Identification of differentially expressed microRNAs in human male breast cancer

<p>Abstract</p> <p>Background</p> <p>The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.</p> <p>Methods</p> <p>The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.</p> <p>Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.</p> <p>Results</p> <p>Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.</p> <p>Conclusions</p> <p>Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.</p

LINE-1 hypomethylation in human hepatocellular carcinomas correlates with shorter overall survival and CIMP phenotype.

Reactivation of interspersed repetitive sequences due to loss of methylation is associated with genomic instability, one of the hallmarks of cancer cells. LINE-1 hypomethylation is a surrogate marker for global methylation loss and is potentially a new diagnostic and prognostic biomarker in tumors. However, the correlation of LINE-1 hypomethylation with clinicopathological parameters and the CpG island methylator phenotype (CIMP) in patients with liver tumors is not yet well defined, particularly in Caucasians who show quite low rates of HCV/HBV infection and a higher incidence of liver steatosis. Therefore, quantitative DNA methylation analysis of LINE-1, RASSF1A, and CCND2 using pyrosequencing was performed in human hepatocellular carcinomas (HCC, n = 40), hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5), and corresponding peritumoral liver tissues as well as healthy liver tissues (n = 5) from Caucasian patients. Methylation results were correlated with histopathological findings and clinical data. We found loss of LINE-1 DNA methylation only in HCC. It correlated significantly with poor survival (log rank test, p = 0.007). An inverse correlation was found for LINE-1 and RASSF1A DNA methylation levels (r2 = -0.47, p = 0.002). LINE-1 hypomethylation correlated with concurrent RASSF1/CCND2 hypermethylation (Fisher's exact test, p = 0.02). Both LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation were not found in benign hepatocellular tumors (HCA and FNH). Our results show that LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation are epigenetic aberrations specific for the process of malignant liver transformation. In addition, LINE-1 hypomethylation might serve as a future predictive biomarker to identify HCC patients with unfavorable overall survival

Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing

<div><p>Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies) was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17) as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10). Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41) were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43) could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., <i>TET2</i>, <i>ASXL1</i>, <i>DNMT3A</i>), demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons strongly argue for replacing Sanger sequencing by this semiconductor-based targeted resequencing approach.</p></div

Comparison of sequencing performance of the corresponding fresh and archival patient samples.

<p>Mapped reads, reads on target, mean coverage, and mean read length of the sequenced sample sets (each n = 10) of the aspirate DNA and three isolation methods of FFPE samples are shown. Additionally total reported variants are shown and the number of C>T/G>A variants below 10%.</p

Verification of mutations from validation cohort by Sanger sequencing.

<p>(A) Mapped reads of a Patient with RCMD loaded in the IGV browser (Version 2.3.34). <i>TET2</i> p.L541X (c.1620delT) variant with 29.1% variant frequency in 592 reads is shown (B) Results from Sanger sequencing of <i>TET2</i> Exon 3. (C) Mapped reads of a Patient with AML loaded in the IGV browser. <i>NPM1</i> p.W288CfsX12 (c.860_863dupTCTG) mutation with 43.7% variant frequency is shown (D) Results from Sanger sequencing of <i>NPM1</i> Exon 12. Please note that Sanger sequencing shows the reverse sequence.</p

Technical run data from Torrent Server.

<p>(A) Loading intensity of 318 v2 chip with 6 aspirate samples. (B) Loading intensity of 318 v2 chip with 6 FFPE samples isolated with the standard protocol. Read length histogram represent selected samples from aspirate (C), standard protocol (D), GeneRead Kit (E), and standard protocol + UNG pre-treatment (F) sample sets.</p

Estimation of the turn-around time and the costs for 12 samples processed with the AML panel and IonTorrent PGM semiconductor sequencer.

<p>Reagent costs are calculated from net list prices for Germany.</p

Overview of verified known pathogenic mutations and additional detected variants in the validation cohort (n = 41).

<p>All variants are reported as mutations by Cartagenia Bench Lab NGS software (Version 3.1.2).</p

Verification of mutations from training cohort by Sanger sequencing.

<p>(A) Mapped reads of Patient 10 loaded in the IGV browser (Version 2.3.34). <i>CBL</i> p.T377A (c.1129G>A) variant with 50.6% variant frequency is shown. (B) Results from Sanger sequencing of <i>CBL</i> Exon 8. (C) Mapped reads of Patient 2 loaded in the IGV browser. <i>TP53</i> p.S241T (c.721T>A) with 24.7% variant frequency is shown (D) Results from Sanger sequencing of <i>TP53</i> Exon 7.</p

Comparison of sequencing performance of the corresponding fresh and archival patient samples.

<p>Mapped reads, reads on target, mean coverage, and mean read length of the sequenced sample sets (each n = 10) of the aspirate DNA and three isolation methods of FFPE samples are shown. Additionally total reported variants are shown and the number of C>T/G>A variants below 10%.</p