Researchers from the Jackson Laboratory have begun using targeted sequencing to profile the lab's growing cohort of mouse avatar models — populations of mice engrafted with samples of a single patient's tumor.
The lab's patient-derived xenograft, or PDX, mouse program includes tumors from about 500 separate cancer patients of which more than 100 have been successfully grown in mice populations. The group initially collected genomic data from the first grafted mouse tumor samples using microarrays and other tools. Recently, the program has begun supplementing that by sequencing the same samples using Illumina's TruSeq Amplicon - Cancer Panel on the MiSeq, starting with 20 tumor samples as a pilot study of the approach.
According to Neal Goodwin, the PDX mouse project's leader, and Doug Hinerfeld, a member of the project, the plan is to now run the sequencing panel on all the remaining samples. The group is also beginning early experiments with RNA-seq methods as well.
Hinerfeld told Clinical Sequencing News that mutational information gleaned from the sequence results will help potential customers or collaborators choose PDX mouse models to test molecularly targeted cancer therapies.
"The idea is that the data can be used for the purpose of selecting which tumor models are best for which drugs … so people can then access the [sequencing data] and find a cancer with a particular mutation … [that] might be a good model to use in [a particular] drug study," Hinerfeld said.
Goodwin told Clinical Sequencing News this week that the mutations are "probably the number one thing that people look at when determining which models they want to use for which purpose."
Tumors for the PDX mouse populations have come from a consortium of institutions and represent a wide range of tumor types. Hinerfeld said the group has identified a number of potentially useful mutations after sequencing 20 samples in its pilot effort, including EGFR, KRAS, and p53.
The Illumina TruSeq cancer panel, launched earlier this year, covers mutational hotspots on 48 genes. Hinerfeld said the JAX team is able to sequence up to 20 samples per run on the MiSeq instrument at a cost of about $200 per sample.
Cost was a significant concern for the group, and a large reason why the team started with the targeted approach, rather than sequence exomes or whole genomes, he said. "One of the unique aspects of doing genomic analysis of these tumors, and one of the things about this that’s challenging is that a significant percent of the nucleic acids you extract are going to be of mouse origin. So you're going to have mouse blood vessels, mouse cells, mouse stroma that are going impregnate this tumor."
"We knew upfront this would be a challenge. Because the sequence you are going to get is going to be a mixture. We also knew we wanted to be able to sequence deeply so we could identify low-level somatic mutations," he said. "The lab is funding this ourselves right now, so really deep exome sequencing and genome sequencing was not in the cards for us financially.
The Illumina cancer panel offered the group a way to measure mutation status in a more affordable way, he said. "We're sequencing these 48 genes, identifying all the mutations that pass filters and then whittling those down to ones that are likely impactful in those genes," Hinerfeld said.
"That data is then available to anyone who might want access to these models – to see if they have mutations in P53, or KRAS, EGFR, or whatever their interest is."
In the team's pilot group of 20 samples, results of the cancer panel marked 12 samples as having potentially damaging mutations — several in genes that are the subject of targeted drug development research.
The group's mouse models are developed by grafting tumors into larger and larger groups of immune-deficient mice. The ranks multiply as tumor tissue grows in each iteration or generation of animals.
Goodwin said that the team is currently working particularly with lung cancer samples from UC Davis, examining drug response and resistance in EGFR- and KRAS-mutated PDX models.
The group has also created model populations with EGFR-mutated glioblastoma to test EGFR-targeting molecules being developed as potential treatments for brain cancer.
Goodwin said the program has been "very well received" by the biopharmaceutical industry. He said the Jackson lab currently distributes PDX models for groups to use in their own research and also contracts with private sector partners to do in-house studies through the JAX cancer services lab. Right now, he said most of that research is related to testing efficacy or mechanism of action.
According to Hinerfeld, the eventual hope is to gain a more comprehensive genomic picture for the mouse PDX mouse populations. He said that Edison Liu, the director of the Jackson Lab, eventually hopes for the mouse avatars to be able to serve as a testing ground for real-time personalized medicine, as a way of identifying the best therapies for patients currently undergoing cancer treatment.
"People are beginning to live long enough that the PDX models could catch up to the disease and be used predictively," Goodwin explained.
On the sequencing side, Hinerfeld said, "ideally … we want to do genome sequencing. We want to be able to identify structural variants."
"We would be doing exome sequencing on all these right now if we could, but [because we] have to sequence quite deeply, it gets too expensive. 30X is not going to be sufficient for identifying lower-level somatic SNPs."
Sticking with the targeted approach for the time being, Hinerfeld said that the group hopes Illumina might expand the cancer panel.
The researchers are also planning to experiment with RNA-seq and have just begun running some initial samples. Hinerfeld said the group expects the confounding influence of mouse RNA to be a problem with this as well, and plans to test some established methods for informatically dealing with the two-species issue as well as work on an in-house developed software solution.
Hinerfeld also said the group may experiment with other sequencing technologies, like the Ion Torrent sequencing platform from Life Technologies. "We are looking at their platform, particularly when the [Ion Proton] comes out," he said.
Goodwin said that he has been impressed with the TruSeq cancer panel. "It's not comprehensive but it covers … very popular and important onco-mutations."
He also said the group is planning to add more robust mutation detection for the PDX models with a higher-throughput system, but declined to say what that system would be.