ALBERT

Description: ATP binding cassette (ABC) transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes and confer drug resistance against a wide range of chemotherapeutic agents. We recently found that WT1, which is regularly overexpressed in AML and interact with the splicing machinery, modifies the splicing of ABC transporters A2, A3, A5, and C2. For ABCA3, WT1 knock-down in three AML cell line coupled with Affymetrix HTA2 exon arrays analysis confirmed by exon-specific PCR revealed that WT1 influences the skipping of exon 19. ABCA3 belongs in the ABC subclass and induces a significant reduction in cytotoxicity observed following exposure to DNR, mitoxantrone, etoposide, Ara-C and vincristine. The ABCA3 domain encoded by exon 19 (amino acid 805-847) is localized at the junction of the first nucleotide-binding domain and the second transmembrane domain, and is involved in ATP hydrolysis. In silico, skipping of exon 19 deletes a sequence of 32 amino acids rich in positively charged residues and is thereby assumed to increase drug efflux through increased ATP hydrolysis. The effects of the skipping of exon 19 on chemoresistance and DNR efflux are currently investigated while for the present study, we hypothesized that skipping of exon 19 of ABCA3 might negatively influence outcome in AML patients. Analyzing 132 bone marrow AML samples harvested at diagnostic confirmed the statistically significant correlation between WT1 expression and ABCA3 splicing in vivo (p

Description: Introduction: in large unselected series, the median age of Primary CNS Lymphoma (PCNSL) patients (pts) is about 70 years. In one USA cancer registry study for PCNSL patients (pts) older than 65 years, 14% of them are older than 80 years. Data on clinical characteristics, therapeutical management, toxicity of treatment and outcome of these very elderly pts are limited. Methods: We reviewed PCNSL pts aged of 80 years or older included in the database of the French Oculo-Cerebral lymphoma (LOC) network. From January 2011, this network prospectively recorded all newly diagnosed PCNSL from 22 regional expert centers in France. For this study, 110 PCNSL pts with a histological diagnosis of diffuse large B-cell lymphoma (DLBCL) aged of 80 years or older were analyzed. All medical records were reviewed for clinical and biological characteristics, modality of treatment and supportive care, toxicities and outcome. Results: 110 pts with a DLBCL PCNSL aged of 80 years or older were diagnosed between January 2011 and January 2018 representing 8% of pts available in the LOC database. The clinical characteristics were as follows: 63% of females; median age: 83y (80-92); performance status (PS) according to EORTC scale: 1, 24% of pts, 2, 21% of pts, 3-4, 55% of pts. 23.6% of pts had a CIRS-G grade 3 or 4 in at least one category and 40.9% had a cumulative CIRS-G score more than 6. Diagnosis procedure was biopsy (87%), tumor resection (5%), vitrectomy (4%), CSF cytology (4%). At diagnosis, 9/68 (13%) of evaluable pts had ocular involvement, 13/61 (21%) cerebrospinal (CSF) involvement, 79/110 (72%) involvement of the deep structures of the brain and 35/86 (41%) had elevated LDH level. Median creatinine clearance (CKD.EPI) was 70ml/min (min: 24, max: 102). Treatment was initiated either by a neuro-oncology or a hematology team in 35% and 65% of cases, respectively. Median delay between first symptoms and treatment was 60 days. First line treatment was high-dose (HD) methotrexate (MTX) based chemotherapy (CT) in 85 pts (77%), other chemotherapy regimen in 13 pts (12%) and palliative care in 12 pts (11%). Median number of CT cycles was 3 (1-11) with a median dose of MTX of 3g/m2 (0.5-5.0). Interestingly, no difference of distribution for the main clinical and biological characteristics (median age, PS, symptoms, tumor localization, albumin level, creatinine clearance) was observed between these three groups. Rituximab was used in combination with CT in 53/98 treated pts (54%). After first-line induction chemotherapy, response rate for evaluable patients (n=85) were as follows: 37% of complete response, 9% of partial response, 54% of stable or progressive disease. Finally, 27 pts (32%) received consolidation treatment with high-dose cytarabine after MTX-based CT. For toxicity, among the 351 infusions performed for the 85 pts who received MTX-based CT, grade 3-4 toxicities were: 46% of any events, 15% of infection, 13% of cytopenia, 10.5% of acute renal failure and 8% of elevated liver enzymes. 13% of pts presented toxic death. Median progression free survival (PFS) and overall survival (OS) were 3.9 months and 7 months, respectively. Pts treated with MTX-based CT had a significantly prolonged PFS and OS as compared to patients treated without MTX or with palliative care (Figure 1A, 1B). In the univariate analysis performed for the 85 pts treated with MTX-based CT, no initial clinical and biological characteristics (age, PS, type of symptoms, CIRS-G, tumor localization, LDH level, albumin level, hemoglobin level, lymphocyte count, creatinine clearance) influenced PFS or OS. The initial dose of MTX did not influence outcome but intravenous rituximab used in first line therapy significantly improved PFS and OS (Figure 1C, 1D). Conclusions: to the best of our knowledge, this is the largest series of consecutive PCNSL pts aged of 80y or over prospectively recorded in a national database. This study showed that the prognosis remains poor with major toxicity under conventional treatment. No clinical predictor of survival was highlighted in our series but patients initially treated with MTX-based CT in combination with rituximab had an improved outcome. The development of target and innovative therapies is needed for this category of patients representing 8% of all PCNSL in the database of the LOC network. Disclosures Houot: Celgene: Honoraria; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Novartis: Honoraria.

Description: Background: AZA is the standard of care for patients with either HRMDS or AML with 20-30% blasts (Oligoblastic AML). The response rate is ~50% with a median response duration of 12-15 months. The mechanisms underlying primary and secondary resistance are poorly understood and it remains difficult to accurately predict which patients will respond and in responders, to predict secondary resistance. The main factors demonstrated or suggested to interfere with response to AZA and/or survival include marrow blast percentage, performance status, IPSS cytogenetic risk, presence of peripheral blasts, transfusion dependency, IPSS-R risk, mutations of TET2, P53, IDH1/2, and DNMT3A, elevated expression of BCL2L10, Fas, or PI-PLCbeta1. Deregulation of microRNAs is a hallmark of MDS, yet its consequences on AZA efficacy have not been specifically addressed in HRMDS. Methods: We measured the expression of 754 miRNAs in SKM1 MDS cells resistant or sensitive to AZA. miRNAs of interest were ectopically expressed or specifically inhibited in HEK293T cells which were assayed for AZA sensitivity (MTT). Seven miRNAs were quantified in bone marrow mononuclear cells deriving from 75 patients with HRMDS (n= 50) or oligoblastic AML treated with AZA [median age 74, 24 females, median cycle number 6, (1-39)]. Results: Seven miRNAs (miR-125a-5p, miR-99b-5p, miR-126, miR-126*, hsa-let-7c, miR-34b-3p, and miR-10b*) that include 6 tumor-suppressor miRNAs, were found differentially expressed between AZA-sensitive versus -resistant cells; all being transcriptionally repressed in resistant cells. In silico, 5 of these miRNAs were found to target the 3' UTR of DNA methyltransferase 1 (DNMT1) while Zhao et al. [Arthritis & rheumatism, 2011; 63(5)] have shown that miR-126 directly inhibits DNMT1 translation. DNMT1 is one of the main AZA molecular target, the higher the level of DNMT1 expression, the lower the AZA sensitivity [Li et al., Cancer biology & therapy, 2010; 9(4)]. Microarray and western blotting showed that levels of DNMT1 protein, but not mRNA, were significantly higher in AZA-resistant cells. In HEK293T cells, specific endogenous miR-126/126* inhibition significantly augmented DNMT1 protein expression and triggered AZA-resistance, as measured through MTT proliferation assay. In the same cells, ectopic expression of miR-126/126* decreased DNMT1 protein expression in a concentration-dependent manner. In the 75 patients treated with AZA, a decreased expression level of all but miR-10* was associated with decreased response rate (IWG 2006 criteria) and poor outcome; miR-126/126* having the strongest prognostic impact. When compared with miR-126*High MDS, miR-126*Low MDS had significantly lower overall response rate (37% versus 60%, p=0.04), higher relapse rate (100% versus 71%, p=0.034), shorter progression-free (PFS) (8±8% versus 49±12% at 3 years, p=0.004, log rank test), and overall survival (OS) (16±6% versus 46±9%, p=0.004). Baseline patient characteristics of the 2 groups were similar. Multivariate analyses were performed using the Cox proportional hazards model. Table 1 shows that age, miR-126* expression, and IPSS-R risk independently predicted PFS and OS. miRNAs expression was analyzed over time in 15 patients. The mean expression level of 5/7 miRNAs increased over time in the 10 responders without statistically significant difference (DNS) between the first and latest values. In contrast, the expression of 7/7 miRNAs decreased over time in the 5 patients with treatment failure, the difference being statistically significant for 2 miRNAs (p²0.043, Wilcoxon test). Secondary resistance occurred in 7/10 responders and was found associated with a decreased expression of 6/7 miRNAs (p²0.044 for 4 miRNAs) versus 2/7 (DNS) in the 3 long-term responders. Conclusion: Our results suggest that in HRMDS and oligoblastic AML, i. primary or secondary resistance to AZA is associated with the decreased expression of anti-DNMT1 tumor-suppressor microRNAs that trigger AZA resistance in vitro; ii. measuring miRNAs expression before and under treatment might help to predict primary or secondary AZA resistance and thereby to rapidly offer alternative treatment; iii. increasing AZA exposure might help to circumvent AZA resistance in case of either low or decreased anti-DNMT1 miRNA concentration while using anti-DNMT1 microRNAs as a therapeutic tool might increase or restore AZA sensitivity. Disclosures Fenaux: Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Wattel:AMGEN: Consultancy, Research Funding; PIERRE FABRE MEDICAMENTS: Research Funding; CELGENE: Research Funding, Speakers Bureau; NOVARTIS: Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding.

Description: Background. The ATP binding cassette transporter 3 (ABCA3) has been recently found to induce a significant reduction in cytotoxicity following exposure to anthracyclines, mitoxantrone, etoposide, Ara-C, vincristine, and rituximab. ABCA3 acts through the modulation of multivesicular bodies (MVB) and contributes to drug sequestration in late endosomal organelles, i.e. MVB and lysosomes. Studies having investigated the prognostic impact of ABCA3 expression in AML have yielded conflicting results as ABCA3 expression has both been reported to exert unfavorable or neutral effects on patient outcomes. In addition, the small sample size of these studies precluded the use of multivariate analyses. Methods. Our goal was to investigate the prognostic impact of ABCA3 expression in adult patients with AML treated with IC with or without gemtuzumab ozogamicin (GO). To this end we investigated the relationship between ABCA3 expression and EFS in a representative series of 221 AML homogeneously treated in the ALFA-0701 trial. qRTPCR amplification of conserved ABCA3 mRNA sequences, as identified with FasterDB database, was performed with GUS and ABL as reference genes. Primer sets were complementary to conserved ABCA3 exons 6-7 and exon 19-20 junctions. Patients were given a 3+7 induction course without (control group, n=110) or with fractionated intravenous GO (n=111) (Castaigne S, Lancet 2012; 379:1508-1516). Results. Among the 278 randomized patients, 221 had available bone-marrow diagnostic samples with high-quality RNA. The same benefits associated with GO were observed in the 221 patients from the present study as in the entire trial population. Overall, median age, CR rate, relapse rate, median follow-up, 3-years EFS were 62.1 years, 76.5%, 66%, 47.45 months, 28±3%, respectively. There was no significant difference in the level of ABCA3 expression between responders and non-responders. In the 169 responders, ABCA3 expression at diagnosis was more than 3-fold higher in the 111 remitters who subsequently relapsed than in the 58 patients who remained in persistent CR (p=0.033). The level of ABCA3 expression was significantly lower in ELN favorable group than in intermediate and adverse risk AML (p= 0.004) and negatively correlated with CD33 expression (R=-0.272, p

Description: The nucleoside analogue cytarabine (AraC) has served as the backbone of acute myeloid leukemia (AML) treatment for nearly forty years. About one-third of expressed genes are abnormally spliced in AML yet alternative exon usage (AEU) plays a role in the plasticity of tumor cells and may influence the response to treatment. Here the exon expression profiles of the erythroleukemia K562 cell line were compared to that of its AraC-resistant variant K562/AraC through Affymetrix HTA2 exon arrays. 5140 exon events harbored by 2583 genes distinguished the 2 cell lines. Among these, the skipping of TET2 exon 2 was identified in K562 cells sensitive to AraC whereas TET2 gene expression remained unchanged at the whole transcript level. The results were confirmed by exon-specific RTPCR (ESPCR). Microarray analysis did not evidenced any significant change in mRNA splicing for the 10 remaining exons of the TET2 gene. TET2 is a dioxygenase that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and promote DNA demethylation. TET2 somatic mutations occur in about 25% AML, distributing across the whole coding sequence without obvious hot spots. These mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. TET2 exon 2 is spliced in a mutually exclusive manner with exon 1 yet it is used as an alternative promoter (https://fasterdb.lyon.unicancer.fr/). However, TET2 exon 2 is not translated into protein and its role in TET2 regulation is still unknown. Having found that AraC sensitive cells harbor the spliced TET2 isoform, we investigated whether or not skipping of TET2 exon 2 correlate with disease outcome in AML patients treated with AraC-based intensive chemotherapy (AraC-IC). The discovery cohort included 106 consecutive AML patients treated with AraC-IC (median age 57.91, 64 males). RNA was extracted from bone marrow MNCs and assayed for TET2 exon 2 skipping through real-time quantitative ESRTPCR (qESRTPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2 exon 2 skipping was quantified by calculating the ratio E1E3/E2E3. For statistical analysis, the ratio E1E3/E2E3 was dichotomized using median value as the cutoff value. Skipping of TET2 exon 2 was associated with a significantly lower response rate: 65% vs 92%, p = 0.001 but with a significantly lower relapse rate: 39% vs 85%: p

Description: Introduction: standard treatment for relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) is high-dose chemotherapy followed by autologous stem-cell transplantation (ASCT) but this strategy is not appropriate for elderly DLBCL patients (pts) related to a high risk of toxicities. Multiple chemotherapy regimens had been developed for heavily pretreated elderly DLBCL patients such as R-bendamustine, R-gemcitabine-oxaliplatin (R-GEMOX) and pixantrone; the median progression free survival (PFS) of these regimens were 2, 4 and 3.5 months, respectively in prospective phase II studies for patients previously treated with R (Sehn 2017, Mounier 2013, Pettengel 2016). Adapted dose of ifosfamide and etoposide was firstly developed as sequential consolidation regimen after high-dose CHOP (ACVBP regimen) in first line therapy of young DLBCL patients (Tilly 2003). This regimen with a safe toxicity profile was then used in combination with R in Lyon University Hospital in elderly R/R DLBCL ineligible to intensive strategy. Methods: we retrospectively reviewed the efficacy and the safety profile of this regimen performed in two Lyon University Hospitals (Centre Hospitalier Lyon Sud and Leon Berard Cancer Center). Between June 2004 and March 2017, 75 pts with R/R DLBCL (63 de novo DLBCL, 12 transformed DLBCL) received R (375 mg/m2) in combination etoposide (300 mg/m2) and ifosfamide (1500 mg/m2) on day 1 (N=72, 96%) and on days 1-2 (N=3, 4%) at 2 (N=46, 61%) or 3-week (N=29, 39%) intervals. All medical records were reviewed for clinical and biological characteristics, modality of treatment and supportive care, toxicities, responses and outcome. Results: the median age was 79 years (range, 64-92) at the beginning of R-ifosfamide/VP16 treatment with 46% of the patients over 80 years. 13% of pts had a CIRS-G grade 3 or 4 〉2 categories and 35% had a cumulative CIRS-G score more than 6. The performance status according to EORTC scale was 2-4 in 37% of the pts and 93% had III-IV Ann Arbor stages. Age-adjusted IPI were 0-1 in 20 pts (27%) and 2-3 in 55 pts (73%). All patients were previously treated in first-line therapy by R in combination with chemotherapy (CHOP, N=56, 75%, low-dose CHOP, N=14, 19%, other, N=5, 6%). The patients received a median number of 1 previous line (range, 1-8) and no patient was previously treated by ASCT. The median time between initial diagnosis and R-ifosfamide/VP16 was 20 months (range 4-187). The median time between the last treatment and R-ifosfamide/VP16 was 5 months (range 0-181). A refractory disease to first-line treatment was showed in 14 pts (19%). 31% of the patients had a refractory disease to the last regimen performed before R-ifosfamide/VP16. Patients received a median of 6 cycles (1-12). At the end of treatment, the overall response rate (ORR), defined by the rate of complete response (CR) and partial response (PR) was 37%, with 18% of CR. Evaluations were assessed for 29% of the pts by TEP scanner. For toxicity, among the 387 cycles, 10 patients developed febrile neutropenia (2.6%); 15 (20%) a grade 3-4 neutropenia; 7 (9%) a grade 3-4 thrombocytopenia; 5 patients needed platelet units and 16 patients received packed red blood cell units. No grade 3-4 non-hematological toxicity was observed and no toxic death occurred. With a median follow up of 31.3 months (range, 5.0-202.8), the median progression-free survival (PFS) was 4.3 months with a 1-year PFS rate of 26.0% (95%CI, 17.7-38.3) (Figure 1A). The median overall survival (OS) was 8.2 months with a 1-year OS rate of 40.8% (95%CI, 30.9-54.0) (Figure 1B). The median duration of response was 4 months (range 1-97). The median PFS was adversely affected by response (refractory versus CR/PR) to the last treatment (3.0 months versus 5.5 months, P=0.001) (Figure 1C). Conclusions: in this retrospective study, R-Ifosfamide/VP16 regimen provided effective results in R/R DLBCL transplant-ineligible pts with 37% of ORR and a median of PFS of 4.3 months with a safe toxicity profile. This regimen could also be considered as a platform for combinations with novel targeted agents in these categories of patients. Disclosures Karlin: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sarkozy:ROCHE: Consultancy. Bachy:Gilead Sciences: Honoraria; Takeda: Research Funding; Sandoz: Consultancy; Amgen: Honoraria; Roche: Research Funding; Celgene: Consultancy; Janssen: Honoraria. Salles:Abbvie: Honoraria; Epizyme: Honoraria; Amgen: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Other: Advisory Board; Acerta: Honoraria; Novartis: Consultancy, Honoraria; Servier: Honoraria; Servier: Honoraria, Other: Advisory Board; Takeda: Honoraria; BMS: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Janssen: Honoraria, Other: Advisory Board; Merck: Honoraria; Morphosys: Honoraria; Pfizer: Honoraria. Ghesquieres:Sanofi: Consultancy; Gilead: Consultancy; Celgene: Consultancy.

Description: Purpose Programmed death 1 (PD-1) protein is a key immune-checkpoint receptor expressed by activated T cells and it mediates immunosuppression. It has been recently shown that the blockade of PD-1 or its ligand, PD-L1, by monoclonal antibodies may lead to significant antitumor effects. In diffuse large B-cell lymphoma PD-L1 has been reported expressed by tumor cells and PD-1 by tumor-associated T cells. The dosage of soluble programmed death ligand 1 (sPD-L1) protein in the blood of adults with cancer has never been performed during a clinical trial. We evaluated the clinical impact of PD-L1 level measured at the time of diagnosis for newly diagnosed aggressive diffuse large B-cell lymphomas. Methods Translating a previous study (NEJMed2004; 350: 1287) in the rituximab era, the Groupe Ouest-Est des Leucemies et des Autres Maladies du Sang (GOELAMS) 075 study is a multicenter randomized trial in which adults 18 to 60 years old with untreated histologically proved CD20 positive diffuse large B-cell lymphoma were randomly assigned to receive 8 courses of R-CHOP or, high-dose chemotherapy associated to rituximab followed by autologous stem cell support (clinicaltrials.gov: NCT00561379). Patients were required to have an Ann Arbor stage of I or II with bulk greater or equal to 7 cm or, III or IV with 0 to 3 adverse prognostic factors as defined by the age-adjusted International Prognostic Index. The population of interest consisted of 288 of the 340 consecutively enrolled patients with available plasma collected at the time of diagnosis before any treatment. Soluble PD-L1 was measured using an ELISA assay. The median follow-up was 41.4 months. Results sPD-L1 levels were found significantly increased in the plasma collected at diagnosis for patients compared to healthy -gender and age matched- subjects’ levels (P

Description: Background. Despite progress in the molecular and genetic classification of pediatric acute myeloid leukemia (AML), the prognosis remains heterogeneous. The ATP-binding cassette transporter A3 (ABCA3) seems specifically involved in the resistance of pediatric AML to intensive chemotherapy. However, studies having investigated the prognostic impact of ABCA3 expression have yielded conflicting results with respect to patient outcomes while the small sample size of these studies precluded the use of multivariate analysis. Here we investigated the prognostic impact of ABCA3 expression in a representative series of homogeneously treated pediatric AML. Methods. Samples derived from 233 patients with available high-quality RNA and enrolled in the ELAM2 protocol (NCT00149162). qRTPCR amplification of 2 conserved ABCA3 mRNA sequences was performed with GUS and ABL as reference genes. Primer sets were complementary to exons 6-7 and exons 19-20 junctions. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups (Rubnitz JE, Blood 2012;119:5980-5988, Creutzig U, Blood 2012;120:3187-3205). Treatment consisted of 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); all children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant (HSCT) in complete remission (CR) after 1 to 2 consolidation courses. Results. The discovery cohort included 120 patients. Median age, median WBC, CR rate, relapse rate, median follow-up, 5-years EFS, DFS, and OS were 9.4 years, 19.3 G/L, 95%, 29%, 60 months, 58±6%, 61±6%, and 71±5 months, respectively. The two primer sets yielded consistent results (R=0.9, p

Description: Background. We have recently investigated transcriptional and posttranscriptional changes in AML cells (Oncotarget, 2016, 7, 2889-909) and identified ABCA3 expression, TET2 expression and TET2 exon 2 skipping (TET2E2S) as prognostic factors in pediatric AML (See companion abstracts from Ceraulo et al.). The present study was conducted in order to test whether the combination of these prognostic factors might help stratifying patients according to their relapse risk. Methods. Samples derived from 120 patients with available high-quality RNA and enrolled in the French ELAM2 protocol. Patients were classified according to their standardized cytogenetic and molecular (NPM1 mutations, FLT3-ITD, CEBPA double mutations) risk subgroups. Treatment consisted of 1 induction course (AraC and mitoxantrone) and 3 consolidation courses (course 1 and 3 with high dose AraC); all children with either intermediate or high-risk disease were candidates for hematopoietic stem cell transplant in complete remission (CR) after 1 to 2 consolidation courses. qRTPCR amplification of 2 conserved ABCA3 (exons 6-7 and exon 19-20) and TET2 (exon 4 and exon 2-3) mRNA sequences were performed with GUS and ABL as reference genes. TET2E2S was quantified as already described (Oncotarget, 2016, 7, 2889-909). The concordance index (c-index) was used to compare the relative statistical power of the new model compared with the standardized cytogenetic and molecular-based scoring system. Results. ABCA3 expression [HR 2.6 (95% CI 1.3-5), p=0.006], TET2 expression (HR 0.5 (0.2-0.9) p=0.04, and TET2E2S (HR 2.5 (1.2-5.1), p=0.01) represented independent prognostic factors for EFS. To design a convenient prognostic score, continuous variables were dichotomized using cutoff points that were based using the Maximally Selected Rank Statistics (maxstat R-statistics package, from the R-Project). One point was attributed for lower TET2 expression (n=85), higher ABCA3 expression (n=44), and TET2E2S (n=61). Three groups were formed: lower-risk patients (0 point, n=33), intermediate-risk patients (1 point, n=41), and higher-risk patients (either 2 or 3 points, n=46). The present score identified subgroups of patients with significantly different outcome (p