Cypriot molecular diagnostics company NIPD Genetics has passed another important hurdle toward marketing a blood-based prenatal diagnostic test for trisomy 21, publishing research demonstrating that the variability of free fetal DNA in maternal plasma does not affect the performance of its assay.
In addition, the research indirectly demonstrated the adequacy of the diagnostic formula used in the assay, which measures differential methylation of seven regions on chromosome 21 in maternal peripheral blood using a technology called methylated DNA immunoprecipitation, or MeDIP, combined with real-time quantitative PCR.
The company is now focused on developing and validating an easier and cheaper version of its test with the hope of offering it as a service in Europe by the end of this year, and eventually as an assay kit for use in any clinical laboratory, CSO Elisavet Papageorgiou told PCR Insider.
NIPD Genetics' test involves collecting 10 mL of peripheral blood from a pregnant woman, isolating the DNA, and performing MeDiP, which uses an antibody specific for 5-methylcytidine to capture methylated sites and enrich for DNA regions that are specifically hypermethylated in the fetal DNA and hypomethylated in the maternal DNA.
After methylation enrichment of the fetal DNA in maternal circulation, the researchers use qRT-PCR to quantify the amount of fetal DNA in the test sample. By comparing the amount of fetal DNA present in an unknown sample to that of a control sample from a pregnant woman known to carry a normal fetus, the technique can quantify the extra copy of chromosome 21 and distinguish Down syndrome pregnancies from normal pregnancies.
NIPD Genetics combines the enrichment ratios for seven differentially methylated regions on chromosome 21 into an aggregate score called the D-value, which is what is ultimately used to make a diagnosis.
The company has published several peer-reviewed studies demonstrating the clinical performance of its test, including a landmark study published in Nature Medicine in March 2011 demonstrating 100 percent sensitivity and specificity in diagnosing fetal trisomy 21, or Down syndrome, from maternal blood samples, albeit in a relatively small patient cohort.
Since that time, NIPD Genetics embarked on an even larger validation study of 1,000 pregnant women from various European countries to fully validate its assay in anticipation of a commercial launch later this year (PCR Insider, 11/29/2012).
Papageorgiou told PCR Insider last week that the company has collected the samples necessary for such a study and is currently in the middle of testing them with the MeDIP-qPCR assay with the same goal in mind. However, the company first wanted to address an issue that has thus far cropped up with other molecular prenatal trisomy 21 tests, most of which are based on next-generation sequencing.
"Other studies that have been performed for [non-invasive prenatal diagnosis], which are mainly based on NGS, have shown that the fetal amount that is present in maternal circulation affects the actual results," Papageorgiou said.
"Some the studies that have been done with NGS have actually set a threshold or a cut-off ... where they exclude cases [with a] low fetal DNA amount," she added. "Because we haven't seen any variability in our [assay] results, we decided to actually measure the amount of fetal DNA beforehand. We wanted to prove that the fetal DNA amount really doesn't affect our results. That's why we did this study."
In their study, published last month in Prenatal Diagnosis, Papageorgiou and colleagues at NIPD Genetics and the Cyprus Institute of Neurology and Genetics used real-time qPCR to absolutely quantify free fetal DNA, total DNA, and "fetal fraction" in maternal plasma in 83 samples, to investigate whether these amounts influence the enrichment ratios of its seven differentially methylated biomarkers and the overall correct classification of trisomy 21.
Of the 83 samples examined, 68 were normal and 15 were trisomy 21 cases. The researchers found that there was a significant difference in free fetal DNA and total DNA between normal and trisomy 21 cases. They also found that normal samples and trisomy samples exhibited similar fetal fraction, which is the ratio of free fetal DNA to total DNA — not surprising since previously published research has shown that, as free fetal DNA increases in trisomy 21, so does total DNA, thus resulting in an equivalent fetal fraction.
When they performed correlation studies to see how free fetal DNA and total DNA affected the D-value and enrichment ratios of the MeDIP-qPCR test, they found that no correlation existed in any of the samples considered — trisomy 21 or normal.
"This is important because when a lab uses our method it would not need to restrict [it] to a certain group of women, depending on the amount of fetal DNA present in their bloodstream," Papageorgiou said.
Further, the group "indirectly proved" the robustness of NIPD Genetics' diagnostic formula, Papageorgiou said. The enrichment ratios of a number of differentially methylated regions exhibited moderate correlation with free fetal DNA. Meantime, one differentially methylated region had a weak correlation with total DNA, and one had a similarly weak correlation with fetal fraction. Nevertheless, the sensitivity and specificity of their test was not affected.
This suggests "that D-value is not influenced significantly by the maternal background" and that "the diagnostic formula determining the D-value combines [an] adequate number of DMR markers to allow correct classification independently of the presence of moderate correlation of some markers with ffDNA," the researchers concluded in their study.
Moving forward, NIPD Genetics will use its collection of 1,000 samples to investigate the affect — or lack thereof — that free fetal DNA, total DNA, and fetal fraction have on the ability of its test to make a correct trisomy 21 diagnosis.
In addition, "we're doing other pilot studies with minor modifications to our protocol to make it even simpler and cheaper than it is now," Papageorgiou said.
The current protocol, she explained, is a three-step procedure with various incubation steps, and the group is angling to combine these steps into one protocol that would shorten the turnaround time and reduce the cost of the assay.
NIPD Genetics used a Life Technologies Applied Biosystems 7900HT instrument to conduct its studies, but Papageorgiou said that the test can be performed by any lab with a standard real-time PCR instrument and a sonicator for freeing DNA from the blood sample.
Regarding the planned availability of its test in Europe by the end of this year, Papageorgiou said it was "very possible, because we are very close to finalizing optimization of the test. We would initially offer this as … a service provider, and of course eventually aim to provide it to laboratories so each individual laboratory can conduct the test."
To these ends, NIPD Genetics is in the final stages of raising approximately €4 million in private funding. Last year the company raised about €2.5 million of this goal, and currently it has brought in around €3.2 million of the €4 million. "Now we have potential other investors, and we hope we will have an update soon," Papagirogiou said.