DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20°C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples

Mutational analysis of Polycomb genes in solid tumours identifies <i>PHC3</i> amplification as a possible cancer-driving genetic alteration.

Background: Polycomb group genes (PcGs) are epigenetic effectors implicated in most cancer hallmarks. The mutational status of all PcGs has never been systematically assessed in solid tumours. Methods: We conducted a multi-step analysis on publically available databases and patient samples to identify somatic aberrations of PcGs. Results: Data from more than 1000 cancer patients show for the first time that the PcG member PHC3 is amplified in three epithelial neoplasms (rate: 8–35%). This aberration predicts poorer prognosis in lung and uterine carcinomas (Po0.01). Gene amplification correlates with mRNA overexpression (Po0.01), suggesting a functional role of this aberration. Conclusion: PHC3 amplification may emerge as a biomarker and potential therapeutic target in a relevant fraction of epithelial tumours

Integrated analysis of the prostate cancer small-nucleolar transcriptome reveals <i>SNORA55</i> as a driver of prostate cancer progression

Metastasis is the primary cause of death in prostate cancer (PCa) patients. Small nucleolar RNAs (snoRNAs) have long been considered "housekeeping" genes with no relevance for cancer biology. Emerging evidence has challenged this assumption, suggesting that snoRNA expression is frequently modulated during cancer progression. Despite this, no study has systematically addressed the prognostic and functional significance of snoRNAs in PCa. We performed RNA Sequencing on paired metastatic/non-metastatic PCa xenografts derived from clinical specimens. The clinical significance of differentially expressed snoRNAs was further investigated in two independent primary PCa cohorts (131 and 43 patients, respectively). The snoRNA demonstrating the strongest association with clinical outcome was quantified in PCa patient-derived serum samples and its functional relevance was investigated in PCa cells via gene expression profiling, pathway analysis and gene silencing. Our comparison revealed 21 differentially expressed snoRNAs in the metastatic vs. non-metastatic xenografts. Of those, 12 were represented in clinical databases and were further analyzed. SNORA55 emerged as a predictor of shorter relapse-free survival (results confirmed in two independent databases). SNORA55 was reproducibly detectable in serum samples from PCa patients. SNORA55 silencing in PCa cell lines significantly inhibited cell proliferation and migration. Pathway analysis revealed that SNORA55 expression is significantly associated with growth factor signaling and pro-inflammatory cytokine expression in PCa. Our results demonstrate that SNORA55 up-regulation predicts PCa progression and that silencing this non-coding gene affects PCa cell proliferation and metastatic potential, thus positioning it as both a novel biomarker and therapeutic target

Divergent Genomic and Epigenomic Landscapes of Lung Cancer Subtypes Underscore the Selection of Different Oncogenic Pathways during Tumor Development

For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC