ALBERT

Description: Cell lines are commonly used as cancer models. The tissue of origin provides context for understanding biological mechanisms and predicting therapy response. We therefore systematically examined whether cancer cell lines exhibit features matching the presumed cancer type of origin. Gene expression and DNA methylation classifiers trained on ~9000 tumors identified 35 (of 614 examined) cell lines that better matched a different tissue or cell type than the one originally assigned. Mutational patterns further supported most reassignments. For instance, cell lines identified as originating from the skin often exhibited a UV mutational signature. We cataloged 366 “golden set” cell lines in which transcriptomic and epigenomic profiles strongly resemble the cancer type of origin, further proposing their assignments to subtypes. Accounting for the uncertain tissue of origin in cell line panels can change the interpretation of drug screening and genetic screening data, revealing previously unknown genomic determinants of sensitivity or resistance.

Description: More than 90% of patients with myelodysplastic/myeloproliferative neoplasms (MDSs/MPNs) harbor somatic mutations in myeloid-related genes, but still, current diagnostic criteria do not include molecular data. We performed genome-wide sequencing techniques to characterize the mutational landscape of a large and clinically well-characterized cohort including 367 adults with MDS/MPN subtypes, including chronic myelomonocytic leukemia (CMML; n = 119), atypical chronic myeloid leukemia (aCML; n = 71), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T; n = 71), and MDS/MPN unclassifiable (MDS/MPN-U; n = 106). A total of 30 genes were recurrently mutated in ≥3% of the cohort. Distribution of recurrently mutated genes and clonal architecture differed among MDS/MPN subtypes. Statistical analysis revealed significant correlations between recurrently mutated genes, as well as genotype-phenotype associations. We identified specific gene combinations that were associated with distinct MDS/MPN subtypes and that were mutually exclusive with most of the other MDSs/MPNs (eg, TET2-SRSF2 in CMML, ASXL1-SETBP1 in aCML, and SF3B1-JAK2 in MDS/MPN-RS-T). Patients with MDS/MPN-U were the most heterogeneous and displayed different molecular profiles that mimicked the ones observed in other MDS/MPN subtypes and that had an impact on the outcome of the patients. Specific gene mutations also had an impact on the outcome of the different MDS/MPN subtypes, which may be relevant for clinical decision-making. Overall, the results of this study help to elucidate the heterogeneity found in these neoplasms, which can be of use in the clinical setting of MDS/MPN.

Description: Background: Whole genome amplification (WGA) has become an invaluable method for working with small amounts of starting DNA and for preserving limited samples of precious stock material. Next-Generation Sequencing (NGS) techniques can benefit from WGA, but due to their high sensitivity, WGA reliability needs to be certified to ensure an unbiased and accurate amplification of whole genomes. Myelodysplastic Syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. We have performed whole exome sequencing (WES) and targeted deep sequencing in tumoral samples from MDS patients. With the aim to determine if Multiple Displacement Amplification-based WGA can be applied to perform NGS in these type of samples and to obtain valuable results, targeted deep sequencing was performed on both fresh-DNA and WGA-DNA from the same patients. Mehtods: Whole bone marrow samples from four MDS patients were included in the study. WGA was performed in tumoral DNA samples with REPLI-g (Qiagen). WES libraries were generated in tumoral-control paired samples using the SureSelect Human Exome Kit 51Mb v4 (Agilent) and sequenced on an Illumina HiSeq2000. Targeted sequencing libraries were prepared for fresh-DNA and WGA-DNA following the manufacturer specifications for TruSight Myeloid Sequencing Panel protocol (Illumina), and then sequenced on one single run on an Illumina MiSeq. WES sequencing data was analyzed using an in-house pipeline, as previously reported. Targeted sequencing data analysis was performed with theMiSeq Reporter Software (Illumina). Filtering was performed in all cases by eliminating sequencing and mapping errors and by discarding intronic or synonym variants, variants located at highly variable regions or with low coverage, as well as know polymorphisms. Additional filtering was performed by visualization on Integrative Genome Viewer Software v.2.3.72. Results: Regarding targeted sequencing, fresh-DNA samples generated 6 million reads (SD = 1.9 million), with 98.5 % (SD = 0.8) of the mapped reads on-target and a mean target coverage of 12148.8 (SD = 3872.9). WGA-DNA samples yielded about 5.2 million reads (SD = 1.5 million), with 98.3 % (SD = 0.4) of the mapped reads on-target and a mean target coverage of 10447.5x (SD = 2946.3). A mean of 77% of total bases displayed a Q score ≥30, which did not differ between fresh and WGA-DNA. Comparison of all filtered variants within the four pairs revealed a high level of discordance between fresh/WGA samples (Figure 1A). A mean of 86% of the detected variants, considering both fresh and WGA-DNA, were detected at a low frequency (

Description: Cytogenetic abnormalities are found in around half of MDS patients (pts) and have both clinical impact and may be subtype-defining, e.g. in 5q-syndrome. Interstitial deletion of the long arm of chr.5 [del(5q)] is the most common aberration (almost 20% of cases with abnormal cytogenetics). Del(5q) is heterogeneous, occurring as a sole abnormality or in combination, with the deleted region often truncated within or extended and/or beyond the CDR boundaries. Isolated del(5q) is frequently shorter and confers a more favorable prognosis with regard to survival and lenalidomide (LEN) responsiveness, while del(5q) in the context of a complex karyotype (CK) imparts a poor prognosis. In addition to chromosomal lesions, somatic mutations can contribute to the pathogenesis of MDS, including del(5q). We theorized that recognition of molecular defects in MDS with del(5q) may clarify the pathogenic mechanisms behind this lesion and help explain the clinical heterogeneity. We analyzed 225 pts with myeloid neoplasia and del(5q) using WES (n= 107 samples) and targeted multiplexed PCR (top 60 most frequently mutated genes) (n =133 samples); serial analysis was performed in 15 pts studied at ≥2 time points, 11 during LEN therapy and 4 upon relapse/progression. A total of 116 samples had a CK with other lesions such as -7/del(7q) found in 31% cases, and 18% had -17/del(17p). WES (average depth 〉60x) was followed by a bioanalytic pipeline, detecting ≥1 mutated gene in 71% of cases. Candidate somatic alterations were found in 357 genes and selected for further analysis. When focused on hemizygous mutations within the retained 5q allele, CSNK1A1 mutations were the most common, found in 4 pts, while other genes were only sporadically affected. Among heterozygous mutations on the non-deleted portion of del(5q) and other chromosomes (Chr), we found several novel mutations, in addition to TP53 (n=26), DNMT3A (n=8), PRPF8 (n =8), RUNX1 (n=5), TET2 (n=5), and ASXL1 (n=4), among others. Furthermore, LOH/haploinsuffciency of genes on 7q (e.g., LUC7L2, CUX1, EZH2 and MLL3) appears to be a common defect seen in pts with non-isolated del(5q), suggesting synergistic functional defects. When functionally grouping gene mutations, DNA methylation family (8 cases) and transcription factor mutations (29 cases) were associated with advanced disease (AD) and a CK. Heterozygous mutations in TP53 (34%) or deletions involving the TP53 locus (23%) resulted in total of 42% of cases carrying either TP53 LOH or mutation. TP53 lesions were more common in pts with AD vs. low risk. (21 vs. 5 p =.0008). In contrast, TP53 mutations are found in 8-10% of cases of MDS. A total of 34 pts were treated with LEN and subgrouped into responders (n=17) vs. refractory (n=9) with an overall response rate of 65%. When mutational profiles were compared, the presence of TP53 mutations did not preclude responsiveness to LEN. CK was present in 12% of responders vs. 67% of refractory pts. The most frequent Chr abnormalities were -7/7q (0% vs. 67% in responders vs. refractory) and 17p-(6% vs. 67% in responders vs. refractory) suggestive of their role in LEN resistance. In addition to cross sectional analysis, our WES study using paired Germline/tumor samples followed by deep sequencing facilitated analyses of clonal architecture by examining clonal dynamics over time. Assessment of del(5q) clone size by allelic imbalance combined with clonal burden by VAF allowed us to reconstruct the clonal hierarchy: in 73% of cases, del(5q) appeared to be the initial defect followed by subsequent mutations (e.g., TP53, DNMT3A, IDH2). In contrast, in 24% of cases, TP53, RUNX1, JARID2, were the primary defect followed by a subclonal del(5q) events. Serial samples collected before and after therapy demonstrated that responses were associated with decreased clonal burden for del(5q) but persistence of certain mutations. In refractory cases, persistent subclonal lesions and the appearance of new lesions were associated with progression. For example, pts with TP53, LAMB4, EPHA6 progressed and acquired additional lesions such as CSMD2 or KCND2, and did not see the disappearance of TP53 alterations upon treatment. In conclusion, no unifying somatic defect was found in pts with del(5q) regardless if the deletion event was primary or subclonal. Most commonly associated lesions were not present on the retained 5q alleles but rather other chr yet modified clinical behavior, including responsiveness to LEN. Disclosures Bejar: Celgene: Consultancy, Honoraria; Alexion: Other: ad hoc advisory board; Genoptix Medical Laboratory: Consultancy, Honoraria, Patents & Royalties: MDS prognostic gene signature. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: Myelodysplastic syndromes (MDS) are clonal stem cell diseases of the bone marrow (BM) characterized by a dysfunction of hematopoiesis commonly resulting in cytopenias, dysplasia and an increased risk of acute myeloid leukemia (AML) development. In up to 90% of MDS cases acquired somatic mutations can be identified. Additionally, these aberrations constitute important markers for diagnosis and risk stratification. Currently, the use of BM aspirates is the gold standard for cytogenetic and molecular genetic analysis. However, frequent analyses of peripheral blood (PB) samples are of special interest for monitoring the natural course and therapy response in MDS since this might not be feasible with BM specimens due to ethical and other reasons. The aim of this study was to investigate whether the high sensitivity of targeted deep sequencing (TDS) allows reliable detection of somatic variants also from PB samples and whether the molecular profiles are comparable between these different sources of material. Additionally robustness, feasibility and comparability of the method were verified by inter-laboratory comparisons. This study included 31 patients from two centers, in Barcelona (Spain) and Göttingen (Germany) (12x RCMD, 4x RAEB-2, 4x sAML, 4x CMML-1, 2x MDS-RA, 1x MDS-U, 1x RAEB-1, 1x RCMD-SA, 1x RARS, 1x CCUS). For all patients, genomic DNA was extracted from concurrent mononuclear or total bone marrow cells (BMC), mononuclear peripheral blood cells (PBMC) and immunomagnetically (MACS) enriched circulating CD34+ cells (CD34+). Library preparation was performed using a custom hybridization-probe based panel (Nimblegen SeqCap EZ, Roche) including 83 myeloid-related genes (Barcelona) or the TruSight Myeloid Sequencing-Panel (Illumina) including 53 target genes (Göttingen). Sequencing was performed on MiSeq instruments. Reads were analyzed using local bioinformatic pipelines. Variants were filtered according to read depth (〉 100x), population frequency (〈 1%), their impact on protein integrity or function and evaluated by visualization on the Integrative Genome Viewer Software. Somatic origin of the variants was confirmed by sequencing of control samples from MACS enriched circulating T-lymphocytes (CD3+). Somatic mutations were discovered in 29 of the 31 analyzed patients. Overall we identified 76 aberrations in 22 genes (12x TET2, 7x SF3B1, 7x SRSF2, 5x EZH2, 5x ASXL1, 5x U2AF1, 5x RUNX1, 4x ZRSR2, 4x TP53, 3x NRAS, 3x STAG2, 2x DNMT3A, 2x IDH1, 2x BCOR, 2x KRAS, 2x PTPN11, 1x PDGFRB, 1x IKZF1, 1x IDH2, 1x ATRX, 1x CSNK1A1, 1x ETV6). Literally all variants were found in BMC (n=74) as well as in circulating CD34+ cells (n=72) and PBMC (n=73). The discordance between the three sample types was random (5x RCMD, 2x sAML, 1x MDS-RA, 1x RARS). However, for five variants the allele frequency (VAF) values, which correlate with the clone size of the malignant cell population, were below 5% in PBMC samples. Therefore these variants were likely to be overlooked in case only PBMC would have been tested. There was no significant difference (p=0.774) between the VAF values measured in BMC (average: 40.0%) and enriched CD34+ cells (average: 41.3%). In contrast VAF values of PBMC (average: 30.1%) deviate significantly from both, BMC (p=0.007) as well as circulating CD34+ cells (p=0.027) (Figure 1). Our findings indicate that TDS enables the adequate detection of somatic mutations from BM, circulating CD34+ cells and for the most part also in PBMC preparations. However, the malignant cell population seems to be less abundant in the PBMC fraction and therefore the detection and especially the quantification of clonal somatic variations is more challenging in this sample type. In the present study we demonstrate that enrichment of circulating CD34+ cells from PB can overcome this problem and provide data that are equivalent to the results obtained from the analysis of BMC, especially for follow up or minimal residual disease analyses. We conclude that enrichment of CD34+ cells from PB constitutes an appropriate alternative for the reliable detection and quantification of somatic aberrations in MDS patients. Usage of circulating CD34+ cells in routine diagnostic next generation sequencing applications would significantly reduce invasive clinical interventions and could therefore improve diagnostics and disease monitoring. Support: PI 11/02010, PI/14/00013, RD12/0036/0044, AR 14/34, R14/03, SGR225 Disclosures No relevant conflicts of interest to declare.

Description: Background: Lenalidomide (LEN) is an immunomodulatory drug which binds cereblon through a glutarimide ring modulating the substrate specificity of CRL4CRBNE3 ubiquitin ligase complex, resulting in the proteosomal degradation of specific disease-related proteins. LEN is approved for the treatment of RBC transfusion-dependent (TD) low and int-1 risk MDS patients with del(5q), 70% of whom reach RBC transfusion-independence (TI) and 50% complete cytogenetic remission. It is also under investigation in RBC -TD low and int-1 risk MDS without del(5q) resistant to erythropoietin stimulating agents and with 20-30% of patients reaching RBC-TI. Herein we aimed to study whether molecular mutations in MDS patients with and without del(5q) influenced the response to LEN treatment. Methods: We collected 95 MDS patients treated with LEN, 72 with del(5q) and 23 without del(5q) (23/23: normal karyotype, presence of ringed sideroblasts and SF3B1MUT). Retrospective clinical information was available for 65 patients. To characterize the mutational spectrum of patients with del(5q) and non del(5q), we combined results from multi amplicon targeted sequencing Ion Torrent (44 cases) and from captured-based targeted deep sequencing (51 cases). The Ion Torrent panel included 39 of the most frequently mutated genes in MDS (ASXL1, BCOR, BRAF, CBL, CDKN2A, CEBPA, DNMT3A, ETV6, EZH2, FLT3, GNAS, IDH1, IDH2, JAK2, KIT, KRAS, LUC7L2, MPL, NF1, NPM1, NRAS, PHF6, PTPN11, RAD21, RPS14, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SMC3, SPARC, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2). The Ion Torrent panel was then extended for the captured-base sequencing and updated with the addition of 43 genes including BCORL1, CALR, CSNK1A1 and KDM6A. The average depth per gene was 780x per sample. Results: Patients with del(5q) vs. non del(5q) had an equal risk distribution (LR: 46/55 vs. 21/22; HR: 9/55 vs. 1/22). We restricted our analyses to LR patients. Median age was 69 yrs (34-90) and M:F ratio was 15:50. A significantly higher median of hemoglobin (Hb) levels were found in del(5q) in comparison to non del(5q) patients (9 g/dL (6-13) vs. 8 g/dL (7-9); P=.001). Molecularly we found that a slightly lower number of mutations occurred in del(5q) [2 mutations (0-6)] compared to the number of mutations in non del(5q) [3 mutations (1-7); P=.001]. WHO 2008 distribution was significantly (P