ALBERT

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: 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: 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.

Description: To date, several malignancies of the lymphoid lineage have been linked with microbial infection. Indeed, lymphomas have been associated with the bacteria Helicobacter pylori, and T-cell leukemia and Burkitt's lymphoma have long been known to be commonly caused by viral infections (human T-cell lymphotropic virus 1 and Epstein-Barr virus (EBV), respectively). However, metagenomics research in myeloid malignancies has primarily focused on the roles of gut flora rather than on the tumor site itself (blood and bone marrow). Traditionally, human blood and bone marrow was considered to be normally sterile, and therefore microbiome analysis of these entities would only be performed when deleterious infection was suspected. However, evidence is now accumulating for a normal blood microbiome in healthy individuals. Given the growing number of established relationships in other malignancies between the microbiome at the tumor site and clinical characteristics, we hypothesized roles for the bone marrow microbiome in MDS patients. To this end, we extracted bacterial, fungal, and viral sequence from the bone marrow and blood of 640 MDS patients taken at diagnosis. Briefly, whole-genome sequencing (minimum coverage 100x) was performed on DNA extracted from each patient sample. Reads not aligning to the human genome were aligned to NCBI's database of known microbial reference genomes, and microbial species were assessed for presence and abundance in each sample. As expected, bacterial reads dominated, comprising 99.8% of all microbial reads, while viral reads were 2.73-fold as prevalent as fungal reads. We calculated microbial diversity (Shannon diversity index) on the genus and phylum levels for each patient. As has been reported in solid tumors, higher alpha-diversity is associated with favorable overall survival (age- and sex-adjusted P = 8.6 x 10-3; Figure 1A). Interestingly, individuals with normal karyotype had significantly higher alpha-diversity at the phylum level (Wilcoxon P = 0.002; Figure 1B), while those with deletion of chromosome 5q had significantly lower alpha-diversity (P = 2.2 x 10-5; Figure 1C). Among viral infections, EBV (formally Human gammaherpesvirus 4) was the most common, with 192 patients (30%) harboring EBV DNA sequence. Human cytomegalovirus (HCMV; formally Human gammaherpesvirus 5) and Human betaherpesvirus 6 infections were also observed, in 23% and 2% of patients, respectively. EBV infection was associated with worse overall survival (age- and sex-adjusted P = 0.037; Figure 1D). There was also a substantial dose effect, with higher EBV levels corresponding to poorer outcomes (P = 1.96 x 10-6). Mining the paired-end DNA reads for human-viral chimeras, we found multiple instances of EBV integration into the patient genome, suggesting a possible mechanism for EBV infection impacting patient outcomes. Bacterial composition was also analyzed, showing strong concordance between several pairs of phyla, suggesting synergy between these bacterial communities (Figure 1E). On the other hand, the Proteobacteria and Actinobacteria phyla were strongly discordant with one another (Figure 1E). Multiple studies have shown dominance of Proteobacteria in the circulating bacteriome of healthy individuals. In contrast, the MDS patients in our study showed a continuum between complete dominance of the Proteobacteria phylum and complete dominance of the Actinobacteria phylum (Figure 1F). Intriguingly, lower levels of Actinobacteria was strongly associated with the presence of various chromosomal aberrations, while the opposite was largely true for Proteobacterial abundance. As a result, the ratio of these two phyla abundances was strongly positively associated with normal karyotype and negatively associated with del(5q) (Table 1). Overall, our study provides evidence for roles of the bone marrow microbiome in phenotypes and may even influence outcomes of patients with MDS. Further research is required to determine: a) whether microbial content is cause or consequence of patient characteristics, and b) what the mechanisms are for these microbiome-clinical associations. Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

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