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Wash U Med School Offers 28-Gene Cancer Dx Panel on HiSeq through CLIA Lab

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By Monica Heger

This story was originally published Nov. 22.

Washington University School of Medicine has begun offering a targeted sequencing-based diagnostic cancer panel on the Illumina HiSeq through its CLIA-certified laboratory.

The cancer panel is the first offering from Wash U's new Genomics and Pathology Services Laboratory — a collaboration between the department of genetics and the department of pathology and immunology.

The lab will offer a comprehensive 28-gene cancer panel, as well as separate tests that will sequence a subset of genes for specific cancer types including brain, colon, lung, ovarian, and endometrial cancers.

Shashikant Kulkarni, medical director of Genomics and Pathology Services, or GPS, said Wash U plans to launch additional targeted sequencing tests for cardiomyopathy, hearing loss, mitochondrial disorders, as well as diseases in which clinicians at Wash U have expertise, such as club foot and scoliosis.

The team also plans to launch a clinical exome sequencing test for inherited disorders, he said.

Aside from next-gen sequencing-based panels, GPS will offer a clinical genomics training program that will offer fellowships and a Master's program in clinical genomics.

The Wash U team chose the 28 genes for the cancer panel because they are known to be mutated in various cancers and there are drugs available to target all of them, Kulkarni said. "We could have easily done 200 or 300 genes," he said, "but we wanted to get our feet wet and go from there."

"These are genes that are already well known to be clinically actionable. … We want to provide meaningful information to the clinician that can be used with the patient."

As new cancer genes are discovered and validated, those can be added to the panel, Kulkarni said.

The lab will launch the test on the HiSeq, but Kulkarni added that the group is in the process of validating it on the MiSeq as well. The genes will be sequenced to an average of 4,000-fold coverage.

Turnaround time is still a moving target, but ideally will be around three to four weeks, which includes an 11-day run time on the HiSeq, plus sample collection, library construction, and analysis, Kulkarni said.

"We are working aggressively on this," he said. He added that when the test is launched on the MiSeq, turnaround time will be reduced significantly, to about one week.

Price will vary, depending on whether a clinician orders the 28-gene comprehensive panel or a set of genes specific to a patient's cancer type. For instance, the brain cancer panel includes just four genes, while the myeloid disorders panel includes 12 genes.

Also, next-gen sequencing does not reliably detect some mutations, such as indels and translocations, Kulkarni said. As a result, the lab will likely use other methods, such as fluorescence in situ hybridization, karyotyping assays, or PCR-based assays, to find these mutations.

The team is also developing a reimbursement strategy. Payors currently reimburse single-gene Sanger-based sequencing tests for all the genes included in the panel. Kulkarni said this should help his lab make the case that reimbursement of a single test for multiple genes will be more cost effective than multiple single-gene tests.

Targeted vs. Whole-Genome

Kulkarni said that while there is significant overlap and collaboration between Wash U's Genome Institute and the School of Medicine, the diagnostic sequencing offered through GPS is separate from the research-oriented cancer sequencing activities underway at the Genome Institute.

One difference is that researchers at the Genome Institute have so far focused on whole-genome sequencing and transcriptome sequencing of cancer patients, arguing that targeted sequencing approaches tend to miss relevant mutations (CSN 10/5/2011).

The Genome Institute team last year published a study in the Journal of the American Medical Association about the feasibility of doing whole-genome sequencing of a cancer patient for diagnostic purposes, and concluded that it was feasible (CSN 4/26/2011). The team even found information relevant for the patient's treatment.

Kulkarni said that the medical school opted for the targeted approach for its first NGS-based test for a number of reasons.

First, he said, a targeted approach is more cost-effective for deep sequencing. The 28-gene panel will have an average coverage of several thousand-fold, he said, compared to whole-genome sequencing, where 60-fold coverage of both the tumor and normal genome can run upwards of tens of thousands of dollars per patient, which is "not ideal for patient care."

Additionally, the higher-fold coverage "allows us to really get a good look at the minor cell population," which is especially important in cancer diagnostics because not only are oncogenic mutations often present at very low frequencies, but samples often contain a mixture of both tumor and normal DNA, further diluting the relevant mutations.

"The key is to find a solution that gives a deep coverage in a cost effective way, and only targeted panels can do that," he said.

CLIA Validation

In order to run its next-gen sequencing test in a CLIA-certified setting, the lab needed to validate both the instrument and the test, said Kulkarni.

CLIA validation refers to the standards by which the laboratory itself is run. The instruments and tests aren't CLIA-certified, but to operate them within the CLIA lab they must hold up to the standards of the lab, he said.

Instrument validation on a next-gen platform is done by sequencing a "gold standard set," Kulkarni said, such as a HapMap sample or cell lines that have been extensively sequenced and characterized. The CLIA lab at Wash U has 10 such samples that it runs when a new instrument is brought into the lab. "We do extensive bioinformatics analyses to make sure our data is concordant with what is known in the database and what we have found."

He acknowledged that certifying a specific test is tricky with next-gen sequencing "because there are not many guidelines." In general, though, Kulkarni said the group tries to follow recommendations from the American College of Medical Genetics and the College of American Pathology about genetic tests.

ACMG issued guidelines in 2007 regarding the reporting of sequence variations identified in the course of clinical lab testing but has not yet issued formal recommendations for next-gen sequencing-based tests. However, ACMG, CAP, and the Association for Molecular Pathology are currently working together to develop an accreditation checklist for labs running NGS-based clinical tests, which they plan to have in place by the end of 2012 (CSN 9/14/2011).

Validating the cancer test has posed unique challenges, Kulkarni said. For instance, there is a lot of variability in terms of the sample itself, whether it is from formalin-fixed paraffin-embedded tissue, fresh frozen, or a fresh tissue sample. A test must be able to produce the same results no matter the sample type, Kulkarni said.

To validate the 28-gene cancer panel, the team made use of its large repository of around two million clinically annotated samples, including running samples extracted from fresh frozen and FFPE tissue from the same patient.

The entire validation process took about one year on the HiSeq, he said, and the team is still in midst of doing validation studies on the MiSeq.


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