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Natera Describes Massively Multiplexed PCR Technology, Liquid Biopsy Pipeline at ASHG

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NEW YORK (GenomeWeb) – At the American Society for Human Genetics meeting last week, Natera presented research results in line with purposing the firm's core non-invasive prenatal testing technologies for liquid biopsy.

This marked one of the first times that Natera has publicly provided an overview of the methodology, and the firm also showed preliminary clinical data, Matthew Hill, vice president of research at the San Carlos, California-based company told GenomeWeb in an interview.

Natera described its entrance into the oncology market last year. "We seek to make an impact through early detection of cancer at stage 1, when it's curable," CEO Matthew Rabinowitz said at that time.

And in August of this year, the firm told GenomeWeb that initial products could be used as reflex tests, or for disease load and recurrence monitoring, but ultimately the firm would be developing assays for early detection of cancer.

For NIPT testing currently the firm uses approximately 14,000 primers targeting SNPs across the relevant chromosomes, Hill said.

In the company's Panorama test, for example, this is used to detect aneuploidies of chromosome 13, 18, 21, as well as determining gender and discovering sex chromosome abnormalities.

And because the technology detects SNPs, the firm can determine fetal fraction, "which is widely agreed to be a crucial component in the most accurate NIPTs," Natera's Hill said.

"We've developed our own proprietary multiplex PCR — we call it massively multiplexed because we're able to go up to tens of thousands of primers in a single reaction," Hill explained.

The secret sauce for the multiplexing is proprietary, but Hill described it as "the outcome of a highly optimized primer design and high-performance chemistry and protocol, all built with multiplex in mind."

Previously, GenomeWeb reported that the technology also used chemistries that reduced formation of primer dimers.

This massive multiplexing also allows the firm to achieve high signal-to-noise ratios, he noted.

The single-tube multiplex reaction is then followed by next-generation sequencing, and the outcome of that data is "comparable to SNP microarray data but with the precision and reproducibility of an NGS platform."

In the presentation, the firm presented data on the performance of the Panorama test relative to public data on tests from competitors in the field. The firm also compared a Panorama panel add-on for assessing microdeletions — which, unlike some other factors, are independent of maternal age — to a competitor's assay, showing superior performance, Hill said.

Now, the firm is pivoting the technology into cancer, focusing on the three molecular signatures relevant to cancer DNA analysis: single-nucleotide variants (SNVs), copy number variants (CNVs), and fusions.

Hill said that this leverages essentially the same workflow — collect the sample, extract cell-free DNA, and perform a library prep procedure — but the library can be split into multiple different workflows which are modifications of the existing multiplex PCR to detect these three signatures.

The SNV analysis, for example, can be used to look at particular base positions in the genome. "We can use this to interrogate exons, or genes relevant to cancer," Hill said, adding that the firm is also adapting its NIPT algorithms to interpret the resulting data.

Interestingly, at ASHG Hill presented some preliminary evidence that the same approaches can be applied to single cells and formalin-fixed, paraffin-embedded material.

He noted that DNA in FFPE tends to be broken into small fragments, and, "since cell-free DNA is also broken up into small fragments, by design our approach works very well."

The presentation also showed validations of the overall methodology, demonstrating limits of detection for calling mutations at sites. Specifically, in a model based on 810 insert bases among 45-plex PCR amplicons using 35 negative samples, the firm showed a 0.05 percent limit of detection for A:G or C:T transitions and 0.01 percent LOD of transversions between a purine and pyramidine.

The firm also reported detecting  CNVs in cancer patient plasma with a sensitivity of less than 0.5 percent, and SNVs as low as 0.01 percent. 

"For CNVs, we're leveraging the same multiplex PCR targeted SNPs that we use to detect fetal aneuploidies in a mixture sample — cell-free DNA that is a maternal, fetal mixture — we can leverage that exact same approach to detect copy number variations in the context of liquid biopsy where you have a mixture of somatic tumor copy number events in an otherwise normal germline background," Hill said.

At ASHG, the firm also presented preliminary results from small cohorts of ovarian, breast, and lung cancers.

In some cases, the firm titrated two matched samples at a level to simulate a 0.5 percent tumor fraction with a single copy deletion event, identifying this "most challenging context" CNV down to 0.5 percent.

It also showed a small set of five ovarian cancer samples in which there was a 100 percent detection rate of CNVs from 1 ml plasma with a 0.45 percent cutoff, noting that all five had matched CNVs in tumor biopsies.

Using cfDNA from 21 stage I–IIIB breast cancer patients, CNVs were called with an allelic imbalance of less than or equal to 0.45 using as few as 62 heterozygous SNPs, and all had matching CNVs observed in tumor tissue. The presentation also touched on data showing detection of SNVs in plasma from breast cancer patients.

Finally, Natera presented data showing SNV and CNV detection in plasma in 24 lung cancer samples using a proof-of-concept panel, in which it detected 92 percent of stage I-III matched tumor samples in plasma.

The company touched on a further study using the technology to look at circulating cell-free DNA in non-small cell lung cancer, called Tracking Cancer Evolution through Therapy (TRACERx), which was announced in June of this year, and in the presentation the firm detailed upcoming collaborations with other institutions related to ovarian, breast, and lung cancers.

Natera recently raised $180 million in an initial public offering. It reported a 26 percent increase in revenues in the second quarter of this year, although it was also affected by an industry-wide stock price drop in September.

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