Racial differences in endometrial cancer molecular portraits in The Cancer Genome Atlas.

Endometrial cancer (EC) is now the most prevalent gynaecological malignancy in the Western world. Black or African American women (BoAA) have double the mortality of Caucasian women, and their tumours tend to be of higher grade. Despite these disparities, little is known regarding the mutational landscape of EC between races. Hence, we wished to investigate the molecular features of ECs within The Cancer Genome Atlas (TCGA) dataset by racial groupings. In total 374 Caucasian, 109 BoAA and 20 Asian patients were included in the analysis. Asian women were diagnosed at younger age, 54.2 years versus 64.5 years for Caucasian and 64.9 years for BoAA women (OR 3.432; p=0.011); BoAA women were more likely to have serous type tumors (OR 2.061; p=0.008). No difference in overall survival was evident. The most frequently mutated gene in Caucasian and Asian tumours was PTEN (63% and 85%), unlike BoAA cases where it was TP53 (49%). Mutation and somatic copy number alteration (SCNA) analysis revealed an enrichment of TP53 mutations in BoAAs; whereas POLE and RPL22 mutations were more frequent in Caucasians. Major recurrent SCNA racial differences were observed at chromosomes 3p, 8, 10, and 16, which clustered BoAA tumors into 4 distinct groups and Caucasian tumors into 5 groups. There was a significantly higher frequency of somatic mutations in DNA mismatch repair genes in Asian tumours, in particular PMS2 (p=0.0036). In conclusion, inherent racial disparities appear to be present in the molecular profile of EC, which could have potential implications on clinical management

COSMIC mutations called in the primary tumor and axillary lymph node.

<p>Mutations were not present in the normal blood sample. Three mutations were unique to the tumor whilst the node harbored a single unique mutation: a frameshift in the coding sequence of <i>PDS5B</i>, a gene that interacts with the cohesion complex to maintain accurate sister chromatid segregation during mitosis and meiosis and suggested previously as a tumor suppressor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115346#pone.0115346-Denes1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115346#pone.0115346-Kim1" target="_blank">[28]</a>.</p><p>COSMIC mutations called in the primary tumor and axillary lymph node.</p

Known and novel variant counts at a read-depth of ≥100 that overlapped genes and their flanking regions.

<p>Variants were judged as known by their being listed in dbSNP. Variant counts for those unique to both samples are also shown. The lowest frequency variant detected for each variant type (SNV, insertion, deletion, and substitution, respectively) in each sample was 0.88%, 3.7%, 10.07%, and 4.57% in the tumor, and 7.41%, 3.01%, 10.07%, 2.78% in the node.</p><p>Known and novel variant counts at a read-depth of ≥100 that overlapped genes and their flanking regions.</p

Total output (Gb) and depth of coverage for each sample.

<p>The amount that was successfully mapped to the reference genome for each sample was >97%, with a mean of 92.7% of each base achieving ≥40x coverage (or 96.4% for the exome fraction).</p><p>Total output (Gb) and depth of coverage for each sample.</p

Comparison of number of reads between two different extraction kits using the Ion AmpliSeq™Cancer Hotspot Panel v2.

<p>Analysis of 4 plasma samples extracted using both the QIAamp^® DNA Blood Mini kit and the QIAamp^® CNA Kit. In each trace, the number of reads is shown on the Y-axis. Red lines indicate the QIAamp^® CNA Kit and the blue lines represent the QIAamp^® DNA Blood Mini kit. For in depth analysis see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077963#pone.0077963.s003" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077963#pone.0077963.s004" target="_blank">S2</a>.</p

Bioanalyzer analysis of circulating miRNA iolated with different kits.

<p>A-C. Representative Bioanalyser traces of cell-free RNA obtained from 1 ml of plasma using 3 commercial kits. The kits used and RNA integrity (RIN) values are highlighted above the trace and representative gels are shown below each trace. A = extractions using the miRNeasy Serum/Plasma kit, B = <i>mirVana</i> microRNA Isolation kit and C = Circulating Nucleic Acid kit. Quantities obtained are shown in D. Error bar = ±SEM, n = 3 from 3 independent experiments. </p

Recovery of cfDNA and miRNA from plasma: effects of increasing centrifugation speed.

<p>A. Quantitation of cfDNA using qPCR for <i>GAPDH</i>. Blood was processed within 2 hr or 6 hr post-venepuncture and cfDNA isolated from 1 ml of plasma. Whiskers of the box-plot show minimum and maximum values. B. Comparison of miRNA profiles from plasma processed 6 hr post-venepuncture compared to samples processed at 2 hr. Results presented as -ΔC_T (change from 2 hours. Error bar = ±Std Dev, n = 5. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. C. Two-tailed Spearman’s correlation analysis of ΔC_T values obtained from TLDA A cards for duplicate miRNA samples following centrifugation at 1000g and 2000g. Dotted lines represent the 95% confidence interval. D. Two-tailed Spearman’s correlation analysis of ΔC_T values obtained from TLDA A cards for duplicate miRNA samples following centrifugation at 1000g and 10000g. Dotted lines represent the 95% confidence interval. E. Two-tailed Spearman’s correlation analysis of ΔC_T values obtained from TLDA A cards for duplicate miRNA samples following centrifugation at 2000g and 10000g. Dotted lines represent the 95% confidence interval. For C - E, Rho (ρ) and <i>p</i>-values are indicated on each graph. </p