Ch. 2, Part 9: What MaterniT21, Harmony, Panorama, and verifi are actually testing

In the course of Chapter 2’s exploration of prenatal genetic testing, we finally arrive at cell free DNA screening and what exactly tests like MaterniT21, Harmony, and Panorama are actually testing.

NIPT, cell free fetal DNA screening

At the end of the 1990’s, a team in China led by Dennis Lo, identified free floating DNA in the mother’s bloodstream from the pregnancy. Other teams had been trying to identify whole cells from the pregnancy. Similar to whole cells from the pregnancy, the free floating DNA theoretically held the promise of diagnosing the genetic make up of the fetus without the risk of miscarriage associated with amnio and CVS tests. Cell free DNA screening would go on to be hailed as the “Holy Grail” of prenatal testing: a diagnosis based just on a blood sample from the mother without any risk of miscarriage. It became referred to as “Non-Invasive Prenatal Diagnosis” or “NIPD.”

The discovery sounded a starter’s pistol for the race for labs to be first-to-market with NIPD. Sequenom, a publicly-traded San Diego-based laboratory, obtained an exclusive license with Lo to develop NIPD. Investors put up money on the gamble that Sequenom would be the first to develop NIPD.

On the eve of its launch in 2009, however, Sequenom had to admit it had falsified its data, which had artificially inflated its stock price. An SEC-inquiry and shareholder class action lawsuit ensued. Heads rolled, with CEO Harry Stylli and Sequenom’s senior vice-president of research, Dr. Elizabeth Dragon, being fired, along with other employees. Just five-months before being sentenced, facing up to 25 years in prison and fines of up to $250,000 for having pled guilty for securities fraud, Dr. Dragon died at the age of 62 of reportedly natural causes.

The race for market dominance

Alas, NIPD was not to be.

Instead, in October 2011, Sequenom commercially launched its “MaterniT21” test. In its press materials and journal study that accompanied the launch, Sequenom touted that MaterniT21 would test “cell free fetal DNA” providing the most accurate results of any screening test–note, not diagnostic.

Before the end of 2011, two other laboratories would join the marketplace. Ariosa launched its “Harmony” test. The lab was originally named “Artemis” for the Greek goddess of childbirth (and, somewhat fittingly, the goddess of the hunt, as well). Re-named, Ariosa advertised Harmony as a “targeted” screening test, testing only what are considered the “major trisomies”: Trisomy 21 (Down syndrome); Trisomy 18 (Edwards syndrome); and, Trisomy 13 (Patau syndrome). Ariosa also highlighted that Harmony was the lowest charged cost of any test like it.

Another lab, Verinata launched its “verifi” test. In an effort to distinguish itself from Sequenom and Ariosa’s offerings, verifi tested for the major trisomies, but also claimed it could also detect conditions associated with the sex chromosomes, like Monosomy X (Turner syndrome).

Not to be outdone, soon after verifi entered the market, Sequenom retitled its test as “MaterniT21 Plus,” adding the sex chromosomal aneuploidies. The last to join the competition was Natera with its test, Panorama, distinguishing itself by using a different testing methodology involving single nucleotide polymorphisms or “SNPs”.

A competition it certainly was, given that the prenatal genetic testing market has been valued in the billions. At the 2012 ISPD Annual Meeting (referenced earlier), there was a tense, awkward breakout session where representatives of Sequenom and Verinata presented one after the other. The industry of prenatal genetic testing is a relatively small one. Having a focused expertise within the larger fields of genetics and obstetrics, the main players have attended the same conferences for years, served on committees with one another, and, as in this breakout, presented in the same section with one another. The presenters refer to each other knowingly, having followed each other’s professional accomplishments. Plus, now big money was involved. The breakout had all the feel of a family divvying up a relative’s estate, politely, but snippily explaining why they had the better claim. Following these presentations of why Sequenom’s testing was better or Verinata’s was best, a graduating student at Stanford, an outsider, as though to complete the contentious probate analogy, detailed the varying lawsuits and claims each had filed against the other for patent infringement.

NIPD falls short of being “D”

If Sequenom et al. were not offering NIPD, then what did MaterniT21, verifi, etc. do that was worth all the hoopla (and money)?

Falling short of the “D” in NIPD, these tests were referred to as “Non-Invasive Prenatal Testing” or “NIPT.” This is how they remain referred to most often. However, as more than one observer noted, all non-diagnostic prenatal testing is “non-invasive.” The NT-combined and Quad are only “invasive” for the needle that draws the mother’s blood sample, which is still required by NIPT. Plus, if it was not NIPD, then NIPT was just screening, meaning its results were non-diagnostic. Like NT-combined and Quad screening, NIPT just provides a recalculation of the mother’s chances for carrying a child with a tested-for condition.

The ACMG sought to emphasize the screening aspect by officially referring to it as “Non-Invasive Prenatal Screening” or “NIPS.” The acronym, however, never really caught on outside of official ACMG publications, possibly because it induced snickering.

Why, though, was this new testing not diagnostic? After all, it was testing free floating DNA from the pregnancy and all the labs said it was fetal DNA. Shouldn’t that mean it can diagnose the DNA of the developing fetus?

When cell-free fetal DNA isn’t

I wondered this, as well.

In 2013, I attended the ACMG meeting in Nashville, my submitted abstract on “cell free fetal DNA” was again relegated to the domino row of posters. One of the first attendees to visit me at my poster said matter-of-factly “Everyone knows its placental DNA.” Clearly, not everyone.

I asked what he meant. He was a geneticist from Washington state who worked with genetic counselor Katie Stoll, a colleague of mine. He explained that what was being tested was DNA that had come from cells of the placenta, not the fetus. Sure enough, following that meeting, ACMG issued its position statement on NIPS noting that the DNA was placental DNA, not DNA from the fetus.

Following the meeting, I reviewed the materials passed out at the exhibit booths of the labs. Almost all of them had quietly dropped “fetal” from “cell free DNA” in their printed materials. But, for years on, the reference to fetal DNA could still be found on their websites.

In 2015, this time at the ACMG annual meeting in Salt Lake City, a platform presenter provided a helpful graphic (see above) for those not schooled in obstetrics or embryology, or for whom terms like “cytotrophoblast” or “syncytiotrophoblast” sound quite alien in nature. The image showed how when the embryo attaches to the uterine wall, there are cells that anchor it. It is from these cells which become the placenta that DNA is passed through to the mother’s bloodstream and, hence, cell free DNA from the pregnancy.

Then, in 2017, I was on the train back from the last meeting for a work group for The Hastings Center. The group was addressing how to ethically implement the next generation of prenatal genetic screening. My fellow group member, and former chief medical officer for Natera, Dr. Sue Gross, and I were having a conversation, when, in some bi-annual revelation of what is being tested in cell free DNA, Dr. Gross shared the following: the DNA tested by Panorama, etc., comes from the “outside” of the placenta, the side that anchors to the womb wall, whereas the cells that develop into the fetus are from the “inside” of the placenta, i.e. the side towards the open womb into which the fetus grows.

While this is perhaps more than anyone needs to know about where the DNA comes from, it is significant for the intuitive understanding a layperson would have. Consider that it took me–someone who writes and presents regularly on prenatal genetic testing–years before discovering that it is not fetal DNA, it is placental DNA, and that the source of the DNA is from the polar opposite side of the placenta than those cells that develop into the fetus.

If an expectant mother was counseled that tests like Harmony test “placental DNA” or “DNA from the placenta” and not “fetal DNA,” then most would understand their test result is not from the fetus, but their placenta. Intuitively, they would understand that a screen-positive test result does not necessarily mean their fetus actually has the tested-for condition.

While the DNA from the placenta and the fetus are mostly the same, there is a phenomena known as “confined placental mosaicism” or “CFM.” CFM means that groups of cell within the placenta may have an extra or missing chromosome. This is why a test of cell free placental DNA may return a screen result for conditions like Down syndrome or Trisomy 18, but the fetus does not in fact have Down syndrome or Trisomy 18. Instead, the test found cell free DNA from a region of the placenta with CFM for Down syndrome or Trisomy 18 (or the other cited tested-for conditions the labs say they can screen for). But, the labs, marketing their tests based on their accuracy, made a choice out of the gate to represent they were testing “fetal” DNA, even though that wasn’t accurate. It wasn’t true.