MassGenomics

The Genome Center at Washington University is currently hosting a remarkable two-day event focused on the study of cancer genomics.  Yesterday there was a symposium on the School of Medicine campus, featuring speakers from major genome centers around the world, who delivered an excellent series of talks on recent advances in cancer genome research.  Here were the highlights.

Mutational Signatures in Lung Cancer (Peter Campbell, WTSI)

First up was Peter Campbell from Wellcome Trust Sanger Institute, who presented their first cancer genome – a small lung cancer cell line called NCI-H209.  Using the ABI SOLiD platform, they sequenced NCI-H209 and a matched B-cell sample from the same individual to 30-40x coverage.  Extensive PCR-based validation yielded almost 23,000 somatic substitutions and over a hundred structural events (indels and rearrangements).  As expected, the mutational spectrum was enriched for G->T and C->A changes associated with adduct formation on guanine nucleotides induced by benzopyrene, the chemical mutagen found in tobacco smoke.  Dr. Campbell also described some of the complex rearrangements observed in the paired-end sequencing data, which were particularly convincing when overlaid with spectral karyotyping images.

Next-Gen Sequencing Strategies to Study Cancer Genomes (Elaine Mardis, WU)

Next was our own Elaine Mardis, who gave an excellent overview of the strategies developed here to apply NGS to cancer genomes.  She described five key elements to success in this arena:

1. Genomic characterization prior to sequencing. For example, at WashU we type tumor and normal samples on genome-wide SNP arrays, which yield tumor purity/ploidy estimates, LOH information, and a dense set of SNPs for tracking the coverage of genomes by Illumina sequencing.
2. Resource characterization. The tissue preservation method, DNA/RNA quality and quantity, and pathology information are all critical components.  Also important are high-quality clinical data (diagnosis, chemotherapy/radiation protocols, and outcome), informed consent, IRB approval, and additional cases of the same cancer subtype for recurrency screening.
3. Data production capacity.  US genome centers seem to have this, either in the form of Illumina (WashU and Broad) or ABI SOLiD (Baylor).  It’s not just the throughput of the machines, either – it’s the ability to construct sequencing libraries from ever-shrinking DNA inpus.  Tumor samples are precious, and the ability to use only a tiny amount of DNA or RNA while achieving informative results is one of the key areas of focus of tech development groups.
4. Informatics and bioinformatics.  We have entire groups devoted to LIMS, pipeline automation, medical genomics, and sequence data submission.  Other important elements of bioinformatics that Elaine touched on were data display interfaces for collaborators and high-end data storage and computational infrastructure.
5. Validation and recurrent site screening.  This the essential coup de grace for tumor genome characterization, in which we validate somatic mutations and identify those that are recurrent in other samples of the same subtype, the best indication that we currently have of pathological relevance.

Elaine also discussed the rapid scaling up of TCGA (which is adding 20 tumor types thanks to ARRA funds) and other projects, which will only exacerbate the challenges of scale that NGS platforms have already presented.

Integrating Genomics with Biology (Richard Gibbs, Baylor)

Richard Gibbs gave an action-packed talk of some relevant work going on at Baylor, both for cancer and inherited diseases.  They are applying an intriguing if controversial multiple-platform strategy for whole genome sequencing: deep (20-30x) coverage on ABI SOLiD and light (6-10x) coverage on 454. “We’re just telling people that if you do it twice, you’ll get it right,” Dr. Gibbs said.  One interesting project is an investigation of Charcot-Marie Tooth (CMT) syndrome, a recessive inherited disorder where the locus is unknown.  Whole-genome sequencing of an affected individual on ABI SOLiD identified a few dozen novel missense mutations; among them lurked the causal variant, which was found to segregate with the disease in a family cohort.

Dr. Gibbs also gave an overview of their investigations into heritable variants in pediatric cancers (in collaboration with MD Anderson).  There’s also a lot of work under way for TCGA, not just the 6K capture project, but also adjunct analyses of gene expression, DNA copy number, microRNA, and DNA methylation data being generated on TCGA samples.

Insights into Rare Tumors (Steven Jones, BC Cancer Agency)

Steven Jones from BC Cancer Agency retold the story of the rare tongue adenocarcinoma that I heard at AGBT 2009.  What I didn’t know about BCCA is that under the Canadian universal healthcare system, they see all of the cancer patients in the surrounding population of over 4 million citizens.  One of these was a rare one – an 80 year old man with adenocarcinoma of the tongue.  It was removed surgically, of course, but in a short time metastasized to the lungs.  The clinician prescribed erlotinib, an EGFR inhibitor, but unfortunately the patient did not respond.  To help the patient, and also make some advances in tech development, Jones and his colleagues did whole-genome and RNA-Seq of the tumor samples and matched normals.  There were just four somatic mutations: two in known cancer genes and two in zinc finger proteins (these remain unexplained).  Transcriptome and copy number analysis showed that the tumor had loss of PTEN and down-regulation of SMAD4.  Unfortunately, it had recently been shown that tumors lacking PTEN and TP53 don’t respond to TK inhibitors like erlotinib.  However, this particular tumor showed an amplification of Ret, and as it happened, the drug bank had a single drug, sunitinib, that was known to inhibit Ret.  The patient’s response, initially, was quite dramatic – all of the metastases vanished.  Sadly, several months later they turned up again, and this time were resistant even to sunitinib.  Still, the results of this effort were promising, because genomic information was used to keep cancer at bay, if only for a short time.

Genomic Medicine in Pediatric Brain Tumors (Chinc C. Lau, Baylor)

Ching Lau of Baylor presented genomic studies of medulloblastoma (MBM), which accounts for 20% of all brain tumors and has a 60% survival rate.  Classification of MBM patients in the past was relatively crude – based on the amount of residual tumor post-surgery and metastatic status. Using gene expression profiling, Lau and colleagues identified 4-5 distinct clusters.  Two clusters were associated with known cancer pathways – SHH signaling and WNT activation. The same four clusters could also be isolated by unsupervised miRNA clustering. Also, gene expression analysis showed that ERBB2 expression correlates with outcome (higher expression = poor prognosis).

Finally, Dr. Lau mentioned some future directions for targeted cancer therapy.  One of these that I readily admit I don’t understand: cytotoxic T-cells with Chimeric TCRs. Evidently these are T-cells that recognize and attack cancer cells in the body.  There was a short movie, courtesy of Dr. Lau’s collaborators, in which we saw these specially programmed immune cells recognizing and attacking a tumor that was roughly four times their size.  It was like watching ants swarm a piece of fruit on the sidewalk, and very compelling.

Evolution of a Breast Cancer Tumor (Samuel Aparicio, BC Cancer Agency)

Dr. Aparicio presented a study recently published in Nature and already discussed on Massgenomics.  However, he did discuss the continuing challenge of mutation heterogeneity in tumors – we can no longer refer to mutations as present or absent, but instead should report their frequency, which represents the proportion of clones with each mutation.  The question of how deep we need to sequence to find the very rare variants has yet to be answered.

Breast Cancer Genomics (Matthew Ellis, Siteman Cancer Center)

Matthew Ellis, our collaborator from the Siteman Cancer Center, presented very recent work we’ve done on a basal subtype breast cancer.  A quartet of samples were sequenced in this study – the primary breast tumor, the matched normal tissue, the brain metastasis (from which the patient died), and finally, a mouse xenograft model developed in “humanized” NODSCID mice.  We validated some 50 tier 1 mutations, all of which were detected (at some level) in all four samples.  Deep read counts for these mutations in each sample revealed some interesting stories about the progression of the cancer from tumor to metastasis.

Genomic Signatures and Cancer  (Todd Golub, Broad / Dana Farber)

Todd Golub of the Broad Institute and Dana Farber Cancer Center presented his group’s work on Hepatocellular Carcinoma (liver cancer), which is the fifth most common cancer worldwide.  It’s a disease of growing concern on the African and Asian continents, and presents numerous challenges.  Molecular classification “is a mess,” Dr. Golub said, and recurrence is common.  The problem is that there are few frozen samples with long-term outcome information.  Thus, Dr. Golub and his group applied the Illumina DASL assay – which enables very small, highly multiplexed, locus-specific PCR – to perform expression profiling in formalin-fixed paraffin-embedded (FFPE) samples.  They achieved up to 90% success across 6,000 genes in samples that were 25 years old.  Doing so opened up a vast bank of viable samples for gene expression profiling, from which Dr. Golub and colleagues made some interesting findings.

The AML Genome (Tim Ley, WashU and Siteman Cancer Center)

Tim Ley gave the last talk, which highlighted the work that he and colleagues at WashU began around a decade ago on the disease acute myeloid leukemia (AML).  Our goal, he said, was to find 95% of the mutations that occur in at least 5% of AMLs.  To do so will require whole genome sequencing of at least 30 genomes, according to statistics from my colleague Mike Wendl.  Two of these (AML-1 and AML-2) are already done and published, and a number of others are currently under way.  One intriguing bit of work that Dr. Ley described was on the “Mouse APL” project, a knock-in mouse with the PML-RARA gene fusion backcrossed 10+ generations to CBL/BL6 mice.  This yielded inbred strains of mice, some of which developed AML after ~6 months, presumably after acquiring “cooperative” mutations.  One mouse was sequenced to 15x coverage, and among the handful of somatic nonsynonymous mutations found, one was recurrent, not only in the APL mice, but also in the same gene in human tumors.

Bio-IT World’s Kevin Davies has a nice interview with David Dooling, who heads informatics here at the Genome Center and still finds time for his PolITiGenomics blog.  Dooling joined the center in 2001, as the Human Genome Project was wrapping up.  Now, he oversees about half of our informatics group – including IT personnel as well as the developers of our LIMS and automated data pipelines.

All three groups, now that I think about it, have had to address significant challenges during our transition to a next-generation sequencing center.  Our LIMS deals with tens of millions of transactions per month, with a back-end database whose tables sometimes have billions of records.  Our automated pipeline (or APIPE) group develops all of the data pipelines that make whole-genome sequencing feasible – primary data analysis, alignment, coverage reporting, mutation detection, etc.  And the IT group must address the exponentially growing needs of data transfer and compute time for all of it – not an easy job.

Despite these monumental tasks, under the leadership of David and others we’re currently “on a good path” to handle the current generation of sequencing tools.  Of course, that may change in the next couple of years, when technologies like Pac Bio’s SMRT platform begin cranking out single-molecule sequences 1,000 bases long or longer.

In-House and Open Source

Bio-IT World is heavily read by providers of commercial informatics tools, and this is reflected somewhat in the interview.  Davies often asks whether we’re working with any specific vendors, or considering any commercial tools.  Often enough we are – certainly for storage and data transfer systems, things that can’t be built from the ground up.  Yet whenever possible, we opt for the open-source solution.  Every workstation here, for example, is Linux.  We have but one Windows PC, and it’s not allowed to connect to the internet.  Most of our LIMS system and many of our in-house tools were written in Perl.

A Tough Nut for Commercial Vendors

There are, of course, commercial alternatives to anything.  Yet vendors face significant hurdles in marketing products to large genome centers.  The tools that we use are often highly customized, and must continually evolve to address new technological developments.  Take aligners for example.  In the early days of Illumina sequencing, we licensed some commercial software – SLIMsearch and SXOG, for example – because there simply were no good alternatives to ELAND.  Then Maq came along, offering better functionality and performance in a free and open source program (offered, no less, by our trusted friends across the pond).  Exorbitantly priced licenses, needless to say, were quickly not renewed.

Now there are numerous commercial solutions, and we’re often wooed by companies like CLC bio.  Yet for every commercial aligner there’s half a dozen free/open-source alternatives, developed by academic groups that we respect and trust (Maq/BWA from Sanger, Bowtie from UMD, etc.), and many of these tools are pretty damn good.  A commercial option would have to be so incredible, so vastly superior to what’s currently available for us to consider a paid license.  With Bowtie and BWA mapping lanes of 15 million reads in just a couple of hours, the bar is already set pretty high.

Outsourcing Sequencing?

David offers, I think, a polite response to the question of whether we’d ever outsource our sequencing to a third party.  Personally, I can offer two reasons why this will probably never happen.  First, we’re already pretty happy with Illumina, a platform that can deliver whole human genomes at high coverage in just a few weeks.  All available evidence suggests that throughput will only continue to grow, and before long I expect we’ll be doing a genome on a single flowcell or less.  Of course, cost is a consideration (Illumina runs aren’t cheap).  It’s very possible that a company like Complete Genomics might be able to offer similar yields at a substantially reduced cost.  We do use companies like IDT and Agilent, for example, to synthesize oligo sequences that we might make in house.  They can make them cheaper, and faster, than we can.

There is a second, and perhaps more compelling reason to keep sequencing in-house – because we’re in the business of research, and data is precious.  With our current capacity we can track the progress of sequencing runs in real-time, monitor error rates and alignment rates, and assess results the moment data is off of machines.  We maintain a forensics-lab-like “chain of custody” on the data from start to finish.  Doing so offers a certain sense of security, and confidence, when we use the results to tackle some of the most fundamental questions in biology.