ALBERT

Description: Introduction The majority of patients with Essential Thrombocythaemia (ET) have mutations in JAK2, MPL, and CALR, causing activation of the JAK/STAT pathway; but 10-15% of ET patients lack detectable mutations in these genes, so-called 'Triple Negative' (TN). We applied a systematic approach to investigate mutational status and epigenetic signatures in a cohort of TN ET patients. Methods and Results We investigated 46 patients (72% female), median age at diagnosis 35 years (range 8-77 years) including a father and son. All patients were TNusing standard diagnostic assays. We applied deep, error corrected, next generationsequencing (NGS) of 24 genes using the HaloPlexHS platform to peripheral blood samples. Whole exome sequencing was also performed in 23 patients using skin as constitutional control. Overall we identified somatic mutations in 10/46 patients including MPL(3 patients, W515R, W515G, W515S, R537W, VAF 0.02-0.1) JAK2V617F (4 patients, VAF 0.02-0.08); and germline MPLmutations in a further 3 patients (P453R, S505N); including the father and son pair. We selected patients lacking somatic or germline mutations ("true TN") to analyse gene expression using RNA-seq and DNA methlyation status using 850K Epic Arrays. Patients with JAK2V617Fand CALRmutations and healthy donors (HC) were included as controls. Concerning RNA-seq data, we performed multiple differential analysis of HC vs TN, CALRand JAK2V671F; as well as HC vs all ET samples (adjusted for subtype). Each HC comparison highlighted clear differences between gene expression profiles of HC and disease (Figure 1A). The differentially expressed genes (DEGs) in each comparison overlapped significantly, suggesting that all ET samples have consistent gene expression differences to HC samples regardless of their driver mutation status. In total 1444 differentially expressed genes (ET vs HC) were highlighted (figure 1B). Functional analysis identified significant enrichment for genes involved in the MAPK pathway. Addtionally, we noted upregulation of GATA1,ITGA2B and GP6 genes, not previously reported to be dysregulated in ET. Correlation of gene expression data with DNA methylation status identified a consistent signature of 306 hypomethylated genes, showing significant enrichment for genes involved in transcriptional misregulation and upregulation of inflammatory regulators such as TNF and NFκB signaling pathways. Next, we identified which transcription factor motifs preferentially bind within these methylation blocks. The blocks showed an enrichment for 6 key transcriptional regulators: ATF3, ATF4, CEBPA, CEBPB, MAX, and RARA. All 6 were significantly upregulated in all ET samples. To validate the motifs, we processed ChIP-seq data from the K562 cell line and identified a significant proportion of the hypo methylated regions are bound by these, transcription factors: 374/410 (91%) regions; 43/410 (10%) are bound by all 6 transcription factors. Conclusions A significant proportion (22%) of patients assigned as 'TN' ET via traditional diagnostic techniques in fact harbor known driver mutations at a low allele frequency, suggesting that error corrected NGS approaches may be diagnostically useful in this setting. Additionally, for a group of "true" TN ET patients we demonstrate that patterns of gene expression and DNA methylation are more similar to patients with ET with known driver mutations than healthy controls. Among the upregulated genes are key platelet regulatory genes: GP6, GP1BB, ACTN1and ITGA2B. Furthermore, we identify consistently hypomethylated genes with increased expression across all molecular subtypes of ET which are highly enriched for genes involved in proinflammatory pathways and show that binding of 6 key transcription factorsmay underlie these changes regardless of driver mutation status. Our observations suggest that the ET disease phenotype may, at least in part, be driven by transcriptional misregulation and may be propagated downstream via the MAPK, TNF and NFKappa pathways in addition to activation of JAK/STAT pathways. These findings identify novel mechanisms of disease initiation which require further evaluation. Disclosures Dillon: Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria. Mufti:Cellectis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. McLornan:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Harrison:Shire: Speakers Bureau; CTI: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; AOP: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria; Promedior: Honoraria; Gilead: Speakers Bureau; Sierra Oncology: Honoraria; Incyte: Speakers Bureau.

Description: Key Points There is 100% concordance in the cytogenetic and mutation profile between PB and BM in myelodysplastic syndrome.

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 1707 With the advent of high throughput and high resolution techniques, 〉80% of myelodysplastic syndrome (MDS) patients harbour somatic mutations and/or genomic aberrations, which provide diagnostic and prognostic utility; however, frequent bone marrow (BM) aspirates are required. In a significant minority, the BM is hypocellular and fibrotic with suboptimal in vitro growth and, additionally, the procedure causes discomfort particularly in the elderly. This led us to investigate the use of peripheral blood (PB) and serum to identify and monitor BM derived genetic markers using high resolution single nucleotide polymorphism array (SNP-A) karyotyping and 454 parallel sequencing (454-PS) of a 22 gene myeloid panel comprising of all the exons of DNMT3a, RUNX1, CEBPα, TP53, EZH2, TET2 and ZRSR2 and mutations ‘hotposts’ for NPM1, FLT3, ASXL1, IDH1, IDH2, MPL, JAK2, BRAF, cCBL, NRAS, KRAS, C-KIT, SF3B1, SRSF2, and U2AF1. We selected 23 MDS patients with concurrent BM and PB samples and detected 45 mutations in TET2, SF3B1, ASXL1, TP53, DNMT3a, FLT3, U2AF1, NRAS, cCBL, JAK2 and IDH2 in BM and subsequently analysed their PB using 454-PS with independent validation performed by Sanger sequencing (SS). All the mutations identified in BM were detected in PB with the exception of a single NRAS (BM-11%). Nine patients had a single mutation in SF3B1, ASXL1, TP53, TET2, DNMT3a and U2AF1 with the remaining patients having multiple coexisting mutations. Overall there was no significant difference in the mutation burden between the PB (median 25%(1.5%-50%)) and the BM (median 33%(5%-68%)). Concurrent analysis of unamplified and whole genome amplified PB DNA from 3 patients with mutations in TET2, U2AF1, ASXL1 and NRAS showed no difference in their mutation profile. As expected, in three patients post therapy the mutation burden in the PB was lower than in the pre-treatment BM sample. The lower mutation burden and sequence context in the PB contributed significantly to the quality of SS analysis. Prior knowledge of the mutation site resulted in 98% concordance (smallest clone size - 1.5%) between BM and PB, however, a blind approach decreased this to 84% (smallest clone size - 15%). In addition, serum was available from 14 patients (22 known mutations in BM) and SS of serum DNA identified 12 mutations correctly (U2AF1, FLT3, SF3B1, TET2, TP53, ASXL1, DNMT3a and IDH2, 4 as wildtype (TET2, cCBL, ASXL1 and SF3B1) and 6 failed to amplify (ASXL1, TP53 and TET2) without any preference for specific genes and the failure attributed to the quantity of serum DNA. Karyotype aberrations in PB were assessed using Affymetrix SNP 6.0 arrays on 31 MDS patients; normal karyotype (n=11), del5q (n=9), del7q/-7 (n=5), trisomy 8 (n=2), complex (n=2), isodiXq13 (n=1) and t(2:4)(q33;q27). An overall karyotype concordance of 94% was observed in PB with the 2 discordant cases showing normal karyotype in PB and BM by SNP-A but having monosomy 7 (partial cytogenetic remission after 5-azacitidine) and t(2:4)(q33;q27) respectively in their BM by MC. The mean copy number (CN) in the PB was lower than BM (PB vs BM); deletions (CN of 1.8 vs 1.6) and gains(CN of 2.2 vs 2.4), implying a smaller abnormal clone in the PB. Concurrent SNP-A karyotype from BM from 9 patients was concordant with SNP-A karyotype from PB, although a patient with complex karyotype determined by SNP-A in the BM and having 30 aberrations had only 15 aberrations detected in the PB. To determine if the 5q deletion was lineage restricted, we enriched PB for CD3+, CD19+ and CD3-CD19- populations from 4 patients. FISH and SNP-A karyotyping showed the presence of the 5q deletion using both techniques in all three fractions, however at a lower level in PB lymphocytes indicating the presence of a smaller clone. In conclusion, our study showed an excellent concordance between BM and PB, both for karyotype and mutational analyses using high resolution SNP-A karyotyping and 454-PS suggesting its clinical utility as a surrogate for BM, thereby, avoiding the discomfort of repeated BM aspirates and help in monitoring response to therapy more frequently. Disclosures: No relevant conflicts of interest to declare.

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.

Description: AMM and MA – Joint first authors Diagnosis of myelodysplastic syndromes (MDS) relies on demonstrating peripheral blood (PB) / bone marrow (BM) dysplasia and cytogenetic abnormalities, forming the backbone of the revised International Prognostic Scoring System (IPSS-R). However, assessment of dysplasia is operator dependent and metaphase cytogenetic analysis (MC) is often normal or uninformative creating diagnostic challenges, particularly in patients with early or low risk MDS. Impressive advances have taken place in the last decade in the identification and chronicling of the genetic lesions leading to phenotypic diversity of MDS. We sought to evaluate the value of single nucleotide polymorphism array (SNP-A) based cytogenetic assessment and high throughput sequencing of the 24 genes most frequently mutated in MDS to determine if genetic abnormalities in the BM are reflected in the PB thus enabling easy assessment of response to treatment and/or disease progression. A MiSeq based gene panel comprising of 24 frequently mutated MDS genes: ASXL1, CBL, CEBPA, DNMT3A, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, KDM6, KIT, KRAS, NPM1, NRAS, RUNX1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, ZRSR2 and CytoHD/750K SNP-A karyotyping was applied to both PB and BM concurrent samples. Genomic aberrations and non-synonymous variant calls were filtered using public databases to exclude polymorphisms. PB and BM from 201 MDS patients [median 62 years (17-88)] followed up for median 21 months (0.3-171) was analysed. Sixty (30%) patients received supportive care only whilst others were treated with MDS/AML directed therapies. The WHO subtypes were: 5q- syndrome (n=26), refractory cytopenia/s (RA/RCMD, n=53), refractory anaemia with excess blasts/acute myeloid leukaemia (RAEB/AML, n=51), refractory anaemia with ringed sideroblasts (RARS/RCMD-RS, n=20) and other subtypes (including myeloproliferative and hypoplastic MDS, n=51). Based on the IPSS-R risk groups 62% had low risk disease: classified as very low (n=35), low (n=89), intermediate (n=42), high (n=16) and very high (n=19) risk groups. Metaphase cytogenetic analysis was normal (NK-MC) in 113(56%), abnormal (AK-MC) in 65(32%) patients and in 23(12%) MC failed. SNP-A was informative in all patients identifying a normal (NK-SNP) in 93(46%) and abnormal (AK-SNP) in 108(54%) patients, respectively. A comparison of BM and PB by SNP-A, showed 190 patients having an identical karyotype (95% concordance). BM SNP-A identified 36 (32%) patients with SNP-A abnormalities not detected by MC, changing the IPSS-R in 49 (24%) patients overall. Inclusion of SNP-A abnormalities changed the IPSS-R risk group in 28 (25%) of NK-MC patients; from good to intermediate (n=21), poor (n=5) and very poor (n=2) groups, respectively. In 9/11 patients with discordant SNP-A karyotypes between BM and PB, the IPSS-R remained unchanged. We found no difference in the clonal burden between PB and BM for gains and CN-LOH. However, for deletions, the clonal size was significantly lower in BM [median 1.4 (1 – 1.9)], than PB [median 1.5 (1 – 1.95), p

Description: Background The presence of diverse spectrum of mutations in myelodysplastic syndromes (MDS) has largely revolutionized the understanding of the disease. Although certain mutations have doubtful prognostic significance, the adverse impact of TP53 mutation is irrefutable in all publications. We and others have shown the strong correlation between presence of TP53 mutation and overexpression of p53 by immunohistochemistry (IHC). This evidence provides the rationale for the evaluation of p53 overexpression which is available in routine diagnostic workup, as a surrogate marker to predict TP53 mutations. Additionally, we predicted that the overexpression of p53 might be an independent prognosticator, even in the absence of TP53 mutations or RPS14 haploinsufficiency, in low risk MDS. In our study,we analyzed the p53 protein expression in a cohort of 277 'lower risk' MDS patients and compared the clinico-pathological features, overall survival and risk of progression and evolution to AML with respect to p53 IHC status. Methods This study enrolled 277 adult low risk MDS patients seen at King's College Hospital between March 2009 and February 2016. All marrow samples were assessed for p53 expression (intensity and proportion of cells) and scored using a Modified Quick Score (MQS). MQS ≥2 was chosen to define p53-positive staining. The median follow-up was 18.7 months [95% confidence interval (CI), 16.5-21.0 months]. Results According to WHO categories, the predominant group was RCMD (78%). Patients were either IPSS low (n=134, 48%) or int-1 (n=91, 33%). We included 52 (19%) pts for whom the IPSS category was not available, due to the lack or failed cytogenetic data, but these were low-risk WHO categories with no excess of blasts. IPSS-R categories were very low 103 (37%), low 77 (28%), intermediate 38 (14%) and high risk 7 (3%). Overall, 19 (7%) patients progressed to AML and 41 (15%) patients died of which 25 (61%) were disease-related deaths. Of the 277 patients, 148 (53%) showed p53 protein expression with MQS ≥ 2 and were considered "p53-positive" patients (p53+). The p53 stain intensity was negative in 125 (45%), weak in 84 (30%), moderate in 40 (14%) and strong in 28 (11%). Seventy six (28%) patients had 〉5% p53 staining cells. p53 expression correlated with a higher age at diagnosis (median age 64 vs. 60 years, p=0.01). lower haemoglobin levels (9.8 vs 11 g/dL, p=0.002), but a higher platelets count (139 vs 99 x109/L, p =.003) (Table 1). This significance persisted even on exclusion of patients with 5q- and MDS/MPN. P53+ did not correlate with any cytogenetic risk group or degree of fibrosis. Sequencing data was available in 121 patients with 63 harbouring somatic mutations. Among these patients, only 3 showed the TP53 gene mutation; all of them were classified as positive when assessed for p53 stain (MQS≥2), showing that the nuclear staining reflects underlying TP53 mutations in such cases. Additionally, presence of SF3B1 mutation correlated strongly with p53+ (p=0.02). Patients with p53+ had a significantly shorter OS compared with those with p53- (figure 1a) with 2yr and 4yr survival probability of 87% &68% vs 95% & 90% , respectively (p=0.03 ). Analysis of progression-free survival (progression to AML, cytogenetic evolution, increase of blasts or fibrosis) showed that patients with p53 expression had a significantly shorter PFS compared to patients without p53 expression (Figure 1b): 2yr & 4yr PFS 70% & 54% vs 85% &75% respectively, p=0.01). Multivariate analysis confirmed the independent prognostic value of p53 expression for PFS (p=0.013, HR 2.6, 95% CI 1.2-5.2). Conclusion In conclusion, our study showed that p53 overexpression by IHC, represented as MQS cutoff ≥ 2, correlates with features of poor prognosis, such as more advanced age at diagnosis, lower haemoglobin levels and shorter overall survival, adversely impacting progression-free survival. p53 IHC in MDS patients may represent an important, easily available, cheap and applicable prognostic tool and it should be considered for auxiliary analysis when determining the therapeutic options for a patient. The p53+ predicts poor outcome, especially disease progression, independent of the TP53 mutation status, indicating an alternate yet undefined pathway for p53 overexpression. Figure 1(a) Disease-related OS (a) and PFS (b) in patients with low-risk MDS according to their p53 expression-status Figure 1(a). Disease-related OS (a) and PFS (b) in patients with low-risk MDS according to their p53 expression-status Figure 1(b) Figure 1(b). Disclosures No relevant conflicts of interest to declare.

Description: Background MDS is characterized by ineffective haematopoiesis and a propensity to leukaemia transformation, with increasing evidence linking immune exhaustion to disease progression. Immune checkpoints are known to be upregulated on T cells in cancer, and inhibitors of CTLA-4 and PD1/PLD1 axis have demonstrated efficacy with likely clinical benefit in MDS. We have previously shown profound changes in both the number and function of components of the adaptive immune system, particularly Tregs, in MDS (Kordasti, Blood 2007). In order to characterise the immune signature in a wider range of T cell subsets simultaneously, with particular emphasis on cells likely to be affected by checkpoint inhibitor therapy (CPI), we analyse CTLA-4 and PD1 expression in MDS by cytometry by time-of-flight (CyTOF). Additionally, we aim to explore whether these differences are accentuated by the absence or presence of somatic mutations, or in morphologically more advanced disease. Materials and Methods MDS patients (n=56) and age-matched healthy donors (HD, n=6) were stained with two panels of 35 and 34 antibodies for unstimulated and PMA/Ionomycin-stimulated PBMCs, respectively. Samples were run on CyTOF and data analysed using visual stochastic neighbour embedding (viSNE, Cytobank) to generate t-distributed SNE scores by unsupervised multi-dimensional reduction of T cells. Spanning-tree progression analysis of density-normalized events (SPADE), was performed and T cell subsets identified from heat maps based on typical phenotypic markers (Regulatory, Naive, Memory, Effector Memory (EM) Central Memory (CM), Effector, Terminal Effector (TE)). T cells were also clustered based on cytokine secretion (IFN-ϒ, TNF-α, IL-17, IL-2 and IL-10). Somatic mutation analysis was performed on 48 of the MDS patients using our established targeted panel of 24 genes known to be mutated in MDS, for subgroup analysis (Mohamedali, Leukaemia 2015) Results and Discussion Demographics and subgroups are outlined in figure 1. The number of Tregs was significantly higher in RAEB than non-RAEB MDS (9.6% of total CD4+ cells vs 7.5% p=0.02) and in RAEB versus HD (9.6% vs 5.8% p=0.01). There was a significantly higher proportion of Tregs in MDS patients with somatic mutations compared to those without (8.7% vs 7.06% p

Description: Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using “next-generation” sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chroni myelomonocytic leukemia patients (P 〈 .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. “Deeper” sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34+ and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (〉 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Description: Abstract 748 NCL, AA, AK & AS contributed equally to the study A large number of acquired mutations have been identified in haematological malignancies in recent years. An increasing number of targets present a new problem for diagnostic molecular pathology laboratories. It is not practical for a laboratory to design increasing numbers of gene specific assays for these targets. For these reasons a single assay capable of detecting a large number of gene mutations is desirable. Here we describe a next generation sequencing approach using the Roche 454 platform which is capable of the simultaneous mutation analysis of 17 genes at an appropriate sensitivity level in multiple patient samples. This methodology allows mutation scanning of all coding exons of 5 complete genes (TP53, EZH2, DNMT3a, RUNX1 and CEBPa) scanning of the mutational hotspots of 5 genes (ASXL1 exon 12, JAK2 exon 12, FLT3, MPL and CBL) and the analysis of specific mutation hotspots in a further 8 genes (NRAS, KRAS, NPM1, IDH1, IDH2, KIT, BRAF, JAK2 exon 14). In order to cover all these regions, a total of 99 PCR amplicons (average length 300bp range 250–400bp) were amplified for each patient sample. To avoid time consuming quantification and normalisation of individual amplicons downstream of PCR, we developed a simple method to first designate the amplicons into 4 groups based on the efficiency of PCR amplification. Following amplification the 1st round PCR products are diluted according to the grouping assigned to the particular amplicon prior to a second round of PCR in which MIDs or “barcodes” are added in a patient specific manner. The products of the 2nd round PCRs are then pooled for each patient and normalised before pooling the entire library for subsequent sequencing. The sequencing is carried out using the Roche GS FLX titanium reagents. To validate this approach we have prepared libraries from 80 AML patients with normal cytogenetics. Sequencing of these patients was split over 3 runs of the FLX instrument using standard conditions recommended by the manufacturer. The average number of reads per amplicon was 300 with 95% of amplicons having a minimum coverage of 200 reads. The approach allows the detection of mutations at a sensitivity of approximately 5%. Mutations were detected in 14 /17 genes in at least one patient sample. No mutations of BRAF, JAK2 or MPL were detected. In line with previously published data, mutations were found frequently in NPM1 (67%), DNMT3a (55%) FLT3 ITD (44%) and less frequently in the remaining genes. These data correlate well with published data for normal karyotype AML. In 6/17 genes the mutation status of the patient samples had been previously analysed using: Fragment analysis (NPM1 and FLT3 ITD), RFLP (FLT3 TKD), Sanger Sequencing (IDH1, IDH2, ASXL1, TP53). Significantly, 12 additional mutations were detected (NPM1 n=2, FLT3 ITD n=2, FLT3 TKD n=4, IDH1 n=2, IDH2 n=2, ASXL1 n=4) using the new methodology demonstrating an improvement in sensitivity over and above individual gene specific assays. A single run of the FLX instrument enables the analysis of 30 patients in a batch. In our hands library preparation, sequencing and analysis of 30 patients using the workflow described takes a skilled operator 10 full days. With an estimated test price of P650 ($1000), this compares extremely favourably with the price of providing these analyses separately estimated to be in excess of P4000 ($6500). To conclude we have developed a next generation amplicon sequencing approach to assay 17 individual target genes including whole gene coding sequences and more focused mutational hotspots. In addition we have validated a strategy which dramatically reduces the amount of operator time required compared with most amplicon sequencing approaches. This approach offers a highly flexible platform for analysing multiple gene targets in multiple samples. Changes to the targets analysed or the effective sensitivity can easily be incorporated without the need to make major modifications to the procedure. Disclosures: No relevant conflicts of interest to declare.