ALBERT

Beschreibung: The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving the diagnosis, treatment selection, and prediction of prognosis in cancer. Using whole-genome methylation data from The Cancer Genome Atlas (TCGA) and machine learning methods, we evaluated the utility of DNA methylation for differentiating tumor tissue and normal tissue for four common cancers (breast, colon, liver, and lung). We identified cancer markers in a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples. We replicated our findings in a separate TCGA cohort of 791 tumor samples and 93 matched adjacent normal tissue samples, as well as an independent Chinese cohort of 394 tumor samples and 324 matched adjacent normal tissue samples. The DNA methylation analysis could predict cancer versus normal tissue with more than 95% accuracy in these three cohorts, demonstrating accuracy comparable to typical diagnostic methods. This analysis also correctly identified 29 of 30 colorectal cancer metastases to the liver and 32 of 34 colorectal cancer metastases to the lung. We also found that methylation patterns can predict prognosis and survival. We correlated differential methylation of CpG sites predictive of cancer with expression of associated genes known to be important in cancer biology, showing decreased expression with increased methylation, as expected. We verified gene expression profiles in a mouse model of hepatocellular carcinoma. Taken together, these findings demonstrate the utility of methylation biomarkers for the molecular characterization of cancer, with implications for diagnosis and prognosis.

Beschreibung: Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

Beschreibung: Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p 〈 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p 〈 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Beschreibung: Abstract 969 Selection of the appropriate therapy for patients with myelodysplastic syndromes (MDS) depends heavily on the predicted prognosis of each afflicted individual. Prognostic scoring systems help stratify patients into risk groups, but outcomes can be highly variable even within these groups. Of particular concern are patients predicted to have lower risk disease that go on to progress more rapidly than expected. Such patients may not be offered risk-appropriate therapy at a time when it might be of greatest benefit. A prognostic model that better predicts survival in patients believed to have lower risk disease has been proposed by investigators at the MD Anderson Cancer Center, but not yet validated in an independent cohort. Acquired genetic mutations can also identify patients with higher-than-predicted disease risk. We have previously demonstrated that mutations in any of five genes (TP53, EZH2, ETV6, RUNX1, and ASXL1) predict a poorer prognosis independently of the International Prognostic Scoring System (IPSS). In this study, we examined 289 MDS patients with Low or Intermediate-1 IPSS risk for mutations in 21 genes, including two genes that have recently been reported to be frequently mutated in MDS: DNMT3A and SF3B1. We validate the ability of the Lower-Risk MD Anderson Prognostic Scoring System (LR-PSS) to more finely risk-stratify patients using an independent cohort and identify gene mutations independently associated with clinical features and overall survival. Patients were stratified into one of three risk categories using the LR-PSS shown in the Table. The 58 patients (20%) assigned to Category 1 had a median survival of 5.19 years (95% confidence interval in years [CI] 3.05–10.34), compared to 2.65 years (CI 2.18–3.30) for the 160 patients (55%) in Category 2, and 1.11 (CI 0.82–1.51) for the 71 patients (25%) in Category 3. Differences in survival were significant between all three categories (p 〈 0.001 for all comparisons). Point mutations were identified in 63% of samples, including 64% of those with a normal karyotype. The 10 most frequently mutated genes were TET2 (23% of cases), SF3B1 (21%), ASXL1 (15%), DNMT3A (14%), RUNX1 (9%), EZH2 (8%), JAK2 (3%), NRAS (3%), TP53 (2%), and ETV6 (2%). Mutations of SF3B1 were highly enriched in cases of refractory anemia with ring sideroblasts (RARS; 32 out of 43, 74%), associated with normal blast percentages (p = 0.04) and neutrophil counts (p = 0.002), and more likely to be present in cases with platelet counts greater than 450,000/μl (p 〈 0.001). We extended our analysis of SF3B1 mutations by adding a separate cohort of 98 RARS patients with Low or Intermediate-1 IPSS risk for a total of 141 cases. In this extended RARS set, SF3B1 mutations were associated with improved survival even after adjustment for IPSS risk group or LR-PSS category (hazard ratio [HR]=0.49; CI 0.29–0.81, HR=0.35; CI 0.21–0.58, respectively). SF3B1 is the first gene mutation independently associated with a favorable prognosis in non-CMML MDS. In contrast, DNMT3A mutations were not associated with differences in overall survival in the 289 patients with lower IPSS risk MDS. In a model generated from stepwise Cox regression analysis that considered LR-PSS risk categories and the mutation status of the 13 most frequently mutated genes as candidates, only EZH2 mutations emerged as a LR-PSS-independent risk factor associated with a poor prognosis (HR=2.90; CI 1.86–4.53). In a similar model using IPSS risk groups, EZH2 (HR=2.85; CI 1.78–4.57), NRAS (HR=2.78; CI 1.35–5.72), and ASXL1 (HR=1.60; CI 1.09–2.34) were significant IPSS-independent risk factors. Mutations in genes such as ASXL1, RUNX1, NRAS, and ETV6, which are associated with poor survival in unselected MDS patients, were most common in patients assigned to the LR-PSS risk Category 3, indicating that this prognostic model may capture more clinically relevant information associated with adverse gene mutations. In conclusion, this study validates the LR-PSS 's ability to identify higher risk MDS patients among those with lower IPSS risk and demonstrates that point mutations are common in this cohort, associate with specific clinical features, and independently provide both favorable and adverse prognostic information.Table:Table:. The Lower-Risk MD Anderson Prognostic Scoring System (LR-PSS) Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 311 Background: The clinical heterogeneity observed in patients with myelodysplastic syndromes (MDS) is reflected in the wide range of molecular abnormalities than can be acquired by their diseased cells. Somatic mutations in specific genes have been associated with clinical manifestations of MDS, yet several of these genetic lesions have prognostic significance that is independent of the International Prognostic Scoring System (IPSS).1 Some genetic abnormalities can predict response to treatment, but are found only in the minority of patients. Somatic mutations are more common than karyotype abnormalities and can be identified in over 75% patients with MDS.2 One small study noted that mutations in TET2 appear to predict response to azacitidine.3 However, other mutations were not explored in this study. Similarly, certain karyotype abnormalities have been shown to predict outcomes in MDS patients undergoing stem cell transplantation, but somatic gene mutations have not been explored in this context.4 Here we report on gene mutations detected in over 200 MDS patients prior to treatment with hypomethylating agents (Cohort 1) and in 76 patients prior to undergoing stem cell transplantation (Cohort 2). Methods: Cohort 1 is comprised of MDS patients treated with azacitidine or decitabine at the Dana-Farber Cancer Institute (n =48), the MD Anderson Cancer Center (n =109), or in the ADOPT clinical trial (n =83). A total of 47 patients received azacitidine, 164 received decitabine, and 29 patients received combination therapy. Bone marrow mononuclear cell DNA was obtained from patients prior to treatment with a hypomethylating agent. Responses were assessed using the International Working Group (IWG) Response Criteria for MDS revised in 2006.5 DNA was sheared, ligated to adaptors and barcodes, quantified, and then pooled prior to hybrid-capture enrichment of the target genomic regions. Cohort 2 includes 76 patients with MDS who underwent stem cell transplantation at the Dana-Farber Cancer Institute between 2004 and 2009. Patients without a pretransplant biopsy sample or who died within 30 days of after transplant were excluded. Bone marrow mononuclear cell DNA was obtained from patients prior to transplant and enriched for target genomic regions using the HaloPlex PCR system. For both cohorts, the target genomic region consisted of 74 genes including all genes reported to be mutated in MDS. Target enriched sample pools were sequenced on an Illumina HiSeq 2000. Sequence data was aligned and mutations called using the analysis pipeline at the Broad Institute. Variants previously shown to be germline or present in databases of normal variants (dbSNP 132 or NHLBI Exome Sequencing Project) at a population frequency of 1% or more were discarded. Remaining variants were considered candidate somatic mutations. Candidate mutations were validated in tumor DNA by Sanger sequencing and examined in germline DNA when a matched sample was available. Results: Candidate mutations were identified in over 90% of patients in each cohort. The most frequently mutated genes in Cohort 1 were ASXL1 (39%), SF3B1 (24%), TET2 (23%), RUNX1 (19%), SRSF2 (18%), and DNMT3A (15%). The rate of complete response, partial response, or hematologic improvement (ORR) was 47% and did not differ between sites (ORR for DFCI, MD Anderson, and ADOPT were 42%, 45%, and 53% respectively). Nor was there a significant difference between hypomethylating agents (ORR for decitabine and azacitidine were 44% and 48% respectively). The most frequently mutated genes in Cohort 2 were ASXL1 (25%), TP53 (22%), DNMT3A (17%), and RUNX1 (14%) with 37% of patients carrying a mutation in a splicing factor gene (SF3B1, U2AF1, SRSF2, or ZRSR2). The mutation profile in this higher prognostic risk group includes a high frequency of mutations in genes associated with a poor prognosis, particularly TP53. In contrast, the frequency of mutations in genes with a neutral or favorable association with prognosis (SF3B1 and TET2) was lower. Analyses comparing mutation status, response to treatment, and overall survival are in progress for both cohorts. Conclusions: Abnormalities in recurrently mutated genes can be found in most MDS patients using a pooled next-generation sequencing approach. Single genes are mutated frequently enough in our cohorts to allow us to determine if they can predict response to treatments such as hypomethylating agents and stem cell transplantation. Disclosures: Bejar: Genoptix: Consultancy. Kantarjian:Genzyme: Research Funding. Ebert:Celgene: Consultancy; Genoptix: Consultancy.

Beschreibung: Key Points Higher abundance TET2 mutations are associated with increased response to hypomethylating agents, particularly when ASXL1 is not mutated. TP53 and PTPN11 mutations are associated with shorter overall survival after hypomethylating agent treatment, but do not predict response.