ALBERT

Description: Introduction 5-azacitdine (aza) treatment in myelodysplastic syndrome (MDS) induces a response rate of 40-45% and prognostic factors for response and survival remain still largely unknown. Presence of mutant TET2 has been shown to predict better response to aza and the recently described French Azacitidine prognostic score segregates patients into 3 groups with varying median overall survival. Although a plethora of somatic mutations have been described in MDS, none has been consistently shown to be prognostically important in the context of response to hypomethylating agent(s). Patients and methods To identify the mutation signature associated with aza response, we undertook screening of 24 myeloid genes (splicing, epigenetic, transcription factors, STAG2, TP53) in 66 MDS patients treated with aza at our institution over a period of 2004-2012. Mutation analysis was done by deep (454 FLX) and Sanger sequencing. SNP-6 karyotyping was also performed in a subset to correlate with mutation status. Responses were assessed as per the international working group for MDS criteria. The median age was 67 years (range 36–87 years), median number of courses 7(range 2–42), 79% of patients belonged to int-2/high risk IPSS category. WHO category subtypes were; RA/RARS-2; RCMD-9; RAEB-39; s-AML (evolved from pre-existing MDS) -5, therapy related myeloid neoplasm (t-MDS/t-AML) -8 and CMML-3.IPSS cytogenetic subgroups were, good risk: 22, intermediate: 7, and poor risk: 37. One fourth of patients had received prior therapy, with only two receiving low dose cytarabine. Median time from diagnosis to aza treatment was 8.6 months. The overall response rate (ORR) to Aza was 47% (31/66) with complete response (CR) 17%, partial response (PR) 11%, marrow CR (mCR) 12% and stable disease with hematological improvement (SD-HI) 7%. Results Candidate mutations were seen in 82% (54/66) of patients, with the more than half harboring ≥2 mutations each. The most frequently (≥5 %) mutated genes being ASXL1 (29%), TP53 (23%), TET2 (14%), DNMT3A (12%), SRSF2 (12%), EZH2 (11%), NRAS (8%), U2AF1 (8%), IDH2 (8%), RUNX1 (8%), CCBL (6%) and FLT3-ITD (5%). On univariate analysis presence of EZH2 mutations predicted for a better ORR compared to wild type EZH2 (21% vs. 3%, p

Description: Abstract 2729 Acute myeloid leukaemia with a normal karyotype (AML-NK) is a heterogeneous clonal disorder of haematopoietic progenitor cells with an intermediate prognosis. Acquired mutations of FLT3, NPM1 and CEBPA as well as gene expression profiles have recently been used as biomarkers to further stratify prognostic subgroups, especially in AML-NK. We have previously shown that cryptic genomic aberrations in MDS cases are prognostically significant and regions of uniparental disomy (UPD), detectable by SNP array karyotyping, can harbour mutations in specific gene(s). We analysed 83 AML patients (pts), median age 52 (range 8–78) with normal cytogenetics, excluding pts who died within 30 days of entry, who had been treated on one of the UK MRC/NCRI AML clinical trial protocols, using high resolution 250K SNP microarrays to seek genomic aberrations. In addition, we sequenced the ‘mutation hotspot’ for IDH1/2 as well as screened for the presence of FLT3 ITD and NPM1 mutations. Genomic aberrations and mutational lesions were correlated with outcome data. To circumvent the lack of constitutional DNA, all aberrations overlapping by 〉50% with copy number variants from the Database of Genomic Variants as well variations observed in a cohort of 91 healthy subjects were excluded from further analysis. We demonstrate the presence of 69 cryptic genomic aberrations in 43 pts consisting of 12 deletions (del) (8 pts), 10 gains (10 pts) and 47 regions of UPD (35 pts). The median size of del was 1.6Mb (0.18Mb-3.4Mb) with chromosome 4 (q24) and 17 (q11.2 and q22.1) being the most affected. One pt had micro del on chromosome 12p13.2-p12.3 and 21q22.11-q22.12 affecting the ETV6 and AML1 genes respectively, suggestive of a t(12:21) aberration. Gains (median 0.37Mb (0.26–0.71Mb)) were identified predominantly on chromosome 8 (4 pts) with a common amplified region, 8q21.11. The median size of UPD's was 47Mb (2.3Mb-158Mb). Chromosome 13 was the most frequently affected with UPD of the entire chromosome observed in 17 pts. Interestingly, one pt had an interstitial region of UPD on q21.32-q21.33 (4.8Mb). Additional regions affected by UPD included chromosomes 6, 2, 5 in 5 pts and chromosome 11 in 4 pts. NPM1 mutations occurred in 55 pts (65%) and FLT3 ITD's occurred in 44 pts (53%). Of 39 pts with wild type FLT3, 22 had NPM1 mutations; of 28 pts with wild type NPM1, 11 had FLT3 ITD mutations. Conversely of the 44 pts with mutant FLT3, 33 had NPM1 mutations. Six pts had mutations of FLT3, NPM1 and IDH. Of note, only 2 of 17 pts with UPD of chromosome 13 did not have FLT3 mutations, suggesting the presence of additional pathological genes in this region. IDH1 (R130H) and IDH2 (R140Q and R172K) mutations occurred in 3 and 16 pts, respectively and were mutually exclusive. Interestingly, the R172K mutation occurred in only 3 pts. Of the 19 pts with IDH1/2 mutations, 9 and 12 pts respectively, had co-existing FLT3 ITD and NPM1 mutations. The median duration of follow-up from time of diagnosis of the study group was 29 months (range 2–160). There were no significant correlations between the presence of genomic aberrations and age, sex, WBC, secondary disease or IDH1/2 mutations; however, a significant difference was observed in the frequency of UPD in pts with mutant or wild type FLT3 (p=0.01) and NPM1 (p=0.008). A difference in overall survival (OS) was seen on comparison of pts with or without genomic aberrations, (5 year OS 10% vs 46%, hazard ratio (HR) 1.86 (1.01–3.44), p=0.05): significant differences were seen on univariate analyses when analysing gains (11% vs 27%, HR 3.07 (1.09–8.68) p=0.03), or del (0% vs 28%) HR 3.46 (1.02–11.8) p=0.05) separately, with some evidence that UPD also produced worse results (0% v 34% HR 1.82 (0.97–3.45) p=0.06). In multivariate analyses, adjusted for age, WBC, sex, secondary disease, performance status, IDH1, IDH2, FLT3 ITD and NPM1 all SNP array detected aberrations were independently prognostic for overall survival. Analysis combining all aberrations gave a HR of 2.99(1.33–0.75) p=0.006. When a model-building approach was used, after adjustment for baseline covariates, del p=0.005, gains p=0.002 and UPD p=0.02 all entered the model. We show, for the first time, that SNP microarray analysis in AML-NK pts reveals cryptic UPD and CN changes that may allow risk stratification and alter therapeutic strategies for this poor prognostic subgroup of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1977 First 2 authors contributed equally. Background: Genomic-wide association studies have identified the germline 46/1 haplotype as a predisposing allele associated with JAK2V617F positive myeloproliferative neoplasms (MPN). The present study analysed data on 856 JAK2V617F positive patients, 326 of which had complete clinical data. Aims: To evaluate the JAK2 46/1 haplotype frequencies, JAK2V617F allele burden, c-MPL 515 mutation and risk of transformation. Methods: Genomic DNA from whole peripheral blood or bone marrow patient samples was analysed as follows: JAK2V617F allele burden by Q-PCR, JAK2 exon 12 mutations by Q-PCR and PCR fragment analysis, MPL W515 L and K mutations by allele specific PCR. The 46/1 JAK2 mutation susceptibility haplotype (46/1) tagging SNP rs12343867 (susceptibility allele C) were analysed by pyrosequencing. Results: The allele frequency for the 46/1 tag SNP rs1234867 in the 856 patients was calculated for the total JAK2V617F cohort (0.48) and the clinical entities ET (0.34) and PRV (0.44) confirming that the 46/1 haplotype is greatly over represented in JAK2V617F MPD patients as compared to published the control population (Wellcome Trust Case Control Consortium (WTCCC) (0.24). The Analysis of the 856 patients demonstrated that JAK2V617F and c-MPL W515L/K mutations co-existed in 16 patients(1.9%), the incidence of c-MPL W515L being twice as common as the c-MPL W515K mutations. There was no correlation between these mutations and age or 46/1 haplotype status. The JAK2V617F allele burden (AB) was lower in the c-MPL mutant patients, the average JAK2AB 31%. 3 out 4 c-MPL patients for which clinical information was available had a diagnosis of ET. No JAK2 exon 12 mutations were found in any of the 859 JAK2V617F positive samples suggesting that co-existing JAK2 exon 14 and exon 12 mutations are extremely rare. The genotypic data in ET patients showed: C/C 12%, C/T 44%, T/T 44% and their respective JAK2V617 allele burden (AB) were 46%, 32%, 29%. The genotype data in PRV patients: C/C 18%, C/T 53%, T/T 28.6% and their respective AB were 47%, 31% and 39%. The median AB was 32% (n=121) for ET and 37% (n=103) for PRV. Within a cohort of 255 patients (ET=138, PRV=117) 4% of ET and 6% of PRV patients transformed to acute myeloid leukaemia or myelofibrosis with no predominant haplotype association. In the ET patients, the median AB was 35%, there was no significant difference in the JAK2 V617F AB between those who transformed or not (p=0.45). Interestingly, on the whole ET group C/C genotype patients were more likely to have an allele burden 〉50% (p=0.058). In the PRV patients, the median AB was 48%. Again, the C/C genotype, PRV patients were more likely to have an AB〉50% (p=0.06), although not reaching statistical significance. Conclusions: The 46/1 haplotype in both clinical entities ET and PRV demonstrated a higher allele burden in the C/C genotype in comparison to the other genotypes. No predominant haplotype predicted the risk of transformation to a more aggressive disease such as MF or AML. The analysis also showed that c-MPL W515K/L mutations can co-exist with JAK2V617F. The c-MPL W515K/L mutations did not exhibit a positive correlation with a preferential 46/1, but was associated with a lower allele burden. No co-existing exon 12 and exon 14 mutations were found, suggesting the rarity of this occurrence. Disclosures: No relevant conflicts of interest to declare.

Description: Mixed T-cell chimerism is frequent and persists despite discontinuation of immunosuppression after hematopoietic stem cell transplantation (HSCT) for severe aplastic anemia (SAA) using the fludarabine, cyclophosphamide and alemtuzumab/campath (FCC) conditioning regimen. To investigate the basis for long-term co-existence of donor and patient T cells and remarkably low rates of graft versus host disease (GVHD), the lymphocyte composition and lymphocyte subset chimerism of adult patients transplanted for SAA using FCC was assessed. Patients We have transplanted 42 patients (pts) for acquired idiopathic SAA using FCC conditioning from 2007- March 2015 at King's College Hospital. Median age was 33 years (range 15-63). BM was stem cell source in 7(17%) pts and PBSC in 30 (83%). Median cell dose was 6.85 (1.9-12.4) CD34+ cells x106/Kg. Donor was matched sibling in 11(26%) and unrelated in 31 (74%) pts, 6 of whom also received 2 Gy TBI for 9/10 match. Post graft immunosuppression comprised ciclosporin (CSA) alone. Primary graft failure occurred in 1 pt (2%). Acute GVHD was seen in 6 (14%) pts (all grade I-II), and chronic GVHD in 5 (12%) patients (all skin, only 1 severe in a 9/10 match patient). Factors associated with cGVHD were previous aGVHD (p=0.035) and CD3+ chimerism at day+100 ³ 90% (p=0.006). Median follow-up of survivors was 41 months (4.3-96), with 93% overall survival and 90% event-free survival. At 1, 2 and 3 years post HSCT, 4/30, 20/23 and 16/16 patients, respectively, had discontinued CSA. Results Comparison to 11 healthy age-matched individuals showed prolonged lymphopenia and abnormal proportions of T, B and NK cells. T cells were profoundly deficient comprising only 1.76% of lymphocytes at day 30, rising to 43.8% by day 360 but still significantly below normal (66%, p=0.018). CD8 T cells recovered faster than CD4 T cells producing inversion of the normal CD4 to CD8 T cell ratio. Composition of na•ve, memory and effector subsets within the CD4 T cell population was near normal by day 360 and na•ve CD4 T cells expressed CD31 indicative of renewed thymopoiesis. In contrast, CD8 T-cell subset composition remained abnormal at day 360 due to a high proportion of effector T cells (57.2% of CD8 T cells compared to 9.3% for healthy individuals, p=2 years after HSCT showed the mixed chimerism profile within T-cell subsets was stable. A significant correlation was observed between lower donor T-cell chimerism at day 360 and CMV reactivation or EBV viremia early after HSCT (p=0.036), suggesting that antigen-driven expansion of virus-specific patient CD8 effector T cells may substantially contribute to persistent mixed T-cell chimerism. Sustained co-existence of donor and patient T cells and low rates of GVHD indicates conditions favor mutual tolerance. Findings suggested a potential role for cells with immunomodulatory properties. B cell recovery was robust, with normal numbers by day 100. Notably, the B cell population present early after HSCT contained a significantly increased proportion of cells with an immature transitional phenotype (CD24+ CD38+, CD27- IgMhigh IgDhigh 17.1% compared to 2.7% for healthy individuals, p=

Description: The myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal stem cell disorders characterised by ineffective erythropoiesis and peripheral blood cytopenias. However, specific subgroups of MDS with both myeloproliferative (MPD) and dysplastic features are recognized, which include chronic myelomonocytic leukaemia (CMML), refractory anaemia with ringed sideroblasts and thrombocytosis (RARS-T) and MDS/MPD unclassified. In addition cases of 5q- syndrome may present with a marked elevation in platelet count and a hypercellular bone marrow. The molecular events which determine the predominant morphological features however remain unclear. The JAK2 somatic point mutation (V617F) results in constitutive activation of the tyrosine kinase and culminates in both proliferation and differentiation signals to the cell and thus may contribute to the proliferative features identified in MDS as reported in the chronic MPDs. We analysed 194 patients from six centres with a diagnosis of MDS or MDS/MPD for the presence of the JAK2 mutation. 11 cases of RA; 70, RARS; 60, 5q- syndrome; 28, refractory anaemia with excess blasts (RAEB); 9, acute myeloid leukaemia with multi-lineage dysplasia (AML-MLD); 4, MDS/MPD-U; 1, CMML; and 1, hypoplastic MDS were analysed. DNA samples were obtained from bone marrow aspirate or peripheral blood. Ethical approval was obtained. Genomic DNA was prepared using standard methods (Qiagen). A modified allele specific PCR (AS PCR) was used to screen DNA samples. JAK2 mutated DNA samples were further subjected to pyrosequencing for confirmatory testing. The median age of the JAK2 mutant versus wild type (WT) cases was 66years (46–80years) v 67years (30–92years). The JAK2 mutation was detected in 13/194 (6.7%) cases overall. 4/70 (5.7%) RARS, 5/60 (8%) 5q- syndrome, 1/28 RAEB (associated with del 5q), 1/4 CMML, 1 MDS/MPD-U and 1/8 AML-MLD demonstrated the mutation. Within the RARS cohort, the prevalence of JAK2 mutation increased to 4/6 (67%) of cases with thrombocytosis (platelets 〉500x109/l). The presence of the mutation was confirmed in 8/13 cases. In cases analysed, the mutation was detected in CD34+, CD14+, CD15+ and CD61+ cell fractions but not CD3+ or CD19+ cell lineages. On analysis of the haemoglobin (Hb), platelet count and total white cell count (WCC) in the JAK2 mutant versus wild-type groups, there was no difference in median Hb (10.8 v 9.2 g/dl, p=0.275) but a significantly higher median platelet count (526 v 209 x109/l, p

Description: The specificity of the clinicomorphological characteristics in 5q- syndrome has led to a search for putative tumour suppressor genes localised to the critical deleted region (CDR) of 5q-. To date, microarray expression studies in 5q- syndrome patients have been carried out on CD34+ cells, neutrophils or total bone marrow (BM) cells. We have used a Affymetrix microarray approach to investigate allelic imbalance (AI) and the transcription profile of CD61+ and CD34+ cells from patients (n=17) presenting with 5q- of which 10 have an “isolated 5q-“. the rest having additional cytogenetic abnormalities. BM CD34+ and CD61+ cells from normals (n=10) were controls in expression profiling. DNA or total RNA from CD61+ or CD34+ cells and constitutional DNA was prepared and hybridised to the 500K mapping or U113 plus 2.0 arrays. GC-RMA normalized expression data was analysed using non-parametric testing with Genespring GX software to generate lists of differentially regulated genes. DChip was used to analyse SNP data. From a list of changing genes 48/720 occur in both the CD61+ and CD34+ cell profiles. 16/48 of these genes map to 5q of which 10/16 map to 5q31. Of these genes KIF20A showed the most marked change (5 and 3.8 fold down) in CD34+ and CD61+ cells respectively. KIF20A is a microtubule-associated protein that is upregulated in mitosis and localizes to the spindle midzone during anaphase. Knock out of KIF20A protein results in binucleate nuclei indicating its involvement in both cytokinesis and nucleokinesis. Haploinsufficiency of this gene, may account for mononuclear megakaryocytes that are so characteristic of 5q- syndrome. Interestingly other members of the kinesin family expressed in 5q- CD34+ cells are down regulated in 5q- CD61+ cells (KIF21B, KIF2, KIF3A and KIF1B) indicating that in CD34+ cells, a compensatory mechanism for KIF20A haploinsufficiency may exist which is absent in megakaryocytes. The 5q- deletion (q14.2–q33.3) was detected in 6/10 CD61+ and 11/17 CD34+ cell populations by SNP analysis. The disparity reflects the admixture of normal and MDS CD61+ and CD34+ cells. Three patients presented with additional cytogenetic abnormalities, two had −7 or del 7(q22–q36), both being detected with the SNP array. The third patient had trisomy 8 that was not detected. SNP microarray identified 5q31.1–5q33.2 as the minimal deleted segment, which includes previously described CDR. Paired analysis of CD61+ and CD34+ with constitutional DNA revealed only 2/17 patients had additional abnormalities in CD34+ cells. One with a del12(q21.1–22) (21Mb). The 2nd had −13(q12.3–34;83Mb),+ 13(q11–12.3; 12.5Mb) and +11(q22.3-qterm; 30Mb) These changes were not detected by cytogenetic analysis. 2/6 patients shared 13Mb of UPD on chromosome 3(p12.2–q11.2) and chromosome 9(q33.1–q34.1) in CD34+ cells as well as constitutive DNA. We conclude that unlike AML, 5q- Syndrome does not have significant rate of UPD. Only a small number of genes are under expressed in both CD61+ and CD34+ cells and amongst these KIF20A is the most significant change and may be responsible for some of the clinicomorphological features of 5q- abnormality.

Description: Monosomy 7 is the second commonest abnormality in myelodysplastic syndrome (MDS). Recent studies (Cordoba et al 2012, Schanz et al 2012) have shown partial loss [del(7q)] of the chromosome (chr) is associated with better prognosis than total loss (-7). However it is still unclear if the biogenesis of these 2 abnormalities are separate or step wise progression of del(7q) to -7. Moreover monosomy 7 (-7) often occurs in the presence of other cytogenetic abnormalities which further adversely impacts the prognosis. We designed a multicenter study to describe and compare clinical features, bone marrow characteristics, genetic profile and outcome of a large population of MDS patients with del(7q) or -7 as sole cytogenetic abnormality. We retrospectively analysed 224 MDS patients who presented at diagnosis with the loss of chr. 7 as isolated cytogenetic abnormality or acquired it during follow up. We also performed a deep targeted mutational screen of the 24 commonest mutated genes in MDS. Patients were included from the King’s College Hospital of London (n=75), the Spanish MDS group (n=107), the University of Medicine of Göttingen (n=35) and the "Città della Salute e della Scienza" hospital of Turin (n=11). Fifty-five patients presented with isolated del(7q) and 169 with isolated -7. Median age at diagnosis was 69 and 64 years old in the two groups, respectively (p n.s.). According to WHO classification 18 patients had refractory anemia (RA), 3 RA with ring sideroblasts (RARS), 61 refractory cytopenia with multilineage dysplasia (RCMD), 42 RA with exces of blasts type 1 (RAEB-1), 53 RAEB-2, 25 MDS/MPN (MDS/Myeloproliferative neoplasm) and 8 MDS unclassified. Fourteen patients with bone marrow blasts percentage between 20 and 30% were also included. MDS with excess of blasts type 1 or 2 were more frequent in the del(7q) group (56% vs. 42%) whereas MDS/MPN prevailed in the -7 group (14% vs. 4%), p=0.049. At diagnosis, del(7q) patients had a higher platelet count whereas there were no differences in neutrophils count and haemoglobin between the two groups; despite similar basal haemoglobin levels a higher number of patients with del(7q) was transfusion dependent (52% vs. 32%, p=0.015). Regarding the mutational status, we have so far analysed 55 patients, 45 with del(7q) and 10 with -7. Overall we found 118 different allele variants (37 previously described as somatic mutations in cancer) across 24 myeloid genes commonly mutated in MDS. Sixty-four percent of patients had 1 or more previously described mutations, with a range of 1 to 6 mutations per patient (median 1). The genes involved in epigenetic modification were the most commonly mutated (in 36% of patients). Genes encoding for spliceosome components, signalling factors, transcriptional factors and STAG2 were mutated in 29%, 22%, 16% and 2% of patients respectively. There were no differences in mutation distribution between patients with -7 and patients with del(7q). Median survival for the whole cohort was 23 months and was significantly affected by WHO diagnosis and, interestingly, by bone marrow cellularity: patients with hypocellular marrow at diagnosis had a better outcome with a median survival of 38 months, compared to 26 and 23 for normocellular and hypercellular marrow respectively (p= 0.031). Patients with isolated del(7q) had a trend towards longer survival than patients with -7 (32 vs. 23 months), but this difference was not statistically significant. Overall 30% of patients were treated with azacytidine, 20% with intensive chemotherapy, 5% with immunosuppressive drugs and 5% with other therapies, including lenalidomide; 46 patients (20.5%) underwent allogeneic transplantation and this significantly impacted on survival (median survival 35 months for transplanted patients vs. 22 for not-transplanted ones, p=0.002), regardless of induction treatment or cytogenetic status. In conclusion, although patients with del(7q) had worse disease characteristics (excess of blasts and transfusion dependence), they showed a trend towards a better survival than those with -7. Preliminary data on the genetic profile showed a prevalence of mutations in the genes involved in epigenetic regulation with no significant differences between the partial and total loss of chr.7. Disclosures No relevant conflicts of interest to declare.

Description: Kinase domain (KD) mutations in the BCR-ABL1 gene are associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). Next-generation Sequencing (NGS) allows detection of low-level KD mutations but its relevance in clinical practice remains debated, and the incidence and prognostic significance of finding of low-level KD mutation in chronic phase (CP) patients is unknown. We analyzed the outcome for 121 newly diagnosed CP-CML patients treated with TKIs (imatinib 111, nilotinib 7 and dasatinib 3) who we routinely screened for KD mutations using ultra-deep NGS regardless of response to TKI. When a mutation was found by ultra-deep NGS (using Illumina NexteraXT and MiSeq platform) all available previous cDNA samples were analyzed to establish the date of its first occurrence and subsequent kinetics. ABL1 transcripts from 27 healthy donors were sequenced and used as a control. Sequencing for both single and nested PCR products were compared, and samples were re-sequenced in two independent runs in order to analyze reproducibility of variants detection. Median age of patients was 56 years and the Sokal risk score was 'low' in 38% patients, 'intermediate' in 15.5% and 'high' in 46.5%. A mutation was detected in 25 of the 121 patients (20.6%) at a median time of 14 months from starting TKI. 19 different mutations were identified, the most frequents being F317L (n=17), Y253H (n=15), M244V (n=14) and T315I (n=10). All mutations previously detected by Sanger sequencing were also found using ultra-deep NGS. In addition low-level mutations (

Description: Telomerase complex maintains telomeres and protects genomic DNA from degradation during cell divisions. Abnormal telomerase function can result in chromosomal instability predisposing to malignant transformation. Short telomere is a typical feature of inherited bone marrow failures syndromes (BMFs), especially dyskeratosis congenital (DC), caused by mutations in genes encoding components of the telomerase gene complex (TGC), shelterin proteins and DNA helicases. Telomere attrition have been associated with leukemic transformation in myelodysplastic syndromes (MDS), as well as complex cytogenetic aberrations, and also with the development of secondary MDS and acute leukemia (AML) after chemotherapy. However, the incidence of TGC mutations in de novo MDS remains largely unknown. Recurrent somatic mutations in genes involving epigenetic, spliceosome, cell signaling and proliferation pathways are common in MDS and have prognostic significance. Identifying specific associations between mutational patterns helps characterize disease biology and thereby improve the therapeutic strategies To determine the incidence of TGC mutations and study theassociation of TGC mutation patterns with recurrently mutated genes in MDS. To correlate TGC mutations with telomere length, clinical phenotypes and outcome of patients. We undertook a massively parallel targeted sequencing of all 10 TGC, (TERT, TERC, TINF2, NHP2, NOP10, RTEL1, CTC1, DKC1, USB1 and WRAP53) in a cohort of 174 MDS patients. Furthermore, we measured the telomere length (T/S ratio) by a multiple quantitative real-time PCR in bone marrow mononuclear cells. Additionally, in 151/174 MDS patients, we studied 22 recurrently mutated MDS-associated genes (MGP) by targeted sequencing. Among the whole cohort, 61% were male. The median age of patients was 63 years (range 17–87). WHO subtypes were 45 RA/RARS/isolated de5q (26%); 50 RCMD/RCMD-RS, (29%); 41 RAEB 1/2, (24%); 8 AML secondary to MDS, (5%); 8 (5%) MDS/MPD and 3 CMML (2%). IPSS cytogenetic risk groups were: 108 patients with good risk (62%), 21 intermediate (12%) and 32 poor risk, (18%) and cytogenetics failed in 10 patients (6%). IPSS categories were low risk 41(24%), intermediate-1: 54 (31%), intermediate-2: 30 (17%), high risk: 13 (7%) and 10 (6%) patients were not evaluated (proliferative CMML and MPD/MDS). Transfusion dependency was present in 80 patients (46%). Twenty nine TGC mutations were present in 26 patients (15%)(figure 1). Twenty-three patients (88%) had TERT mutations, 3 RTEL1 mutations (13%) and 1 TINF2 mutations (4%) with variant allelic frequency around 50%. Two patients presented more than one mutation in TGC genes. Most of mutations in TGC genes were previously described as germ line variants inpatients with DC and inherited aplastic anemia. All mutations found in TERT gene were missense. In patients with TGC mutations, the median T/S ratio was 1.1 (range 0.4–3.5), shorter than the T/S ratio of age-matched controls, although no statistically significant difference was seen in T/S ratio when compared to wild type. (P=0.527). TGC variations did not correlate with clinical features such as age, cytogenetic risk or IPSS, and had no impact on the overall survival (P=0.659). In 151 MDS patients, 73% (n=110) had at least one known somatic mutation in the MGP (21% TET2, 15% ASXL1, 14% TP53, 11% DNMT3A, 11% U2AF1, 9% IDH2, 9% SRSF2, 6% EZH2, 4% NRAS, 4% CEBPA, 3% SF3B1, 3% RUNX1, 2% JAK2, 2% FLT3, 1% cCBL). Among the MGP mutated patients, 13% carried also TGC mutations concurrently (Table 1). Chromatin remodeling gene mutationswere less frequent in patients with TGC mutations (P=0,001) as compared to patient with wild type TGC. We show TGC mutations are frequent in MDS patients (15%). The presence of known TERT variants seen in our cohort demonstrates a clear pathogenic association between MDS phenotype and telomerase mutations, rather than these being bystander variants. Although the heterozygous nature of these abnormalities indicates an inherited variant, the absence of telomere shortening argues against this concept and needs further evaluation. Chromatin remodeling gene mutations are less frequent in patients with TGC mutations. These findings suggest that defective telomere maintenance through TGC mutations might play an important etiological role in the multistep process in pathogenesis of a subset of MDS. Figure 1 Figure 1. Disclosures Mufti: Onconova Therapeutics, Inc: Research Funding.