Mate pair sequencing yields rich new data

For diagnostic samples, she says, MPseq works well. “But FISH is much more amenable to monitoring minimal residual disease, because the sensitivity for some of our probe strategies is 0.6 percent. Mate pair cannot go that low. We can only detect down to about 10 to 20 percent of rearrangements. So once a patient has been treated and they are expecting very low levels of that rearrangement, mate pair tends not to work very well.”

But NGS is better, Dr. Baughn says, particularly for identifying MYC rearrangements. “The detection of MYC rearrangements is important for several B-cell neoplasms, including high-grade B-cell lymphoma and multiple myeloma. We’ve discovered from validation studies that a commonly used MYC break-apart FISH probe cannot identify approximately four percent of MYC rearrangements in high-grade B-cell lymphomas [King RL, et al. Haematologica. 2019;104(6):e248–e251] and about half of multiple myeloma cases that we are able to identify from the mate pair sequencing.”

“So if we could ignore money completely—which is probably naïve to even think—characterizing abnormalities in newly diagnosed specimens through mate pair or any NGS test that can identify a single nucleotide variant, structural variations, and copy number changes would be the way to go,” Dr. Baughn says.

Nicole Lynn Hoppman, PhD, started Mayo’s cytogenetic next-generation sequencing work in 2012, collaborating with George Vasmatzis, PhD, to improve the MPseq algorithm for detection and develop early visualization software as part of the interpretive phase. The clinical launch was slow, says Dr. Hoppman, another co-director of the genomics laboratory, because the first version of the test involves looking for specific chromosome abnormalities or signal patterns by FISH that would merit investigation by MPseq and there were only one or two a month of those test results to begin with. And there was a need, of course, for a “very robust validation of the algorithm.”

To obtain a billing code, the laboratory had to first have the test live. “After being live with the test for several months,” she says, “we went through the PLA application process with CMS and received PLA codes for the tests—0012U for blood, 0013U for bone marrow, and 0014U for solid tumors.” These three tests are currently available as reflex tests, plus the AML panel developed by Dr. Baughn and colleagues, “which is basically a FISH replacement,” Dr. Hoppman says. The next step is for B-ALL and T-ALL, “where we are taking all the FISH probes we have and adding several targets we don’t have FISH probes for. Our plan is to do this for multiple malignancies.” So across a range of abnormalities, “we can detect structural variations, copy number changes, and we are working on an algorithm to detect loss of heterozygosity.”

While solid tumors can also be tested, Dr. Hoppman says, mate pair doesn’t work well in FFPE samples. “So hematologic malignancies are definitely where we see the most volume for MPseq.”

The number of patients who have benefited clinically from mate pair sequencing so far is small, Dr. Hoppman says, compared with the total number of patients diagnosed with hematologic malignancies. But for those patients individually, the benefit can be significant. “It can lead to a patient receiving a drug that they would not have been previously eligible to receive and that might put them into long-term remission.”

One institution that uses Mayo Medical Laboratories as its reference lab for mate pair sequencing is Legacy Health in Portland, Ore. Yassmine Akkari, PhD, scientific director for cytogenetics and molecular pathology, says it is not likely that they would be able to perform mate pair sequencing at their institution. “I would absolutely love to. But a health system such as ours would not be able to bring something this technically and bioinformatically sophisticated in-house very soon.”

Published reports from the Mayo team have demonstrated the value of MPseq in various clinical situations in which an accurate diagnosis provided value in the clinical management of cancer patients, Dr. Akkari says. “There are cases when we perform traditional tests such as conventional cytogenetics, FISH, and NGS and results come back normal. In these instances, we still cannot tell the oncologist or the referring clinician what the accurate diagnosis is and how the disease will behave in terms of prognosis. In essence, we haven’t helped the patient yet.” Having the option to perform mate pair sequencing, she says, provides an avenue to identify structural rearrangements that may have therapeutic consequences.

Although some NGS-based pipelines can detect a variety of genomic aberrations, “they are not as validated as MPseq,” according to Dr. Akkari.

Genetic diseases can be the result of a variety of mechanisms, she notes. “There could be copy number changes, sequence variation, and/or structural abnormalities including translocations, inversions, or insertions. Many laboratories are equipped to offer several testing modalities, but the ones we have available can get to one of these mechanisms and not the others. FISH, chromosomes, arrays, and sequencing all have a lot of limitations.”

Mate pair sequencing, on the other hand, is able to capture the majority of the mechanisms of disease that are known today, Dr. Akkari says. While it would be inefficient to use mate pair in all cases, in her view, “in cases where diagnosis and, more importantly, prognosis completely eludes us, mate pair sequencing is perfect.”

Dr. Jenkins of Mayo says the bioinformatics pipeline is the most important piece. “The chemistry is pretty straightforward in most places that do NGS. The bioinformatics pipeline is the golden goose; it’s the piece that is Mayo’s intellectual property.” The standard NGS that is done for any kind of whole genome or exome sequencing is called paired-end sequencing; DNA fragments are made and the end of the fragments are sequenced. “Mate pair is just a modification; it involves sequencing the ends of much larger fragments,” he explains.

Most places could perform the mate pair sequencing, but they have not developed the interpretive part: mapping the sequencing data back to the genome and then displaying the rearrangement in a way that makes sense, Dr. Jenkins says. “There are multiple pipelines that can do this. Mayo’s pipeline is just more efficient at mapping these DNA sequences and then visualizing the rearrangements.”

“It is incumbent upon us as laboratorians,” he continues, “to sift through the large amount of data and present back to clinicians a succinct report that describes the alterations that are predictive, meaning actionable, versus prognostic or diagnostic, meaning they have implications for the diagnosis but not necessarily for the treatment indications. Finding an actionable mutation is actually unusual. But that is not the only reason to do these tests. You can also find things to reclassify the tumor, and that is huge.”

The practical impact on patients has been evident at Mayo Clinic, Dr. Jenkins says. “Every week we find something that absolutely makes a diagnosis for a patient, or changes the diagnosis for a patient, or determines whether or not a patient will have a poor or a good prognosis.”

The jury is still out on mate pair sequencing’s usefulness with solid tumors, he says. “Mate pair needs to work in paraffin before we can start. It can work, but not at the same efficiency as some other technologies do. Mate pair is lovely in myeloma and leukemia because bone marrow is relatively easy to get, our blood is relatively easy to harvest, but it is a challenge in most institutions to freeze a piece of tumor for DNA.”

Another part of the reason mate pair has yet to catch on in solid tumors is the number of rearrangements that are present. “Your typical carcinoma has hundreds of rearrangements. So the real problem is how does one find the driver mutation in that pile of rearrangements?”

Dr. Jenkins views mate pair as part of the overall armamentarium of genetic testing. “I don’t think it is going to solve the world’s cancer problems, but it is a very useful test in some very specific situations. For hematologic cancers it has the potential to be a test to replace other more standard tests. The stretch goal is that mate pair could replace FISH and maybe even chromosomes for most of the standard run-of-the-mill leukemias and myeloma.” Whether it is generalizable, he says, depends on how the whole field evolves. “It has the potential, but there are competing technologies.”

The clinical laboratory needs to move past chromosome studies and FISH as a gold standard, Dr. Hoppman says, “because we know now from doing mate pair that we are missing things. Next-generation sequencing is advancing so rapidly, we also know that in the future we will be able to do this more cheaply. We just need to promote awareness of this technology and what it can do in the hope of guaranteeing reimbursement for these types of tests and changing guidelines so that our patients aren’t stuck behind our advancements in the laboratory.”

The genomic laboratory co-directors at Mayo are reaching out to raise awareness of the value of mate pair sequencing. “We try to publish a lot to raise awareness of our findings,” Dr. Baughn says. “We give talks, multiple posters, and we are planning to give a pre-symposium workshop at the American Society of Hematology meeting this year. We are trying to communicate the test to people.”

She and colleagues are “revolutionizing cytogenetic testing,” as she sees it, and bringing it to the molecular level to provide more clarity to rearrangements in hematologic malignancies. “But we are at the beginning stages where we need a lot more data. I am sold already, but the community probably needs more evidence that this is a better approach. We need to do a lot more sequencing and correlation with outcomes.”

Dr. Akkari is cautious about a universal prediction that mate pair sequencing will drive a revolution in diagnosis and treatment, mainly because clinical MPseq is currently performed only at Mayo. But “the more we know, the more powerful we can be against a certain disease. From that perspective, mate pair sequencing has shown a huge amount of value and has the potential to help patients. And I remain hopeful that it will revolutionize the diagnosis of hematologic malignancies.”

Anne Paxton is a writer and attorney in Seattle.