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Mayo Researchers Develop MP-seq Test to Detect Rearrangements in Cancers, Genetic Disease

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NEW YORK (GenomeWeb) – Researchers at the Mayo Clinic have devised a next-generation sequencing technique for detecting rearrangements and are in the process of developing clinical tests for hematological malignancies, breast cancer, and constitutional disease.

George Vasmatzis, co-director of the biomarker discovery program at Mayo's Center for Individualized Medicine, described the strategy at last month's Clinical Genome Conference in San Francisco. He told GenomeWeb in a follow-up interview that Mayo would launch a clinical test based on the technique in the next six months to a year for hematological and constitutional diseases, and a year later for solid tumors.

The technique is a genome-wide approach that is comprehensive and unbiased like whole-genome sequencing, but less expensive, since it focuses solely on detecting structural variants, which can be done with shallower sequencing.

Dubbed MP-seq for mate pair sequencing, the researchers described the application of the technique in lung cancer in a publication in the Journal of Clinical Oncology last year.

The technique uses a mate pair sequencing strategy on Illumina's instruments. Instead of first fragmenting DNA into small pieces — a standard first step for most Illumina NGS protocols — the researchers create long inserts around 5 kb, or 10 times longer than the typical insert. Next, they circularize the DNA, adding a biotin in the junction. The goal is to "breach a break point by enough bases to call it," Vasmatzis said. "You'll miss the intervening sequence, but for our purposes, we didn't care."

During his presentation, Vasmatzis described an application where the strategy could be used to distinguish between multiple lung tumors in one patient. Such patients present oncologists with a clinical dilemma, Vasmatzis said. If nodules are all related — meaning there is a primary tumor and metastatic tumors — that is an indication of Stage III or IV cancer and a patient will typically be prescribed adjuvant therapy plus surgery. But, if the tumors are unrelated, meaning more than one primary tumor, the patient can often be treated without surgery.

By identifying the structural variants specific to each nodule, the researchers can determine whether the tumors are related or not.

For this clinical situation, tumors are first biopsied using laser capture microdissection. Researchers then isolate genomic DNA and perform in situ whole-genome amplification. Then they perform the mate pair sequencing protocol for each nodule and validate the structural variant calls with PCR and Sanger sequencing.

So far, Vasmatzis said that the team has analyzed around 1,600 genomes in a research setting using the MP-seq strategy.

In one example, Vasmatzis described a patient with three squamous cell nodules and one adenocarcinoma. It was unclear if the four different nodules in the patient were related or not. Applying the MP-seq strategy allowed the researchers to look at the lineage of the breakpoints to find that the patient had two unique primary tumors with two related ones.

In another example, pathologists had determined that a patient with two nodules had two independent tumors. But, MP-seq analysis found that those tumors had lots of shared breakpoints, and in fact represented a primary and metastatic tumor.

Vasmatzis said the first clinical application would be in hematological malignancies. Already, gene fusions are used to diagnose certain hematological malignancies —such as acute promyelocytic leukemia, which is characterized by a translocation involving the RARA gene. In addition, targeted therapies exist for other fusions common to hematological malignancies, such as imatinib, which targets the BCR-ABL gene fusion in chronic myeloid leukemia.

Currently, multiple techniques, including FISH, PCR, and array CGH, are used to identify such rearrangements and fusions in blood-based cancers. But, MP-seq "can replace all three techniques and resolve very well what's going on in the genome," Vasmatzis said.

In addition, he said it would be more cost-effective. Since MP-seq is not looking to identify point mutations, sequencing does not have to be done to a high coverage. As such, Vasmatzis said that the reagents cost per sample run is around $500. The clinical test will cost substantially more, although a final price has not yet been set, he said.

For solid tumors, Vasmatzis said that the team is currently using MP-seq in a research setting to identify biomarkers as well as to study tumor lineage and evolution. 

Vasmatzis said that the team is currently in the midst of validation studies for the test to nail down its sensitivity and specificity. Thus far, most false positives occur in areas of low coverage. Increasing sequence coverage can lower the false-positive rate, but also increases the cost. "We're trying to find the right coverage cutoff point," he said. 

The team is aiming for a turnaround time of about one week for the test. Currently, Vasmatzis said they have run it in around 10 days. This one-week turnaround will make it faster than current methods for analyzing structural variants in cancers, Vasmatzis said. Often, oncologists will order a battery of tests one right after the other, which not only takes time, but also uses up the limited tumor sample, Vasmatzis said. With MP-seq, one test will be able to identify all the structural variants for which there are individual tests.

Vasmatzis said he envisions the test eventually being used as a primary analysis for hematological malignancies. For solid tumors, it will likely be used in conjunction with either exome sequencing or a gene panel that would identify point mutations.

While the researchers also considered RNA-seq to identify fusions, Vasmatzis said that a DNA-based test was preferable. Most clinical genetic tests analyze DNA, not RNA, so the MP-seq test should be readily adapted to a clinical setting. In addition, DNA-based tests are "more robust" than RNA and typically provide better sensitivity and specificity, he said. 

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