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TGen Opens CLIA Lab; Aims to Prove Clinical, Cost Benefits of NGS in Rare Disease and Cancer

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The Translational Genomics Research Institute has opened a CLIA lab and is working to validate whole-genome, exome, and transcriptome sequencing protocols on the Illumina HiSeq 2500, Clinical Sequencing News has learned.

On a recent visit to the Phoenix, Ariz.-based laboratory, researchers there told CSN that the institute is focused on driving clinical adoption of sequencing by demonstrating both the clinical utility and cost benefits of the technology for use in guiding cancer treatment and in diagnosing rare disorders.

"A third party payor is going to say, 'Where's the evidence that this is actually something we should pay for?', and I think that's one thing that TGen is being smart about," Johnathan Keats, assistant professor in TGen's Integrated Cancer Genomics Division, told CSN. "We're going to make sure that there's evidence that there is some value to this."

"We want to make the case in a study that shows the economics," added David Craig, co-director of TGen's Center for Rare Childhood Disorders, "so that you don't have to go through preauthorization and fight with the insurance company. … A physician can order this without preauthorization, and it will be reimbursed and they can make decisions, and it's not a four-month fight with an insurance company."

In the field of cancer, the institute is testing sequencing in a number of clinical trials as a means of identifying a drug target, and it is also collaborating with the Mayo Clinic to sequence patients with late-stage metastatic cancer to identify potential treatment.

Its sequencing protocol for these patients combines low-pass whole-genome sequencing, deep exome sequencing, and transcriptome sequencing on the HiSeq 2500, and it is aiming to validate the protocol in the CLIA lab with a turnaround of 15 days.

Separately, through the Center for Rare Childhood Disorders, it is looking to make the case to third-party payors that exome sequencing is a cost-effective test for certain cases, as well as studying the impact of including transcriptome sequencing on improving the diagnostic rate.

TGen is equipped with five HiSeq 2000s, two of which have been upgraded to the 2500, with upgrades for the remaining three machines forthcoming. It also has several of Life Technologies' Ion Torrent PGMs and is getting an Illumina MiSeq.

The HiSeqs are TGen's workhorses, and it uses the PGMs mostly for validation. It plans to use the MiSeq in the CLIA lab for quick screening purposes.

Cancer Trials

TGen is working on validating a cancer sequencing protocol to be run in its CLIA lab that includes low-pass, 5x-8x, whole-genome sequencing with long insert libraries of 100,000 base pairs, plus deep exome sequencing, and whole-transcriptome sequencing. The low-pass, long-insert, whole-genome sequencing enables the detection of structural variants, translocations, and inversions, while deep exome sequencing can identify point mutations, and the transcriptome sequencing looks for gene fusions and aberrant expression.

The center is implementing this protocol in the context of a number of clinical trials. The one that is currently the furthest along is a melanoma trial that it is doing in collaboration with the Barbara Ann Karmanos Cancer Institute at Wayne State University in Detroit, which is being funded by Stand Up to Cancer and the Melanoma Research Alliance.

For the three-year trial, the center is sequencing melanoma patients that have the wild-type BRAF gene, for whom targeted therapy options are limited. The center is sequencing around 150 patients on the HiSeq 2500 looking for mutations that could point to an alternative therapy. The trial is testing prospectively whether sequencing can impact survival by identifying a treatment, Craig said.

For this trial, Craig said that the researchers also have to work with the US Food and Drug Administration, since the trial involves matching patients to therapies.

Currently, the next-generation sequencing is being done in a research setting, with results validated in TGen's CLIA lab, but the center is moving toward validating its NGS protocols in the CLIA lab.

"We're working on analytical validation of the entire pipeline," Craig said, "to basically have a report that is documented and fixed in the CLIA lab so you don't have to do an extra assay to validate." Having the protocol validated under CLIA will require the group to be able to pinpoint sensitivity and specificity for the different types of mutations it reports out.

The melanoma study is the follow-up to a pilot that TGen did in collaboration with US Oncology and Life Technologies. For that study, the group sequenced the whole genomes and transcriptomes of 14 patients with metastatic triple-negative breast cancer (CSN 12/14/2011).

That study proved the concept of using sequencing to identify therapeutic targets, said Craig, but the goal for the melanoma and other studies is to demonstrate that doing this is in a clinically relevant time frame makes a difference to the patient.

As such, one of the main goals of the current studies is to reduce turnaround time to two to four weeks from the two to three months in the US Oncology study.

TGen has been collaborating with Illumina, which is "providing us access to some of the fastest tools," Craig said, and also is working with them on data analysis to help reduce turnaround times.

Another clinical trial, being funded by the Multiple Myeloma Foundation, will involve the sequencing of 1,000 patients in the first phase to identify predictors of drug response. In the second phase, TGen will sequence 800 relapse patients that failed their initial treatment to identify therapeutic targets.

The institute also has an ongoing collaboration with the Mayo Clinic to sequence late-stage metastatic cancer patients (IS 2/22/2011).

So far, the team has sequenced around 21 patients with a variety of cancers including ganglioma, melanoma, pancreatic cancer, lung cancer, and cervical cancer.

Rare Childhood Disorders

Last year, TGen launched its Center for Rare Childhood Disorders (CSN 7/25/2012). At the time, it was using primarily Illumina's RapidTrack service, which delivers a sequenced genome in two weeks, but is now doing around 70 percent of the sequencing in-house. For patients where turnaround time is critical, it will continue to use the RapidTrack service.

The diagnostic rate is similar to others offering the service, between 30 percent and 50 percent.

One main goal of the center is to figure out ways to improve the diagnostic rate, Craig said, and it is testing transcriptome sequencing for this purpose. For childhood disorders, "the RNA can matter too," Craig said. For instance, there are disorders caused from aberrant X-chromosome expression.

"The real challenge for RNA is always tissue," he said. Currently, the researchers are doing RNA-seq from blood, but there is always the chance that the variation won't be picked up in the blood if it is only being expressed in certain tissue. So, they are also looking to test the protocol on fibroblasts and induced pluripotent stem cell lines.

The center is also looking to do a cost-effectiveness study of sequencing for childhood disorders.

Currently, it is working to involve all stakeholders in the trial design — "the people who are going to be the decision makers," said Craig.

Already, the center has several examples where sequencing enabled a diagnosis that led to treatment or helped guide management.

For example, one family had a girl with a neurological condition who was unable to walk and could barely hold her head up. Despite going through a battery of tests, including muscle biopsies, magnetic resonance imaging, and chromosome tests, her doctors were unable to come up with an answer. Whole-genome sequencing identified a variant involved in dopamine production and doctors were able to successfully treat the girl. Her parents removed the wheelchair lift from their house and she is now walking to school.

In another case that did not have a happy ending, the team identified in a three-year old boy two mutations to the POLG gene, indicative of the mitochondrial disease Alpers syndrome. The disease is characterized by rapid decline in health and is ultimately fatal. "That seemed like the most terrible information you could hand back to a family," Craig said, but even in that case, the family was grateful to know the cause of their child's disorder. "They had the ability to not wonder if there was anything more they could be doing." The child ended up passing away several months later.

Despite these two very different outcomes, both can be used to make the case for insurance reimbursement.

In the first case, doing sequencing earlier could have "cut costs of all those tests just by doing one single test," said Keri Ramsey, clinical research coordinator at the TGen Center for Rare Childhood Disorders. "Not to mention, in cases like mitochondrial disorders, [physicians] do muscle biopsies, which are incredibly invasive and a lot of times inconclusive."

In the second case, the diagnosis, while terminal, still enabled the family to make decisions about the boy's care, Ramsey said.

Going forward, the center will continue to focus on fine tuning their methods to improve the diagnostic rate, reduce turnaround time, and demonstrate the cost-effectiveness of such tests, Craig said.

It's "not meant to be high-throughput, necessarily, as much as to be an ambitious, prospective, precision medicine approach," he said.

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