C071
CORRELATIONS BETWEEN MUTATIONAL AND IMMUNOPHENOTYPIC PROFILES IN THE DIAGNOSIS OF MDS
E. Fabiani, L. Maurillo, C. Gurnari, C. Attrotto, G. Falconi, R. Palmieri, E. Buzzatti, F. Esposito, E. De Bellis, F. Buccisano, A. Venditti, W. Arcese, F. Lo Coco, M.T. Voso
Dipartimento di Biomedicina e Prevenzione, Università di Tor Vergata, Italy Introduction: The diagnosis of myelodysplastic syndromes (MDS) is
based on the presence of peripheral blood (PB) cytopenia, bone marrow (BM) dysplasia and of karyotype abnormalities. Flow-cytometry (FC) and FISH are recommended in doubtful cases. The mutational profile by NGS may add information in cases with inconclusive results by stan- dard analyses.
Aims: The aim of our work was to evaluate the usefulness of adding
NGS in the diagnostic algorithm of patients with a suspected MDS, in- cluding evaluation of the Ogata score and expression of LIM (lineage infidelity markers, including CD7, CD56, CD2, CD5 and CD25) on CD34+ cells.
Patients and methods: At the time of diagnosis, we studied BM samples
harvested from 46 patients with MDS and 17 healthy controls using the Ogata cytofluorimetric score, and LIM expression. A subgroup of 24 MDS patients was also studied by NGS using a custom panel of 94 genes known to be involved in myeloid neoplasms.
Results: The diagnosis of MDS was confirmed by an Ogata score ≥2 in
33/46 patients (72%), whereas LIM analysis identified 40/46 patients pos- itive for at least one infidelity marker (87%), with CD7 as the most ex- pressed (41%). Thus, the addition of LIM analysis to Ogata score supported the diagnosis of MDS in 8 of 13 patients with an Ogata score <2, increasing the sensitivity to 89%. NGS screening performed in 24 pa- tients identified at least one mutation in 23 out of 24 samples (96%), with a median number of 3.7 mutation/patient. The most frequently mutated genes were: DNMT3A, SF3B1, SLX4 and SRSF2 (5/24, 20,8% of patients). We found a statistically significant correlation between a high Ogata score (≥2) and >4 mutations/patient (p=0.003). In addition, all 6 patients with an FC score equal to 0 were found to carry at least one somatic mu- tation in critical genes. Looking at associations between the mutational status and IPSS-R (very high/high/intermediate vs low/very low), we found a correlation between the number of mutations in three of the major pathways involved in MDS pathogenesis (DNA methylation, his- tone modification and splicing machinery, figure 1) and IPSS-R grouping (median 2.5 mut in IPSS-R high, vs 1.1 mut in IPSS-R low, p=0.0073). Of note, SRSF2 mutations were significantly more frequent in patients with an Ogata score ≥2 (5/13 vs 0/11, p=0.0411).
Conclusions: NGS combined with LIM analysis and with the Ogata
score may improve diagnostic sensitivity in MDS.
Figure 1.
C072
IDENTIFICATION OF A NOVEL MUTATION PREDISPOSING TO FAMILIAL AML AND MDS SYNDROME BY A NGS APPROACH
S. Bernardi1, C. Zanaglio1, E. Dereli Eke1, F. Cattina2, C. Foroni1,
M. Farina2, S. Masneri3, B. Rambaldi2, A. Turra2, N. Polverelli2,
E. Morello2, L. Gandolfi2, T. Zollner2, E. Accorsi Buttini2,
M. Malagola2, D. Russo2
1Unit of Blood Diseases and Stem Cells Transplantation- Laboratorio CREA
Centro di Ricerca Emato-oncologica AIL- Department of Clinical and Experi- mental Sciences- University of Brescia- ASST Spedali Civili di Brescia; 2Unit of
Blood Diseases and Stem Cells Transplantation-Department of Clinical and Ex- perimental Sciences- University of Brescia- ASST Spedali Civili di Brescia; 3Uni-
versity of Brescia-Department of Molecular and Translational Medicine, Brescia, Italy
Introduction: Recently, revised WHO classification (2016) has recog-
nized familial AML and MDS (FAMS) as a useful model to investigate predisposing genetic mutations. A pivotal importance is given to the identification of patients with germline mutations in genes involved in the familial predisposition to neoplasia. Next Generation Sequencing panels are a well known tool to investigate these cases suggesting they are more frequent than those previously recognized. Even though well- established causative gene mutations are recently discovered, about 80% cases remain unexplained. This suggest that other inherited mu- tations could predispose to MDS/AML. New deep sequencing ap- proaches would help to the identification of more cases, genes, as well as novel syndromes. Our aim is to look for predisposing mutations in patients and relatives affected by AML and MDS with familial history of myeloid malignancies.
Methods: At present, 12 AML/MDS patients have been enrolled in a
multicentric prospective study (Clinical trial.gov NCT03058588). Leukemic (bone marrow) and germline (buccal swab) DNA were ana- lyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 genes associated to FAMS (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA). NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was per- formed by MiSeq (Illumina).
Results: Ten patients did not reveal any germline mutations, one pre-
sented a germline mutation on RUNX1 and one revealed a new muta- tion on ETV6. Particularly, this mutation c.*514C>T in 3’UTR of ETV6, with VAF of 50% on tumor DNA, has never been described before. ETV6 variant was confirmed by Sanger sequencing on the germline DNA in heterozygosis. The same variant in heterozygosis was also con- firmed on 2 affected relatives still alive. In silico analysis, performed on PolymiRST Database, revealed that c.*514C>T in 3’UTR of ETV6 re- sults in a gain of miRNA binding site (hsa-miR-4717-3p and hsa-miR- 942-3p) that seems to repress the gene.
Conclusions: The down-regulation of ETV6 is associate to an alteration
of cell growth and hematopoiesis. This specific miRNA interference have been already described in solid and hematologic tumors. Due to these evidences, NGS approach can help to the identification of new mutations involved in FAMS predisposition.
Figure 1. Case index pedigree (black arrow indicates the case index; MDS=Myelodisplasticsyndrome; AML=Acute myeloid leukemia).
C073
DANAZOL: AN ‘’OLD-FASHIONED’’ DRUG FOR A CURRENT PROBLEM IN LOWER-RISK MYELODYSPLASTIC SYNDROMES
M. Riva1, L. Crucitti1, E. Ravano1, M. Nichelatti1, G. Reda2, R. Cassin2,
C. Frangi1, R. Cairoli1, A. Molteni3
1ASST Grande Ospedale Metropolitano Niguarda; 2Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico; 3ASST Cremona, Italy
Severe thrombocytopenia in lower-risk MDS is an uncommon event, but very significant about prognosis. No specific therapy in this setting are currently available: hypometilating agents are not licensed in Eu- rope, and a trial testing Eltrombopag is ongoing (Oliva 2017). Few data were reported about danazol, an attenuated androgen, that seems to have some effectiveness in this still unmet need. We retrospectively re- viewed 35 thrombocytopenic pts (30 MDS and 5 MDS/MPN) treated with danazol. The initial and maximal dose was 600 mg/day, modu- lated by response and toxicity. The response was evaluated according to the revision of IWG criteria (Platzbecker 2018). Two pts treated for less than 3 months (mo) were not included in the analysis. At baseline, the platelet (PLT) count was lower than 20x103/mL in 11 pts, the me-
dian was 23x103/mL. The response rate was 63,6% (21 pts on 33 evalu-
able). Median time to response was 3.5mo (range 0.3–12.4); the average response time was 5.09mo. In the first year of treatment, the PLT count changed in a significant way (F test after repeated measures ANOVA: p<0.001) Figure 1. Pairwise comparisons of PLT count according to Bon- ferroni showed a significant difference for: baseline (b.) vs 3mo (p=0.0013), b. vs 6mo (p=0.0255), b. vs 9mo (p=0.0047) and b. vs 12mo (p=0.0014). The median and average duration of the response were re- spectively 12,5 and 32,5mo. Within the 21 responders, only 6 (28%) lost the response (the median and average duration of response were respectively 5.8 and 12.9mo); the median PFS was not reached after 24mo. The probability to maintain the response after 50mo was as- sessed at 58.2% (C.I. 24.1% to 81.4%). The OS showed a significant difference (logrank test: p=0.0064) between responders and non-respon- ders. Adverse events recorded were as follows: 2 cases of Grade3 (1 liver toxicity and 1 renal failure) with need to stop drug; 10 cases of Grade1-2 (increase in transaminases in 4 pts and in serum creatinine in 6 pts) with need to reduce danazol to 400 mg/day; 3 cases of reversible cutaneous rash; 1 case of amenorrhea (the only fertile woman in the series); 1 case of weight loss and 1 of weight gain. Even if the mecha- nism of action is unclear, this series confirms the efficacy of danazol and safety to improve PLT count in MDS pts with severe thrombocy- topenia. The response may not be immediate, but reachable after 3-6 mo of treatment. A responsive patient has a good probability to main- tain a long-lasting response.
Figure 1.
C074
INTRACELLULAR RE-LOCALIZATION OF NUCLEOSIDE METABOLIZING ENZYMES CORRE- LATES WITH SENSITIVITY TO AZACITIDINE.
E. Masala,1A. Brogi,1D. Nosi,2V. Santini1
1MDS Unit, Department of Experimental and Clinical Medicine, University of
Florence; 2Department of Experimental and Clinical Medicine, Section of
Anatomy and Histology, University of Florence, Italy
We evaluated the role of nucleoside metabolizing enzymes in mod- ulation of cellular response to azacitidine. Azacitidine sensitive SKM1 (SKM1-S) and resistant (SKM1-R) cell lines were analyzed for expres- sion of UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 by quantita- tive PCR, in parallel with 51 cases of IPSS high risk MDS. We evaluated by NGS the most frequently mutated genes in MDS in 42 different cases of HR-MDS patients before azacitidine treatment. UCK1 and UCK2 expression was blunted by siRNAs in SKM1 sensitive cells to de- termine their role in in vitro sensitivity to azacitidine. UCK1 and UCK2 silencing was obtained with specific siRNAs (OriGeneTechnologies, MD, USA); cells were exposed to azacitidine 0,1 and 1 M. After assess- ment of effective gene silencing, apoptosis was evaluated by Annexin V, and percentage of 5-methylcytosine evaluated by ELISA assay (Global DNA Methylation LINE-1 kit ActiveMotif,CA,USA). UCK1 and UCK2 protein expression and localization was analyzed by confocal laser scanning microscope using antiUCK1(1:200;ThermoFisherScien- tific,USA) and anti-UCK2 (1:200;ThermoFisher Scientific,USA) antibod- ies. Labeled cells were observed under a Bio-RadMRC1024 ES confocal laser scanning microscope (CLSM;Bio-Rad). SKM1-R cells did not ex- press UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2. A reduction of apoptosis was observed in UCK1 and UCK2-silenced SKM-1 S after azacitidine treatment. Azacitidine induced DNA hypomethylation was reduced by defective expression of UCK1 and UCK2. The confocal laser analysis confirmed that SKM-1 R express very low levels of UCK-1 and UCK-2 proteins. UCK1 and UCK2 localization was observed in the cy- toplasm of both cell lines at basal conditions. Exclusively in SKM-1 Sazacitidine in vitro exposure could re-localize UCK1 and UCK2 pro- teins in the nucleus. Gene expression of all metabolizing enzymes in MDS primary cells was not significantly correlated with clinical re- sponse to azacitidine and cellular localization of UCK1 and UCK2 pro- teins is under evaluation. SFR3B1, TET2 TP53 and ASXL1 resulted as most frequently mutated genes in our cohort; mutations do not corre- late with response. UCK1, UCK2 and the corresponding proteins are absent in azacitidine-resistant cell line SKM1-R. Their silencing induced significantly decreased azacitidine effects. SKM-1 S cells, when exposed to the drug, re-localized UCK1 and UCK2 into the nucleus. This event seems to be directly correlated with cellular response to the drug.
C075
CLINICAL FEATURES AND PROGNOSTIC SIGNIFICANCE OF RECIPROCAL BALANCED TRANSLOCATION IN A COHORT OF 79 PATIENTS WITH MYELODYSPLASTIC SYNDROMES
S. Mohamed, S. Nigro, A. Campagna, A.L. Piccioni, I. Carmosino, S. Rosati, M. Ansuinelli, E. Scalzulli, M. Porrazzo, M. Breccia, A. Aloe Spiriti, R. Latagliata, R. Foà, M. Mancini
Dipartimento di Medicina Traslazionale e di Precisione sezione di Ematologia Uni- versità “Sapienza”; Unità di Ematologia Azienda Ospedaliera Sant’Andrea Uni- versità “Sapienza”; Unità di Ematologia Azienda Ospedaliera Sandro Pertini
Chromosomal abnormalities are very common in de novo myelodys- plastic syndromes (MDS) and are represented by losses or gains of ge- netic material. On the contrary, reciprocal balanced translocations (RBT) are rare and their prognostic value is uncertain. In order to highlight clinical features and survival of MDS patients with RBT, we retrospec- tively analyzed a cohort of 79 patients diagnosed between 7/1987 and 2/2018, enlisted in the GROM-L registry, characterized by having a RBT at the cytogenetic analysis. Complete peripheral blood and bone mar- row features were recorded for all eligible patients, with follow-up data and date of leukemic evolution. IPSS and IPSS-R were retrospectively assigned. Cytogenetic analysis was performed at local specialized lab- oratories in each participating centers. Based on karyotype, the entire cohort was divided into three cytogenetic subgroups: 24 patients with an isolated RBT (30%), 23 patients with a RBT associated to another chromosomal abnormality (29%) and 32 patients with a RBT in the
context of a complex karyotype (41%). The clinical features of the en- tire cohort as well as of these cytogenetic subgroups are reported in Table 1. The most frequently involved chromosomes were: chromo- some 1 in 18 patients (23%), chromosome 3 in 22 patients (28%), chro- mosome 5 in 32 patients (41%), chromosome 7 in 29 patients (37%), chromosome 11 in 7 patients (9%), chromosome 12 in 13 patients (16%) and chromosome 17 in 5 patients (6%). During the follow-up, 23 patients (29%) evolved into acute myeloid leukemia (AML), with a median time to evolution of 9.3 months (range 4.2-23.8), without dif- ferences according to the different cytogenetic subgroups (p=0.137). The 5-year overall survival (OS) of the entire cohort was 36.9% (CI 95% 24.9-48.9). According to the cytogenetic subgroups, the 5-year OS was 50% (CI 95% 27.7-72.3) in patients with an isolated RBT, 52.6% (CI 95% 29.6-75.6) in patients with a double abnormality including a RBT and 12.9% (CI 95% 0-28.5) in patients with a complex karyotype including a RBT (p<0.001). According to our data, RBT’s presence in MDS characterizes patients with a significantly more aggressive clinical course and worse prognosis only when associated with a complex kary- otypes. MDS patients with isolated RBT or with RBT associated with a single additional abnormality show a more indolent course, with a median OS exceeding 50 months, thus falling into the intermediate-1 risk group of IPSS score classification.
Table 1. Patient’s clinical features.
Allogeneic and Autologous Transplantation 2
C076
INTERLEUKIN-6 AS EARLY BIOMARKER FOR ACUTE GVHD AND SURVIVAL AFTER ALLO- GENEIC TRANSPLANT WITH POST-TRANSPLANT CYCLOPHOSPHAMIDE
R. Greco, F. Lorentino, R. Nitti, M.T. Lupo Stanghellini, F. Giglio, D. Clerici, E. Xue, L. Lazzari, S. Piemontese, S. Mastaglio, A. Assanelli, S. Marktel, C. Corti, M. Bernardi, F. Ciceri, J. Peccatori
Haematology and Bone Marrow Transplant Unit, IRCCS San Raffaele Scien- tific Institute, Milano, Italy
Although the outcome of allogeneic hematopoietic stem cell trans- plantation (allo-HSCT) has dramatically improved in the past decade, it is still compromised by transplant-related mortality (TRM), mainly caused by Graft-versus-Host Disease (GvHD). We conducted a prospec- tive observational study to ascertain the potential of serum interleukin- 6 (IL6) levels, measured before conditioning and 7 days after allo-HSCT, in predicting acute GvHD, TRM and survival after allo-HSCT with Post-Transplant Cyclophosphamide (PT-Cy), sirolimus and MMF as GvHD prophylaxis. We collected samples from 166 consecutive allo- HSCT patients. Most patients were affected by myeloid malignancies. Most patients (91%) received unmanipulated PBSC and myeloablative conditioning regimen (81%). Stem cell donors were unrelated (n=41), family haploidentical (n=89), HLA-identical sibling (n=36). Median fol- low-up on survivors was 469 days (range 69-1269). By ROC analysis, we identified a threshold of 2.5 pg/ml for pre-transplant IL6 and 16.5 pg/ml for post-transplant IL6. Rates of grades II-IV and III-IV acute GvHD were higher in patients with post-transplant IL6 levels higher than 16.5 pg/ml (47% vs 14 %, p<0.001; 32% vs 3%, p<0.001, respec- tively). Instead, baseline IL6 levels higher than 2.5 pg/ml were associ- ated with grade II-IV acute GvHD (36% vs 26 %, p 0.03). We found a trend towards a worse TRM in patients presenting high post-transplant IL6 (36% vs 23%; p 0.06). Survival analysis confirmed a significantly decreased 2-year overall survival (OS) in patients with baseline IL6 lev- els higher than 2.5 pg/ml (38% vs 79%; p<0.001) and/or post-transplant IL6 concentrations higher than 16.5 pg/ml (47% vs 83%; p<0.001). Both univariate and multivariate analyses confirmed the ability of baseline IL6 levels to predict OS (HR 4.3; p<0.01) and grade II-IV acute GvHD (HR 1.8; p 0.04), and of post-transplant IL6 to identify patients with worse OS (HR 3.3; p<0.01) and higher risk of grade II-IV (HR 5; p<0.01) and grade III-IV acute GvHD (HR 10.2; p<0.01). In multivariate analysis, both baseline (HR 6.7; p<0.01) and post-transplant IL6 (HR 3.5; p 0.02) predicted higher TRM. In this prospective observational study, measurement of plasma IL6 resulted a valuable biomarker in pre- dicting the risk of acute GvHD and TRM, providing a window for ad- ditional prophylactic or preemptive strategies, and potentially improving the final outcome of allo-HSCT.
C077
IMMUNE RECONSTITUTION - BASED SCORE AT DIAGNOSIS OF CGVHD PREDICTS GVHD SEVERITY AND OVERALL-SURVIVAL: A NOVEL PROGNOSTICATION TOOL FOR GVHD TREATMENT TAILORING
M.T. Lupo-Stanghellini1, F. Serpenti1, R. Milani2, C. Messina1,
R. Greco1, S. Girlanda1, D. Clerici1, F. Giglio1, C. Liberatore1, G. Casir-
ati1, F. Farina1, S. Mastaglio1, E. Guggiari1, F. Lunghi1, S. Marktel1,
M.G. Carrabba1, M. Bernardi1, C. Bonini3,4, A. Assanelli1, J. Peccatori1,
C. Corti1, F. Ciceri1,3, F. Lorentino1
1Hematology and Bone Marrow Transplantation Unit; 2Immunohematology and
Transfusion Medicine Unit; 3University Vita-Salute San Raffaele; 4Experimental
Hematology Unit, San Raffaele Scientific Institute
Introduction: Allogeneic stem cell transplantation survivors are at a rel-
evant risk of developing chronic GvHD (cGvHD), which importantly affects quality of life and increases morbidity and mortality. We have previously demonstrated the role of immune reconstitution (IR) as a predictive biomarker of occurrence of cGvHD. The aim of this study was to evaluate the prognostication power of IR at cGvHD onset.
Methods: We analyzed data from 424 adult pts consecutively under-
going 1st allogeneic transplant between Jan-2011 and Dec-2016 at our Institution: 151 pts developed cGvHD (median follow-up 4 y). Pts were
divided into a test cohort (111 pts) and a validation cohort (40 pts). We built a Cox multivariate models for OS (overall survival) in pts with cGvHD of any severity. Variables included in the models were: pts age, R-DRI score, type of donor, GvHD prophylaxis, IR values (CD4, CD19, NK, IgA, IgM) at cGvHD diagnosis, history of prior aGvHD, Karnofsky PS, Plt <100.000/ L, ALC<1000/ L, Eos <500/ L. Once we identified the variables independently predicting OS by multivariate analysis, we elaborated a formula for a prognostic risk index by using the coefficients derived from the model. Each pt was then assigned a score and we de- fined 3 groups of OS risk (low, intermediate and high) by dividing the score into 3 classes using the first and third quartiles. Finally, to evaluate predictive performance of the IR-score, we calculated the receiver op- erating characteristics (ROC) curve via the area under the curve (AUC), to summarize the IR-score ability to correctly classify events and non- events.
Results: Our multivariate model defined the variables independently
predicting OS at cGvHD onset: CD4+ >233/ l, NK <115/ l, IgM <0.45g/L, Karnofsky PS <80%. Final score was calculated as follows: 2,4 (if CD4 >233/ L) + 2,1 (if NK <115/ L) + 2,1 (if IgM <0,45g/L) + 4,3 (if Karnofsky <80). Low risk pts were defined as having a score ≤2.4, intermediate risk >2,4 and ≤4.5, high risk >4.5. The 3y-OS and Trans- plant Related Mortality (TRM) for both cohorts are reported in Table1. The ROC curve analysis supports the validity of the IR-score: OS AUC 85.5% - TRM 76,4% with 95% confidence intervals higher than 50%. Furthermore IR-score was able to stratify across NIH-severity classifi- cation.
Conclusions: IR-score at diagnosis of cGvHD predicts GvHD severity
and overall-survival. IR-score could be adopted to identify patients at high risk and modulate cGvHD treatments accordingly.
Table 1. 3-years Overall Survival and Transplant Related Mortality according to IR-Score stratification.
C078
UNMANIPULATED HAPLOIDENTICAL MARROW TRANSPLANTATION WITH A MODIFIED POST-TRANSPLANT CYCLOPHOSPHAMIDE (PT-CY) REGIMEN: AN UPDATE OF THE GEN- OVA-ROME GEMELLI EXPERIENCE
S. Giammarco1, A. Bacigalupo1, A.M. Raiola2, A. Dominietto2,
C. Di Grazia2, S. Bregante2, M.T. Van Lint2, P. Chiusolo1, L. Laurenti1,
F. Sorà1, S. Sica1, E. Angelucci2
1Università Cattolica del Sacro Cuore; Fondazione Policlinico Universitario
A.Gemelli-IRCCS, Istituto id Ematologia, Roma; 2IRCCS AOU San Martino
IST, Istituto di Ematologia, Genova, Italy
The aim of the study is to update the outcome of our HAPLO pro- gram on 444 patients grafted between 2011 and 2017, in two transplant Units (Genova and Rome). Pts were selected for HAPLO grafts in the absence of a suitable HLA matched related or unrelated donor. Pts me- dian age was 52 years (14-74), and 106 pts were over 60 years of age. Remission status was as follows: CR1 (n=171), CR2 (n=112) and ad- vanced disease (n=161). Median donor age was 34 years (18-67). The diagnosis was AML (n=154), ALL (n=87), MDS (n=83) , myelofibrosis (n=47), non Hodgkin lymphoma (n=31) other (n=44).We used 2 mye- loablative conditioning regimens, one chemotherapy based (n=346) in- cluding Thiotepa, Busulfan, Fudarabine (TBF) and one radiation based (n=99) with full dose radiation (999-1200 rads) (TBI) and fludarabine. The TBF regimen was used with full dose Busulfan 3.2 mg/kgx3, or 3.2 mg/kg x2 , for patients over 60 years of age. Median age for the TBF regimen was 55 years (18-74), whereas for the TBI it was 35 years (14- 64). GvHD prophylaxis was CsA 2 mg/kg i.,v. starting day 0,MMF 2 gr/day p.o, starting day+1 to day+30, PT-CY 50 mg/kg day +3 and day+5. When possible CsA was tapered starting day +100 and discon- tinued day +180. All patients received unmanipulated marrow as a stem