecutive bone marrow (BM) biopsies from patients with MDS at diagnosis, and correlated our findings to other validated prognostic markers and clinical outcome. The DO-7 antibody (DakoCytomation, Denmark), which labels both wild and mutant-type p53 proteins, was used to detect p53 expression (a colorectal carcinoma positive control was included on each slide). Diagnosis according to the 2008 WHO Classification was: isolated del (5q) in 2 cases, RA in 7, RARS in 4, RCMD in 7, RAEB-1 in 12, RAEB-2 in 8. Results: Among patients with strong p53 immunostaining in ≥1% of BM cells, we found significant correlations with BM blasts (75% RAEB-1/2, vs 30% in negative cases). IPSS-R cytogenetic risk was int/poor/very poor in 33% of positive and 0% in negative IHC cases. IPSS score was int-2/high in 50% of patients with strong positivity vs 20% in negative cases. BM fibrosis was present in 70% of positive vs. 30% of negative IHC cases. Transfusion dependency was observed in 50% of patients with p53 strong expression vs. 20% of negative cases. Moreover, we found that strong p53nuclear expression was associated with a significantly worse outcome (87% disease progression plus leukemic evolution vs. 20% in negative cases) and shorter median OS (18 months vs. 30 in negative cases). Conclusions: Our data indicate that IHC p53 protein expression, evaluated in bone marrow biopsies by a widely available method, is a highly predictive marker and thus a helpful tool in risk assessment and the decision making process in MDS.

165 RECURRENT MUTATIONS AS WELL AS CLONAL EVOLUTION ARE COMMON IN PATIENTS WITH LOWER-RISK MDS AND DEL(5Q) TREATED WITH LENALIDOMIDE C. Scharenberg1, M. Karimi1, M. Jansson1, G. Walldin1, P. Woll2, S.E. Jacobsen2, E. Hellström-Lindberg1 1 Hematology and Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden; 2Weatherall Institute for Molecular Medicine, University of Oxford, Oxford, United Kingdom Introduction: Deletion of chromosome 5q in lower-risk MDS is associated with distinct clinical characteristics and sensitivity to the immunomodulatory drug lenalidomide (LEN). Recurrent somatic mutations play a role in the clinical course and prognosis of MDS, however, whether or not these mutations are affected by standard treatment regimens remains currently unknown. Aims: We sought to determine how lenalidomide and EPO treatment are associated with development of the mutational landscape in del(5q). Methods: 26 patients with del(5q) MDS untreated, or treated with EPO or lenalidomide underwent targeted sequencing for 72 genes implicated in myeloid neoplasms using the Haloplex platform. In 12 patients, material was available from more than a single timepoint, which allowed us to study the co-variation between clonal evolution and treatment. Results: The most frequent mutations were found in TP53 (n=6) and DNMT3A (n=6), ASXL1 (n=4), and TET2 (n=3). 8 of 26 patients analyzed at diagnosis or before any treatment had no mutations,

hence a majority of patients actually had recurrent mutations early in the course of their disease. Of the patients that showed mutations at some point during their clinical course, 4 patients had only one mutation and 14 had ≥2 mutations. Clonal evolution was associated with stable disease (n=1), loss of response (n=1) and disease progression to higher-risk MDS or AML (n=2). The 3 patients who progressed to overt AML showed acquisition of TP53 (n=2), DNMT3A and TET2 (n=1). Three patients analyzed at >1 time point showed no mutations and no clonal evolution after 38 months of EPO treatment and 26 and 12 months of LEN, respectively. Of 5 patients without identifiable mutation at the earliest timepoint, 4 were treated with LEN and 3 of 4 showed clonal evolution (40 ±8 months after the first time point; 1 with stable clinical disease acquired mutations in CSF3R, CUX1, and SH2B3, and two exhibited both loss of response and leukemic progression. In 3 patients, leukemic progression was preceded by the acquisition of new mutations: TP53, TP53/TET2, and DNMT3A. In one patient, ASXL1 and IDH2 mutations present at diagnosis completely disappeared during lenalidomide-treatment, while mutations in TET2 and TP53 appeared prior to leukemic progression. Conclusions: Recurrent mutations are common in del(5q) lowerrisk MDS, in particular in patients who are candidates for treatment, and clonal evolution is common during LEN treatment. Our study suggests that lenalidomide differentially affects different clones and that the mutational landscape is clinically relevant.

166 TARGETED SEQUENCING ANALYSIS OF COMMONLY MUTATED GENES IN CHRONIC MYELOMONOCYTIC LEUKEMIA USING NGS: IMPACT AND CLINICAL IMPLICATIONS A. Valencia1, O. Abdel-Wahab2, F. Buchi1, E. Masala1, A. Sanna1, A. Gozzini1, M.E. Figueroa3, E. Contini4, F. Torricelli4, B. Allione5, M. Lunghi6, F. Onida7, A. Polloni8, E. Angelucci9, F. Finelli10, A. Levis11, D. Gioia11, A. Bosi1, V. Santini1 1 Medicina Sperimentale e Clinica, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy; 2Human Oncology and Pathogenesis Program and Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York., USA; 3Department of Pathology, University of Michigan, Michigan, USA; 4SOD Diagnostica Genetica, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy; 5Hematology, Ospedale S. Giovanni Battista, Turin, Italy; 6Hematology, Amedeo Avogadro University, Novara, Italy; 7Hematology, Ospedale Maggiore Policlinico, Milan, Italy; 8Hematology, Università Politecnica delle Marche, Ancona, Italy; 9Hematology and Bone Marrow Transplantation Unit, Ospedale Oncologico di riferimento regionale “Armando Businco”, Cagliari, Italy; 10Hematology, S.Orsola-Malpighi University Hospital, Bolonia, Italy; 11FISM Onlus, FISM Onlus, Alexandria, Italy Chronic myelomonocytic leukemia (CMML) is an overlap myelodysplastic/myeloproliferative neoplasm characterized by a highly variable clinical course and prognostic factors include WBC, splenomegaly, cytopenias, and cytogenetics. Recently, mutations have been identified and preliminary evidence suggests that selected mutated genes may influence prognosis (i.e. ASXL1, SRSF2). The aim of this study is to evaluate the frequency and confirm prognostic value of mutations of different genes in CMML. Fifty-three CMML patients diagnosed according to WHO classification were included. The mutational profile of ASXL1, DNMT3A, EZH2, RUNX1, SF3B1, SRSF2, U2FA1, TET2 and TP53 was characterized using a TruSeq Custom Amplicon panel (Illumina) or a custom design HaloPlex Target Enrichment kit. Around 250 ng of gDNA obtained at diagnosis were used to prepare sequencing libraries. Samples were run on an Illumina MiSeq and data