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Cyprus' NIPD Genetics Validates Updated Trisomy 21 Assay; Eyes 2013 Launch for Testing Services


Cyprus-based molecular diagnostics startup NIPD Genetics has taken another key step toward commercializing its flagship assay, a noninvasive prenatal diagnostic test for Down syndrome.

The company published the findings of its most recent validation study for the test, which combines methylated DNA immunoprecipitation with quantitative real-time PCR, in the October issue of the journal Prenatal Diagnosis, reporting a sensitivity of 100 percent and specificity of 99.2 percent.

NIPD Genetics is now putting together an even larger validation study of 1,000 pregnant women from various European countries with an eye toward completing the trial and offering its assay as a genetic testing service in Europe next year, company officials said this week.

To that end, NIPD Genetics said it is also looking to raise up to €4 million (about $5.2 million) in a second round of equity financing.

NIPD Genetics, a spinout of the Cyprus Institute of Neurology and Genetics, published the first peer-reviewed study of its test in Nature Genetics in March 2011 (PCR Insider, 3/10/2011).

The test involves collecting 10 mL of peripheral blood from a pregnant woman, then isolating the DNA. This is followed by methylated DNA immunoprecipitation, or 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.

In last year's Nature Genetics paper, the company used the method to correctly diagnose 14 cases of Down syndrome and 26 normal cases from maternal peripheral blood samples, achieving 100 percent sensitivity and specificity.

That test began with a group of 12 differentially methylated regions, or DMRs, that the group had identified, eventually yielding a diagnostic formula for eight of those 12 regions.

However, since that time the company dug further into the literature to discover that some of these DMRs are located on high copy number regions of the chromosome, which could have caused some difficulties in classification, Elisavet Papageorgiou, NIPD Genetics' CSO, told PCR Insider this week.

"For the [Prenatal Diagnosis] paper, we did additional studies on the initial 12 markers," Papageorgiou said. "We looked at each one individually to see whether they were located in CNV regions. If we found some of them located in CNVs, then we removed them from the set of DMRs that we actually wanted to test … because this will cause problems in picking up the copy number changes that are associated with trisomy 21. So we wanted to take out those DMRs, and work only with the ones located in normal copy number regions."

To that end, the researchers developed a new formula with seven DMRs by eliminating two from the eight that were used in the previous formula, but adding one DMR from the initial group of 12.

"By removing the DMRs located in CNV regions, you reduce the risk of misclassification," Papageorgiou said. "We didn't have this problem ... but studies that had been done in Europe and around the world … showed that regions with CNVs were located within the DMRs that we had identified. Since we want this method to eventually be applied worldwide, we didn't want to include such regions and increase the risk for misclassification. So we've increased the robustness of the approach."

As described in the Prenatal Diagnosis paper, researchers from the company used this new version of the assay in a blind validation study using maternal peripheral blood from 175 pregnant women between 11 and 14 weeks gestation.

They were able to correctly classify all 50 trisomy 21 cases and 124 of 125 normal cases (identifying one false positive), for a sensitivity of 100 percent and specificity of 99.2 percent.

"The fact that our NIPD for trisomy 21 method was validated with a higher number of samples (175 compared with 80 in the first validation) with satisfactory results demonstrates that it is accurate and reproducible," the researchers noted in their paper.

However, the researchers also noted that their test is currently relatively "technically challenging," requiring multiple steps for ligation-mediated PCR, MeDIP, DNA cleanup, normalization steps, and qRT-PCR. Already the company has made some changes to improve the assay's workflow, Papageorgiou said, in an attempt to address this and make the test easier to implement in a clinical laboratory setting.

Currently, the assay has about a three-day turnaround time – still much shorter than competing prenatal diagnostic tests that are on the market or under development and use next-generation sequencing, which can take up to two weeks.

One major advantage that NIPD Genetics sees for its assay is the fact that it uses common clinical laboratory equipment and thus does not require a lab to make a large capital expenditure.

"We only use equipment that every diagnostic lab has and works [with] every day, like a qPCR or conventional PCR instrument … and compared to NGS, it's much cheaper," Papageorgiou said.

For its published studies to this point, NIPD Genetics has used a Life Technologies ABI 7900 HT real-time PCR system, but Papageorgiou noted that the test is flexible in regards to which instrumentation platform it uses. She added that NIPD Genetics has also been working with a Bio-Rad real-time PCR system.

NIPD Genetics has already begun an even larger preclinical validation of its assay in a 300-woman cohort, which will eventually become part of a 1,000-subject trial for final clinical validation. The company expects to complete this study sometime next year, at which time it will begin offering its test as a service.

"That's how we will start offering this," Papageorgiou said. "After that of course we will look into producing a diagnostic kit that will be commercialized." And further down the road, the company will look into seeking regulatory approval in the US, but thus far has barely scratched the surface in that regard.

Lastly, NIPD Genetics will continue to improve the robustness of its test by seeking additional DMRs to either include in its diagnostic formula, or create a secondary diagnostic formula that might be able to "clarify" results from the first test if it resulted in a "gray zone where you might not be sure of the results," Papageorgiou said. "You may not need to run it with all your samples, but only with a subset of your samples."

NIPD Genetics said that the €4 million it seeks to raise in its second round of financing would likely include follow-on investments from existing backers. The company raised €2.5 million in October 2011 (PCR Insider, 10/6/2011).

The new capital would allow NIPD Genetics to "expand our team and increase our capacity to accelerate completion of our large clinical validation study and commercial introduction of our NIPD21 test," the company said in a statement.