ALBERT

Description: Background: Chromosome 14q32 rearrangements involving the immunoglobulin heavy chain gene (IGH) affect less than 5% of chronic lymphocytic leukemia (CLL) patients. Their clinical course is aggressive and the outcome, worse than other CLL subtypes (Cavazzini et al, 2008; Gerrie et al, 2012). However, the biology of CLL showing IGH rearrangements (CLL-IGHR) is not completely defined. The identification of novel recurrent mutations in CLL by next generation-sequencing (NGS) has offered a more comprehensive view into the genomic landscape of the disease and improved the prognostication of CLL. Thus, mutational analysis might be especially useful in those patients with uncertain prognosis, such as those carrying IGH rearrangements. Aim: To analyze the mutational profile of CLL-IGHR patients by targeted NGS in order to improve our understanding of the genetic underpinnings of this subgroup. Methods: The study was based on 899 CLL patients, well characterized at cytogenetic, biological and clinical level, forty-two of them (4.7%) showing IGH rearrangements. Targeted NGS was performed in 231 CLL samples: 117 with 13q deletion, 27 with 11q deletion, 26 trisomy 12, 42 showing IGH rearrangements and the remaining 19 without any cytogenetic alteration. CD19+ B cells were isolated and DNA extracted. SureSelectQXT targeted enrichment technology and a custom-designed panel (MiSeq, Illumina), including 54 CLL-related and recurrent mutated genes, was carried out. The panel yielded 100x or greater coverage on 97% of the genomic regions of interest and the mean coverage obtained was 600x. Mutations were detected down to 3% allele frequency. Results: The mutational analysis of CLL-IGHR patients identified a total of 72 mutations in 32 genes. Seventy-one percent of patients (30/42) harbored at least one mutation. The most frequently mutated genes in this cohort were NOTCH1 (28.6%), POT1 (14.3%), TP53 (9.5%), SF3B1 (7%), BRAF (7%), EGR2 (7%), IGLL5 (7%) and MGA (7%), followed by BCL2, HIST1H1E and FBXW7 (4.8%), uncommonly mutated genes in CLL at these frequencies (Table 1). In fact, mutations in NOTCH1, BRAF, EGR2, BCL2, HIST1H1E and FBXW7 were significantly associated with CLL-IGHR patients (p=0.013, p=0.003, p=0.021, p=0.038, p=0.038 and p=0.021 respectively). In terms of time to the first therapy (TFT), CLL-IGHR had an intermediate-negative impact (median TFT=24 months) compared to the presence of cytogenetic alterations associated with good prognosis such as 13q deletions (median TFT〉120 months; p

Description: Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26〉80%, 12〉93%, 23〉86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22〉42%, 15〉61%, 50〉96%, 17〉100%), in 2/8 were decreased (60〉37%, 51〉31%), while in the remaining 2 stayed stable (53〉48%, 47〉48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Large-scale next-generation sequencing (NGS) studies have suggested common patterns of co-occurrence or mutual exclusivity between genetic alterations in chronic lymphocytic leukemia (CLL). However, little is known about how most of these alterations cooperate to drive CLL pathogenesis, as well as the impact of these concurrencies in clinical outcome. In this regard, we investigated the clinical and biological impact of the co-occurrence of high-risk lesions such as del(11q)/ATM mutation and del(17p)/TP53 mutation by integrating NGS and CRISPR/Cas9 approaches. To address these questions, we first analyzed the mutational profile of 271 CLLs (17.3% del(11q); 10.7% del(17p)). The most frequently mutated genes were NOTCH1 (20%), TP53 (14%), SF3B1 (11%) and ATM (10%). Within del(11q), 32% showed TP53 alterations (53% biallelic; 47% monoallelic). Interestingly, patients harboring combined del(11q) and TP53 alterations by either mutation or deletion (del(11q) TP53ALT) exhibited significantly shorter overall survival (OS) than del(11q) CLLs without TP53 alterations (del(11q) TP53WT) and those TP53 altered without del(11q) (no del(11q) TP53ALT) (median 17 vs. 88, 36 months; P=0.0004, P=0.02). Conversely, we observed a significant lack of ATM mutations in CLLs with biallelic TP53 alterations (P=0.002) and a mutual exclusivity between biallelic TP53 and biallelic ATM losses (P=0.03)(Fig 1A). Based on the NGS results, we next used the CRISPR/Cas9 system to model monoallelic and biallelic ATM and TP53 loss in vitro. We generated isogenic HG3-Cas9 CLL-derived cell lines harboring monoallelic del(11q) (targeting 11q22.1/11q23.3 regions) and further loss-of-function mutations in ATM and/or TP53 to mimic all the possible combinations observed in our CLL cohort. By proliferation assays, we noted that the introduction of TP53 mutations increased the proliferation rates in both HG3WT and HG3-del(11q) cells. In contrast, the introduction of an ATM truncating mutation on the remaining allele of the HG3-del(11q) TP53MUT clone, suppressed this proliferative advantage, with growth rates comparable to those of HG3-del(11q). Accordingly, DNA content analysis by propidium iodide revealed that cells harboring biallelic ATM and TP53 loss also showed mitotic and cell cycle defects. To further evaluate the implications of these alterations in the clonal dynamics of CLL in vivo, we performed fluorescence-based clonal competition experiments by injecting these edited cell lines intravenously into NGS mice. First, we observed that HG3-TP53MUT cells outgrew HG3WT cells in spleen of xenotransplanted mice 14 days after injection (P

Description: The FDA-approval of potent targeted therapies has led to great changes in the therapeutic landscape of chronic lymphocytic leukemia (CLL). As a key example, venetoclax, a first-in-class BCL-2 inhibitor, leads to response in about 80% of patients with relapsed/refractory (R/R) CLL. Disease progression on venetoclax, however, has been increasingly observed, and better biologic understanding of resistance mechanisms to this agent is needed. To systematically discover the potential mechanisms of resistance to venetoclax, we performed both genome-scale loss- (LOF) and gain-of-function (GOF) genetic modifier screens in the BCL-2-driven OCI-Ly1 lymphoma cell line using CRISPR-Cas9 sgRNA and ORF libraries, respectively. Significant hits from both screens included the BCL-2 family: the LOF screen with pro-apoptotic genes (PMAIP1, BAX, BAK1, BCL-2L11) and the GOF screen with anti-apoptotic genes (BCL2L1, BCL2L2, BCL2, MCL1). In addition, the LOF screen uncovered genes in pathways relevant to lymphoid biology (i.e, NFKBIA) and lymphoid transcription factors and modulators (IKZF5, ID3, EP300, NFIA). The GOF screen also uncovered components of the energy-stress sensor PKA/AMPK signaling pathways (ADIPOQ, PRKAR2B, PRKAA2) and regulators of mitochondrial metabolism. In parallel, we performed an integrated transcriptome, whole proteome and functional characterization of an OCI-Ly1 cell line rendered resistant to venetoclax (OCI-Ly1-R) from the parental cell line (OCI-Ly1-S). RNA-seq and spectrometry-based proteomics revealed coordinated dysregulation of transcripts and proteins in the resistant line originating from genes critical to cellular metabolism, cell cycle, B-cell biology and autophagy. Of the transcripts and proteins significantly associated with the resistant cell line, only MCL-1 overlapped with the gene hits from the genome-scale screens. Treatment of the OCI-Ly-R cells with the MCL-1 inhibitor S63845 synergized with venetoclax. Given the dysregulation of proteins critical to metabolism in both the GOF screen and in OCI-Ly1-R cells, we also evaluated the role of metabolic reprogramming in venetoclax resistance. We first assessed mitochondrial respiration by measuring the oxygen consumption rate. Compared to OCI-Ly-S cells, OCI-Ly1-R cells demonstrated markedly higher respiration levels, suggesting a state of higher oxidative phosphorylation (OXPHOS). More directly, we measured oxygen consumption following venetoclax exposure. Consistent with impairment of OXPHOS by venetoclax, we observed both an immediate decrease in oxygen consumption and an immediate burst of glycolysis following venetoclax in the OCI-Ly1-S cells, but not in the OCI-Ly1-R cells. In line with these findings, the AMPK inhibitor dorsomorphin and mitochondrial electron transport chain (mETC) inhibitors synergized with venetoclax in OCI-Ly1-S cells. Transcriptome related to ID3 (identified as one of the LOF screen targets) was characterized in isogenic ID3-knockout OCI-Ly1 lines. It revealed PRKAR2B overexpression as a key effect, suggesting a role for ID3, and perhaps of other lymphoid transcription factors in regulating metabolic reprogramming associated with resistance. Indeed, exposure of ID3 knockout lines to mETC inhibitors overcame resistance to venetoclax. To determine if there is a genetic basis for the drug resistance seen in OCI-Ly1-R cells, we compared whole-exome sequencing (WES) results of DNA isolated from the OCI-Ly1-R and OCI-Ly1-S cell lines. A clear region was amplified on chromosome 1q23, which includes MCL1 and PRKAB2 (the regulatory subunit of AMPK). Similarly, a WES-based analysis of paired CLL DNA samples isolated from 6 R/R CLL patients just prior to venetoclax initiation and at time of progression on venetoclax was performed. We did not identify any non-silent somatic single nucleotide in BCL2 or its family members at baseline or at progression, despite marked clonal shifts in all patients. We confirmed the presence of the amp(1q23) as acquired at relapse after venetoclax in 3 out of 6 patients. Our study reveals that venetoclax resistance implicates changes not only for outer mitochondrial membrane (MCL-1 expression) but also for inner membrane (oxydative metabolism). Such mitochondrial reprogramming represents a new vulnerability that can potentially be exploited through combinatorial therapy with metabolic modulators to overcome resistance. Disclosures Guieze: abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Thompson:Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davids:Merck: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; MEI Pharma: Consultancy, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brown:Sun Pharmaceutical Industries: Research Funding; Abbvie: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Janssen: Consultancy; Sunesis: Consultancy; Roche/Genentech: Consultancy; Verastem: Consultancy, Research Funding; Boehringer: Consultancy; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Genentech: Consultancy. Wierda:AbbVie, Inc: Research Funding; Genentech: Research Funding. Letai:AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership. Wu:Neon Therapeutics: Equity Ownership.

Description: Abstract 1776 Array-based sequence capture (Roche NimbleGen) followed by next-generation sequencing (Roche GS FLX Titanium sequencing platform) was used to analyze genetic variations in 93 genes relevant in CLL and two chromosomal regions: 13q14.3 and 17p13.1. CD19+ cells from 4 patients with CLL and 4 patients with other hematological malignancies (used as controls) were studied. A custom-made data analysis pipeline was used to annotate detected variants, including known single-nucleotide polymorphisms (SNPs), amino acid consequences, genomic location and miRNA binding sites. The enrichment assay followed by NGS allowed the detection of over 1600 variations/sample (median 1721, range 1618–1823). All putative variants were first compared with published single nucleotide polymorphism (SNP) data (dbSNP build 130) and most of the variants detected were identified as known SNPs. Overall, 10% of variants detected in each sample were variations not previously described. Interestingly, a 4bp insertion/deletion polymorphism (rs2307842) in the 3′UTR of HSP90B1, target site for miR-223, was detected. There is an increasing evidence suggesting that SNPs in the 3′UTR targeted by miRNAs (known as miRSNPs) are associated with diseases by affecting gene expression. We hypothesized that this ‘GACT’ deletion disrupts the binding site for miR-223 thereby increasing the translation of HSP90B1 and we confirmed that miR-223 regulates HSP90B1 expression by 3′UTR reporter assays. The relative luciferase activity of the construct with wild-type 3′UTR (WT-3′UTR) was significantly repressed by 31% following miR-223 transfection (p

Description: Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions, such as transcriptional aberrations, may play a role in its development. Moreover, MDS prevalence is almost exclusive to older patients, suggesting that elderly-related alterations may predispose to the development of this clinical entity. Thus, study of the transcriptional lesions occurring in the aging-MDS axis could shed some light of the molecular bases of the disease. To characterize the transcriptional profile of HSCs in aging and MDS, we isolated CD34+, CD38-, CD90+, CD45RA- cells from 11 untreated MDS patients with unilineage and multilineage dysplasia (median of 75 y/o), as well as from 16 young and 8 elderly healthy donors (median of 21 and 70 y/o, respectively), and their expression profile was analyzed using MARS-seq. Unsupervised principal component analysis demonstrated that the three groups of HSCs clustered separately, indicating that different expression profiles characterize healthy young and elderly, and MDS-associated HSCs. To better understand the gene expression deregulation of HSCs, we analyzed the transcriptional dynamisms along the aging-MDS axis, detecting groups of genes following different patterns of expression. Some gene clusters showed exclusive alteration either in aging or in the progression from elderly HSCs to MDS-HSCs, other groups of genes presented a continuous alteration along the axis, and some displayed opposite regulation in aging and in the transition to MDS (Figure 1). Genes showing specific downregulation in aging were involved in DNA damage sensing and repair, and in cell cycle regulation, whereas genes overexpressed in this process were enriched in apoptosis regulators and in cancer-associated genes, including AML-related factors. These findings indicate that transcriptional changes in aging may predispose for MDS and AML, and potentially other malignancies. Interestingly, we detected a group of genes in which the age-mediated upregulation of gene expression was reversed to that of young HSCs in MDS, indicating a "rejuvenation" profile of malignant HSCs. These genes were involved in response to inflammation, to different types of stress conditions such as hypoxia or radiation, and to cytokines. Elderly HSCs may upregulate such genes in response to the known inflammatory microenvironment of elderly bone marrow. Intriguingly, the decrease in expression detected in MDS suggests that malignant HSCs lose the ability of reacting to such stimuli, possibly favoring their survival in a hostile microenvironment. Finally, the analyses performed allowed for the identification of genes showing MDS-specific deregulation. Genes specifically overexpressed in MDS compared to normal (both young and elderly) HSCs, we enriched in transcriptional and epigenetic regulators, and among them, we detected the presence of DDIT3/CHOP, a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. To determine its potential effects on hematopoietic deregulation, DDIT3 was exogenously overexpressed in healthy HSCs. Notably, its upregulation produced an erythroid bias in an ex-vivo differentiation system, with an increase in the percentage of erythroblasts and a decrease in granulocytes and monocytes compared to HSCs transduced with the empty vector. Transcriptomic analysis of transduced HSCs not subjected to differentiation demonstrated how DDIT3 overexpression produced an erythroid-prone state of HSCs, suggesting it may act as a pioneer factor in MDS-HSCs. Furthermore, gene set enrichment analysis showed that DDIT3 overexpression produced an MDS-like transcriptional profile, suggesting this factor may be key in the acquisition of the disease. Altogether, our results demonstrate that HSCs undergo transcriptional changes in the aging-MDS axis that may alter their intrinsic functions as well as their response to the microenvironment, ultimately contributing to the acquisition of the disease. In particular, our data show that DDIT3 may be a potential driver of MDS transformation. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Díez-Campelo:Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies. To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATMKO, del(11q) BIRC3KO and del(11q) ATMKOBIRC3KO (three clones per condition). In addition, single ATMKO and BIRC3KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition). Functional in vitro studies revealed that del(11q) BIRC3KO cells had increased growth rates compared to del(11q) BIRC3WT clones (P

Description: Clonal evolution is considered as a hallmark of progression in chronic lymphocytic leukemia (CLL). Next-generation sequencing technologies have expanded our knowledge of genetic abnormalities in CLL and enabled to describe marked clonal changes. The acquisition of driver mutations accompanied by selectively neutral passenger changes may be essential to understand the transformation from diagnosis to later more aggressive stages. However, the role of genetic mutations and clonal evolution during the clinical progression prior any therapy is still largely unknown. Longitudinal studies analyzing CLL patients repeatedly before intervening treatment are currently scarce. Patients and methods: We examined the exomes from 35 CLL patients in 2 time-points. Two groups of patients were characterized: (i)patients with progression (n=26) in which we analyzed samples taken from an early stable stage (inactive disease) and during clinical progression (active disease), but before treatment (median of time to first treatment=2.7 years); (ii)patients without progression with a stable inactive disease until last follow-up (n=9) (median follow-up=5.25 years). We also compared patients that gained new cytogenetic aberration detected by FISH in the 2nd time-point with those who did not. Sequencing libraries were prepared using TruSeq Exome Enrichment and sequenced by Illumina HiSeq1000 (84X). Somatic mutation calling was performed by a standardized bioinformatics pipeline. Thereafter, driver mutations were identified using the Cancer Genome Interpreter (https://www.cancergenomeinterpreter.org), a novel tool that identifies variants that are already validated as oncogenic and predicts the effect of the mutations of unknown significance. Results: We identified 397 somatic mutations in 364 different genes, ranging from 4 to 26 mutations per patient. Among them, 58 driver mutations were identified, being SF3B1 (6/35, 17.1%), TP53 (4/35, 11.4%) and NOTCH1 (4/35, 11.4%) the most common mutated genes. Comparing progressive vs. stable group, CLL patients with clinical progression showed a higher intra-tumoral heterogeneity than cases without progression (median of somatic mutations=14[4-26] vs. 9[5-14]). Comparing both tumoral time-points in the same patient, we identified a total of 11 acquired driver mutations and 7 mutations increasing its allele frequency in more than double in the 2nd time-point respect to the 1st one. All of them were detected in patients with clinical progression. Interestingly, TP53 and BIRC3 exhibited recurrently acquired mutations (detected each one in 2 cases). Three driver mutations in cancer genes not yet known for CLL (DHX9, GNAQ and HDAC2) were also acquired. Within CLL progressive patients (n=26), we observed clonal evolution characterized by acquired cytogenetic aberration in 9 cases. In patients with progression but no cytogenetic aberration gained at the 2nd moment (n=17), we detected that almost half of them (7/17) showed clonal evolution by acquired or doubled driver mutations. In the remaining patients with clinical progression but without any clonal evolution (n=10), 6 cases showed a driver mutation of CLL genes associated with bad prognosis (SF3B1, TP53, NOTCH1 or RPS15) already at first time-point. In the stable group (n=9), none acquired or doubled mutation was detected. However, clonal evolution characterized by acquired cytogenetic aberration was observed in 4/9 stable patients: two of them acquired 13q- whereas the other two acquired 11q-. Within stable patients without clonal evolution (n=5), we detected one case with a driver mutation in SF3B1 already at 1st time-point (follow-up=5 years). Conclusion: Clonal evolution represents a central feature of tumor progression in CLL. Our data show that the disease is evolving during time even in stable patients without any clinical signs of disease activity. In progressive patients, the disease evolution is accompanied by new appearance or accumulation of driver mutations and cytogenetic aberrations. Moreover, progressive patients that showed less or no changes during time bore typical CLL drivers at the first time-point. Funding: Seventh Framework Programme (NGS-PTL/2012-2015/no.306242); Ministry of Education, Youth and Sports (2013-2015, no.7E13008; CEITEC 2020 (LQ1601)); AZV-MZ-CR 15-31834A-4/2015 and TACR (TEO2000058/2014-2019); PI15/01471; Junta de Castilla y León (MHS). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4767 Introduction Extranodal NK/T-cell lymphoma, nasal type is an entity of difficult diagnosis. It is characterized by damage and vascular destruction, necrosis, cytotoxic phenotype and Epstein Barr virus association. It is prominent in Asia, Mexico, Central and South America, predominantly in men. Material and methods It is an observational, descriptive and retrospective study, based on medical records of NK/T-cell lymphoma patients of the General Hospital of Mexico between 1992-2009. Results Of 632 non-Hodgkin lymphomas (NHL), 113 had a T cell lymphoma. Only 32 cases (28.3%) were eligible for the study. The age range was 19-56 years with a media of 42. The male:female ratio was 2.2:1. The most frequent clinical presentation symptoms were nasal obstruction, facial swelling and pain. Central nervous system involvement was present only in two cases and bone marrow infiltration in one. Seventy eight percent were on clinical stages I or II and 56% had B symptoms. In 7/10 patients the Epstein Barr association was documented by hystopathological study. The treatment was completed in 31/32 individuals. Fifty five percent showed some response, regardless of the therapy used. The CHOP regimen was the first line treatment in 80.6% of cases and only 13% received fractionated radiotherapy as initial treatment. The CHOP arm had an overall response rate of 60% (complete remissions, unconfirmed complete remissions, partial remissions), and 100% of the patients in the radiotherapy arm presented complete response. Most patients of the radiotherapy arm were on stage IE. The overall survival rate was 63% at 36 months. The average follow-up was 40 months. There were no differences in overall survival rates between the two arms, but the radiotherapy arm had a higher rate of complete remissions. Conclusions In this study we report the clinical features and response to treatment of the patient population treated at the General Hospital of Mexico. The results are similar to other international reports. Although the incidence of NK/T-cell lymphoma is low, it is necessary to standardize its treatment and these preliminary data warrant further trial of radiotherapy as the initial treatment and chemotherapy as consolidation for advanced stages of the disease. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are hematological disorders at high risk of progression to acute myeloid leukemia (sAML). Previous high-throughput sequencing studies have provided insight into the mutational dynamics and clonal evolution underlying disease progression. However, large serial sequencing studies are still required to define which type of mutations alone or in combination contribute to leukemic transformation. To assess the mutational profiles and mutational dynamics underlying progression from MDS to sAML, a targeted-deep sequencing (TDS) of 117 MDS/AML related-genes was performed in 110 bone marrow serial samples from 50 MDS/CMML patients who evolved to sAML and 5 patients who did not evolved (controls), at two different time-points: at the time of diagnosis and at sAML progression or after a median of 3 year follow-up, respectively. A total of 269 mutations in 57 different genes were identified at second sampling. At diagnosis, all patients, progressing and not progressing (controls), presented similar number of mutations (p=0.15). Moreover, patients evolving to sAML were then divided by FAB/WHO subtypes at diagnosis (CMML, low-risk and high-risk MDS subgroups) and no differences were observed in the number of mutations (p=0.71) and variant allele frequency (VAF) between each group (p=0.63). It should be noted that mutations in the splicing pathway were significantly more frequent in low-risk MDS patients (89% low-risk MDS vs. 56% high risk MDS, p=0.038). However, after progression, those patients who evolved to sAML displayed a statistically significant increase of mutations (p=0.001) at the leukemic phase, while controls did not at the follow-up sample (p=0.88). This higher number of mutations at second sampling in patients who evolved to sAML, independently of their diagnostic subtype, may be indicative of a higher genomic instability during disease evolution. To study the mutational dynamics and what mutations could be important during disease evolution, the VAFs of mutations detected at both time-points in each patient of transformation cohort were compared. We observed that some mutations identified at the sAML stage (163 mutations) were already present at the MDS stage, at clonal or subclonal levels, and were retained during evolution, for example in genes such as SRSF2 and DNMT3A. However, 106 mutations increased in clonal size or were newly acquired. Interestingly, most of mutations in Ras signaling pathway showed a same pattern: they were not present at time of diagnosis and appeared at sAML. In fact, mutations in this pathway were detected in 25 of 50 patients (50%) included in this cohort and in 22 of them (88%) mutations displayed this dynamic. Therefore, in this study, Ras signaling was the most common pathway involved in the progression from MDS to sAML. Of note, 9 of these patients (18% of the whole cohort) presented, independently of diagnosis, a co-occurring cohesin mutation, that was already present at diagnosis and, in most cases, markedly increased in clonal size at sAML. Thus, the combination of mutations in these two pathways could play an important role during disease evolution. In addition, 22 of 50 patients were treated with a disease-modifying agent (18 azacytidine and 4 lenalidomide) before they progressed to sAML, while the remaining 28 patients received no treatment or supportive care and were considered as non-treated. Thus, we studied the effect of disease-modifying therapy on mutational dynamics in this cohort of patients progressing to sAML. In the treated patients, a higher proportion of newly acquired or increasing mutations at sAML in chromatin modifiers was observed, while in non-treated patients most mutations remained stable (61% vs. 28.6%, p=0.013). By contrast, regarding treatment, no differences were detected in the mutational dynamics of cohesin (p=0.56) or Ras pathway (p=1.00). MDS progression to sAML was characterized by a higher genomic instability, independently of MDS subtypes of patients at diagnosis. Ras signaling was the most frequent affected pathway during disease evolution in this cohort and, interestingly, the co-occurrence of Ras signaling and cohesin mutations could play an important role in the progression. Moreover, mutations in chromatin modifiers genes could be related to the evolution of patients who received disease-modifying treatment before progression to sAML. Disclosures Olivier: Celgene: Honoraria; Jassen: Honoraria. Díez-Campelo:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.