CHI’s X-Gen Congress is an EpiGenie favorite, partly because of it’s epigenetics track that gets woven together with the latest technology, but also because it’s always a local San Diego affair. Not only did the EpiGenie crew get to take a break from the daily grind to attend some great talks, but thanks to the conference organizers, University of Nottingham’s Haneen Al-Azzawi was able to make it all the way from the UK to cover the meeting, present her poster and even squeeze in a little California tour while she was at it. Not a bad gig! Take a look at some of the conference highlights below:
X-Gen Congress Overview
The Cambridge Healthtech Institute and NGS leaders X-Gen Congress focused on clinical genomics and next-generation sequencing. This meeting gave a chance for academics and industry to share their research with many of the speakers representing well known companies like GSK, EpiZyme and Biobase.
One of the great things about this conference was the combination of talks about clinical targets and therapies for the treatment and prevention of disease and talks about new techniques and their uses in clinical applications.
The clinical genomics tracks included talks on cancer and new therapies for cancer treatment and prevention, using RNA as a target for therapy and copy number and structural variation whilst the NGS tracks were dedicated to techniques and how to use them so as to obtain the most accurate and reliable data possible. Some of the most interesting talks included using heat mapping to accurately analyse methylation epiallelles which also helps to reduce sequencing costs, using expression heat maps to locate infectious agents for the diagnosis of cancer and using paraffin embedded tissues for NGS in clinical application.
Remodeling the Epigenome in Cancer
Terry Kelly, USC
Dr. Kelly did an excellent job of speaking for Peter Jones who is well known for his studies in DNA methylation and its role in cancer and differentiation. Kelly started by explaining the basics of epigenetics, a great start to the epigenetics track.
Kelly explained that it’s important to look at nucleosomes, DNA methylation and the chromatin state concurrently to fully understand the epigenetic state within a cell. To analyse nucleosome occupancy Kelly uses a technique called Nucleosome Occupancy and Methylome sequencing (NoMe-Seq). This technique takes advantage of M.CviPIs ability to methylate GpC dinucleotides in regions unoccupied by nucleosomes, a modification which does not normally occur in mammalian cells.
Using this technique she found that the higher the level of gene expression, the more nucleosome depleted a region and vice versa. Looking at transcription start sites with H3K4me3 modifications, these were linked to low endogenous methylation and high accessibility. Had she just looked at histone modifications and DNA methylation, she would not have realised the low level of nucleosome occupancy.
HDAC inhibitors as Breast Cancer Therapeutics
Peter Ordentlich, Syndax Pharmaceuticals
Dr. Odentlich’s company is based around HDAC and epigenetic targeted therapy. HDAC inhibitors (HDACi) “open up” the chromatin structure of DNA and demonstrate anti-cancer activity. Odentlich focused on Entinostat, an oral HDACi which has shown positive results in both breast and lung cancer treatments where HDACi maintain a drug sensitive state within a tumor.
The most common breast cancer subsets exhibit hormone resistance at the estrogen receptor, however, Entinostat restores estrogen expression and receptor sensitivity, a therapy which is appealing to both physicians and patients. Coupling this HDACi with aromatase inhibitor treatment provides a well tolerated therapy with encouraging survival data in patients.
Cancer Treatment Using Histone Methyltransferase Inhibitors
Roy Pollock, Epizyme
Dr. Pollock is the director of biological sciences at Epizyme, a company involved in the discovery and development of histone methyltransferases inhibitors (HMTi) for the treatment of cancer. This work focuses on DOT1L, a HMT which targets histone H3K79 mono, di and tri methylation.
Histones are subject to posttranslational modifications which control structure, recruit proteins to chromatin and ultimately control gene expression. Certain types of Leukaemia show aberrant H3K79 methylation which is mediated by DOT1L and so the development of a DOT1L inhibitor would be a beneficial treatment for this type of cancer.
Epizyme have discovered EPZ004777, a potent and selective DOT1L inhibitor which selectively blocks cellular H3K79 methylation and over four days treatment, depletes the mark. This also leads to the depletion of a specific subset of genes which are upregulated but does not lead to a global collapse in transcription.This work proves that it is possible to produce highly potent and selective HMTi as targeted therapies for cancer.
Methylation Sensitive HRM for Profiling Epialleles
Alex Dobrovic, Peter MacCallum Cancer Centre
Dr. Dobrovic came all the way from Melbourne, Australia to talk about accurate profiling of methylation epialleles. He began by asking “what are we actually measuring when we look at methylation?”. Methylation is rarely homogeneous and techniques such as pyrosequencing do not quantify epialelles.
Dobrovic uses a digital method called Methylation sensitive- high resolution melting (HRM). This technique still utilises bisulfite conversion but takes advantages of the higher melting temperature achieved by unconverted and therefore methylated cytosines. Using an intercalculating fluorescent dye a melt curve is produced which can be used to determine which sites are methylated and unmethylated. This can also cut sequencing costs as unmethylated epialleles can be counted from the melt curve rather than being sent for sequencing
This may prove useful where genes with low levels of heterogeneous methylation are used as cancer biomarkers e.g. the BRCA1 gene. Here avoidance of a biased representation of the epiallelic profile is essential for accurate disease diagnosis and digital approaches are the solution.
Detecting Infectious Agents in Colorectal Cancer
Richard Moore, Michael Smith Genome Sciences Center
Dr Moore is interested in finding infectious agents involved in cancer, and his talk focused on colorectal cancer and finding bacteria associated with the disease. 20% of cancers are caused by infectious agents and colorectal cancer is a good candidate for infectious agent tumour involvement because of its location within the GI tract and exposure to diverse exogenous organisms.
Getting the data from these tumours isn’t a problem anymore, it’s sifting through the data and making sure sample quality is good. The best sample to use is RNA rather than DNA to prevent focusing on something that is active and not potentially dead.
Expression heat maps looking at 669 bacterial species flagged up Fusobacterium nucleatum, a major constituent of plaque, as highly overexpressed (that’s 86x more than the other bacterial species!) in 11 subjects when compared to normal tissue controls. They have shown a strong association between colorectal cancer and Fusobacterium nucleatum.
NGS-Based Diagnostics Using FFPE Samples
Eric Duncavage, Washington University
Dr. Duncavage’s talk highlighted the main differences between NGS for research and NGS for diagnostic testing where only known mutations are of interest to aid clinicians with therapy. Most surgical pathology samples exist as formalin fixed paraffin embedded tissues (FFPE) and this highlights a study which examines the different variables that may affect the sample quality which in turn can make the difference between accurate testing and a false positive.
FFPE is known to depurinate, cross-link and fragment DNA but despite this FFPE tissues are still suitable for NGS for diagnostic testing as comparison between fresh tissue and FFPE tissues shows similar results. Formalin fixation techniques can vary but running a PCR size control, expecting a band no smaller than 300bp helps to determine whether the sample quality is acceptable.
The Washington University St. Louis Genomics and Pathology services (GPS@WUSTL ) offer a range of CAP/CLIA compliant NGS-based testing and have recently launched a clinical exome sequencing service. To celebrate World Rare Disease day 2012 GPS@WUSTL are awarding 99 whole exomes to groups in support of rare disease research and diagnosis. To find out more about the Rare99X Clinical Exome Challenge check out http://raregenomics.org/rare99x/.
The two most popular posters won a well deserved $500 each:
Melanie Lehman, Queensland University of Technology
Using Illumina RNA sequencing to detect loci in android-sensitive prostate cancer which express sense and antisense transcripts this work highlights the importance of using strand specific RNA sequencing protocols. Incorrectly mapping the strand of transcripts can lead to a misrepresentation in levels of protein-coding transcripts and mask the detection of novel non-coding transcripts in downstream analysis.
Yoichi Gondo, Riken BioResource
Rikens study evaluates the precision of Illumina and SOLiD deep sequencing, techniques which are widely used for genomic analysis in humans. Using a mutant mouse carrying millions of listed SNPs and many newly induced base substitutions they detected exome and near exome mutations, some out of target mutations and false positives. To help mapping and other software development for deep sequencing, Ricken are in the process of making their data set open to the research community.
**EpiGenie would like to thank Haneen Al-Azzawi, who is a PhD student in the Stöger-lab at the University of Nottingham, for providing coverage for this conference, as well as CHI for allowing access to their X-Gen Congress event.