By Monica Heger
Two CLIA-certified labs, one at the Children's Hospital of Los Angeles and another at the University of Pennsylvania, are in the process of developing tests to diagnose retinoblastoma, a rare childhood cancer, on Life Technologies' Ion Torrent PGM.
Both tests are targeted for launch in 2012 and are similar in some ways, but there are some slight differences. For example, the Children's Hospital LA test is based on amplicon sequencing and will sequence around 30 percent of the Rb gene, while the test being developed in Arupa Ganguly's CLIA-certified genetics lab at the University of Pennsylvania will sequence the entire Rb gene using an undisclosed capture-based approach.
Timothy Triche, director of the Center for Personalized Medicine at the Children's Hospital of Los Angeles, spoke about his test at a PGM user meeting at the combined International Congress of Human Genetics/American Society of Human Genetics meeting in Montreal earlier this month.
While retinoblastoma itself is around 90 percent curable, patients with inherited mutations in the Rb gene have a high probability of developing a second malignancy like osteosarcoma, which has a survival rate of around 50 percent, Triche said.
The test is designed to more clearly distinguish those patients with Rb mutations, who typically have an inherited form of the disease, from those patients with a sporadic form of the disease.
The team is currently validating the test on the PGM 314 chip, using an amplicon sequencing strategy and 100-base-pair reads, but Triche said that the team will upgrade to Ion's 316 chip and longer read lengths as they become available and as those protocols are validated in the hospital's CLIA lab.
The test sequences around 50 kilobases, or 30 percent, of the entire Rb gene. "Rb is an extreme example" of a gene, Triche said. It contains 27 exons, spanning 200 kilobases. In between six clusters of exons are several "huge intervening introns."
While it is possible to use Sanger sequencing to sequence just the clusters of exons, Triche said that some introns have been associated with retinoblastoma.
Sequencing the Rb gene in order to identify patients at risk of a second malignancy became an "obvious example of where one could apply targeted next-gen sequencing and change the feasibility of doing whole-gene sequencing — the introns and exons included," Triche said.
Nevertheless, Triche's group will not sequence the entire gene. Instead, the first version of the test will include seven different sized amplicons, encompassing all the exons and many of the intervening introns.
What won't be included is "a huge intron between exons two and three, an intron between six and seven, a monstrous intron between exon 17 and 18, and a small one between exon 23 and 24," he said.
For each amplicon they are generating coverage of several thousand-fold, with the lowest coverage still "hundreds of bases deep," he said.
Triche said his still lab is still determining a price for the test, but "our goal is to get something out the door for under $1,000."
This would be significantly less than current testing for variants in the Rb gene, which varies between around $1,700 and $4,000, depending on how many tests must be run and if validation has to be done on family members, he said.
The test is unlikely to be reimbursed, he added, because it is such a rare tumor. Patient advocacy groups and special interest groups are heavily involved in fund raising for much of the current testing protocols, and that will likely be the case with this test.
The test will also serve as somewhat of a proof of principle for the use of the PGM in a clinical setting.
The Rb test "meets a real clinical need at our facility and prepares us for the broader use of the platform for more common diseases," he said.
Looking ahead, Triche said that the team would aim to develop other targeted sequencing diagnostic tests on the PGM and eventually even do whole-exome sequencing on the platform for diagnostic purposes.
A logical next step would be to develop a test that sequences all the commonly mutated cancer genes, he said.
The Center for Personalized Medicine currently has two PGMs and Triche said it will likely add another two in the near future.
A Capture-Based Approach
Similarly, Ganguly's lab at Penn is developing a test for retinoblastoma on the PGM, which she said she will launch from her CLIA-certified lab in January.
The test is comparable to the one Triche is developing, except the Penn team is sequencing the entire Rb gene and using a capture technology instead of amplicon sequencing.
Like Triche, Ganguly is also launching the test on the 314 chip with 100-base-pair reads and plans to upgrade to the longer reads as they become available and as they are validated in the CLIA lab.
She agreed with Triche that one advantage of the PGM over Sanger sequencing is that it is more cost-effective at sequencing intronic regions. Additionally, she said, it can better detect low-level mutations since sequencing depth is much higher. Her team's test will generate at a minimum 100-fold coverage.
"Some rare mutations present in the introns will be easer to pick up, and also rare translocations," she said.
Ganguly's lab has been offering Sanger-based testing since 2001. With Sanger sequencing, "if you know what you're looking for, it's easier to see if it's present," she said. "But sometimes it's not that obvious."
A test on the PGM will help elucidate the cases where the diagnosis is not obvious.
Converting to the PGM will also reduce the cost of the test, which should make it feasible for developing countries where retinoblastoma is more problematic, she added. She said the initial cost will likely be around $1,000, but expects that price to drop as the costs of sequencing continues to decline.
Ganguly said she chose to use a capture-based approach as opposed to amplicon sequencing because it will be easier to optimize on formalin-fixed paraffin-embedded tissue.
There are patients "who were diagnosed with the disease 20 years ago and are now adults and wanting to have children," she said. "If you can go back to the FFPE samples, it's easier to get a more definite answer," about whether the disease was caused by a somatic mutation or an inherited germline mutation that they could pass on to their children.
Eventually, she plans to convert to the PGM other diagnostic tests that her lab is currently performing with Sanger sequencing. In particular, the lab offers Sanger-based tests for Li Fraumeni syndrome, characterized by mutations to the TP53 tumor suppressor; sporadic uveal melanoma; hemophilia A; and hereditary hemorrhagic telangiectasia.
Additionally, she said she wants to use the platform to sequence and characterize tumors.
"Once I'm happy with the sensitivity and specificity, I'll start doing other tests," she said.
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