NEW YORK (GenomeWeb) – The Mayo Clinic's laboratory medicine and pathology department has taken a step towards making next-generation sequencing a more routine aspect of clinical cytogenetics.
Last week, the Mayo Clinic announced that the laboratory medicine and pathology department's reference lab, the Mayo Medical Laboratories, will now offer a clinical version of a mate-pair sequencing-based chromosomal breakpoint detection test, developed in partnership with the Mayo Clinic's Center for Individualized Medicine. The reference lab performs clinical tests and pathology services for thousands of healthcare centers in around the world that will now have access to the test.
The new mate-pair sequencing test will initially be offered for three clinical indications — congenital disease cases involving balanced chromosome rearrangements, hematologic samples, and other oncological cases — though the team is pursuing additional applications for the test, including cancer lineage testing to track relatedness between multiple cancer nodules from the same individual.
"There are a lot of clinical applications for this technology right now that we anticipate bringing to the clinic," said George Vasmatzis, co-director of the Mayo Clinic Center for Individualized Medicine's biomarker discovery program.
The team expects the second version of the test will replace FISH panels for hematological malignancies, such as acute myeloid leukemia, while the third version is expected to span a broad range of solid tumor types.
"The issue with clinical tests is that there are a lot of performance characteristics [the tests] have to go through," explained Vasmatzis, who worked on the software and sequencing protocols behind the clinical version of the mate-pair sequencing test. "Typical research protocols and analytics don't have everything that is needed to create an extremely sensitive, specific test without too many false-positives."
In general, the mate-pair sequencing approach is "an inexpensive way to cover the whole genome for rearrangements," he noted.
The strategy resembles paired-end sequencing in some respects, he said, though the genome is fragmented into much larger pieces — which lowers the amount of sequence coverage needed and boosts detection of large rearrangements. The fragments are circularized so that they can be assessed using short read sequencing technologies, such as Illumina's.
"This is a whole-genome sequencing approach, the focus of which is to identify large chromosomal aberrations, not single nucleotide mutations, not indels," Vasmatzis said. "Whatever happens in the genome has to be bigger than [10,000 bases]."
Researchers have been relying on mate-pair sequencing as a strategy for profiling large-scale rearrangements, aneuploidies, deletions, amplifications, and the like for several years.Brigham and Women's Director of Cytogenetics Cynthia Morton, for example, noted that even though her center does not offer a clinical service in this area, she and her colleagues have published studies on mate-pair sequencing-based detection of balanced rearrangements since 2011.
While that work has been done in a research context, she explained, there have been instances where rearrangements identified by mate-pair sequencing contributed to patient diagnoses and were entered into medical records.
"Right now, if we have a case [with suspected balanced rearrangements], we can enroll it in our research study and there's no fee," Morton said. "We have the ability already to … receive the samples in a clinical lab like we did with the ones reported in medical records and handle things in a way that is in compliance with CLIA."
Though the Brigham and Women's Hospital group "can turn things around in a clinically-actionable time," she explained, the center does not run a huge diagnostic reference lab, so there are no plans to scale up the test significantly in the near future.
Morton's team has been profiling balanced chromosome rearrangements in individuals with clinical phenotypes related to developmental conditions for around a decade and a half, since securing a grant for the "Developmental Genome Anatomy Project" (DGAP), which kicked off in 2001 — an effort to use balanced chromosome rearrangements as "signposts" for genes that are disrupted or disregulated in a given condition.
Starting with fluorescence in situ (FISH) hybridization mapping and Sanger sequencing, the researchers transitioned to sequencing-based approaches such as mate-pair or "jumping library" sequencing. They have since used the approach to assess rearrangements in a variety of disorders, including autism spectrum disorder cases, in prenatal samples, individuals with congenital abnormalities, and infertility cases.
Over at the Mayo Clinic, Vasmatzis noted that similar experimental and sequencing protocols are being used in the research and clinical setting. Where the real changes were needed en route to a clinical mate-pair test was on the analytic side, he explained.
"The real value is actually in the algorithms, the analytical part, that makes the mate-pair output very clean so we could get to the point where it could become a clinical test," Vasmatzis said, noting that the Mayo team's analytical pipeline is "push button," automated, and includes filters to pick up authentic rearrangements while masking out false positives.
Vasmatzis and his colleagues validated the test extensively with mate-pair sequencing data from hundreds of clinically characterized samples to take it into the clinical lab.
At the moment, the sequencing pipeline of the mate-pair sequencing test is tailored to Illumina instruments, though the general approach could likely be adapted to other sequencing platforms if needed.
"It would need to be re-worked by someone because other sequencing technologies have different specifications, so we would need to adapt these specifications to our analytics," Vasmatzis said, noting that the masks and filters in place to dial down false positives would need to be tailored to fit the instrument at hand.
The Mayo Clinic lab is working to obtain a CPT code for its clinical mate-pair sequencing breakpoint test so it can be reimbursed by payers. The center has also applied for regulatory approval of the test in New York state.
"Excitement from our cytogenetics community about this test is very high," Vasmatzis added. "They think that at times where they could not see anything in the genomes … this technology allows them to see [previously missed rearrangements]."
Morton noted that sequencing-based approaches are beginning to displace FISH- and array-based methods for some cytogenetic tests to find balanced rearrangements that might be missed or take more experimental effort to detect with these approaches, though "changing the landscape of this discipline is not something you can do overnight."
By learning more about the breakpoints of balanced rearrangements, and scrutinizing them with targeted methods such as PCR to get a clearer picture of the genes and nucleotides affected, Morton argued, some individuals may receive better care or more appropriate placement in clinical trials down the road, while their families could get new opportunities to tap into support groups for families with children affected by similar genetic anomalies.
"If there were some gene that might lead to a medical therapy, then you need to know what that is," she said.
"It can be emotionally difficult for families to go on a medical journey and never really arrive at an explanation," Nicole Lynn Hoppman, a molecular cytogeneticist from the Mayo Clinic who specializes in congenital conditions, said in a statement. "This test offers earlier answers for young patients with neurological or developmental delays who have been part of a diagnostic odyssey."