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Menarini Silicon Biosystems Publishes Single-Cell WGA Liquid Biopsy Workflow, Eyes Clinical Market


NEW YORK (GenomeWeb) – Menarini Silicon Biosystems has combined several proprietary and recently acquired technologies into a cell-based liquid biopsy workflow for whole-genome analysis of copy number alterations in single cells.

In a study published this month in PLoS One, the company showed that its workflow, with single-tube library prep, generates whole-genome absolute copy number profiles of single cells and allows alteration calling at resolutions down to 100 Kbp with as few as 200,000 reads.

Menarini Silicon Biosystems is a wholly owned subsidiary of the Menarini Group, a multinational pharmaceutical, biotechnology, and diagnostics company headquartered in Florence, Italy. It was created from the acquisition of Bologna, Italy-based Silicon Biosystems in 2013, and expanded with the acquisition of assets from Janssen Diagnostics related to the CellSearch circulating tumor cell system.

The firm's CSO, Nicolo Manaresi, said that the company is working toward completing CE-IVD certification of the entire WGA pipeline, which includes CellSearch CTC purification; isolation of pure cells with the firm's DEPArray platform; whole genome amplification with Ampli1 WGA; and one-step, single-tube library prep with the firm's Ampli1 LowPass kit to support both Ion Torrent and Illumina sequencing.

The workflow is currently for research use only, but the firm anticipates wider adoption and expanding clinical use. Menarini is also evaluating further steps into the clinical market through lab-developed testing and the potential of regulatory submission to the US Food and Drug Administration.

The lowpass method can hopefully increase the number of researchers who have access to the molecular information hidden in CTCs, Manaresi said, adding, "This is quite exciting for us; this information is very precious because it is intimately linked to tumor biology, and already several indications are emerging in the literature demonstrating the impact of this kind of approach for precision medicine."

In the PLoS Study, Menarini scientists evaluated the workflow on single cells isolated from six aberrant cell lines of the National Cancer Institute H series, comparing their technique to oligonucleotide microarrays. To demonstrate the feasibility of the workflow on clinical samples, the team also analyzed single CTCs and white blood cells isolated from the blood of one patient affected by prostate cancer and two with lung adenocarcinoma, for a total of 15 CTCs and seven WBCs. Data from clinical samples showed a low level of noise, comparable to the cell line data, with different CTCs from the same patient showing highly consistent results and partially divergent profiles ascribed to tumor heterogeneity. 

Toward a goal of regulatory approval, the firm is starting from a solid foundation, Manaresi said, considering that the FDA has already cleared CellSearch for identification and enumeration of CTCs in metastatic prostate, breast, and colorectal cancers.

The workflow has also been adopted in several ongoing efforts, mainly in Europe, Manaresi said, related to molecular characterization of CTCs. For example, a group in Manchester used the workflow to analyze copy number alterations in tumor cells from patients with small cell lung cancer, work that was published in 2016 in Nature Medicine.

Menarini also offers its instruments and kits, as well as services, to the pharmaceutical industry, and Manaresi suggested pharmaceutical companies' adoption of this workflow for use in clinical trials could become a good source of initial uptake.

To get to single CTCs, the CellSearch technology pulls tumor cells from blood samples using extracellular epitopes and immunomagnetic enrichment in an automated way. The DEPArray technology then uses a programmable microelectrode array to remove any contaminating leukocytes, moving the remaining cells across the microarray and placing each one in its own tube for amplification or collecting a pool, as well as collecting white blood cells for an internal control. The newest version of the DEPArray technology allows for collection of up to 96 single cells.

Manaresi noted that the Ampli1 amplification method has also been shown to be "best in class" in several published head-to-head studies. For example, a PLoS One study showed the method gave the highest rate of correct calls when compared to three other techniques. And an Oncotarget study showed the method outperformed two others with respect to SNP, mutations, and indel calling. This is due in part to the fact that the Menarini method used ligation-mediated PCR, Manaresi said, adding that generating whole genome sequencing libraries with Ampli1 LowPass is faster than other methods because it does not require removal of adaptors, fragmentation, or other enzymatic steps.

The single-tube library prep in turn supports wider adoption, Manaresi suggested, both in translational research and, eventually, in clinical use. "Other techniques are also able to detect copy number alteration, but they are labor-intensive and more costly," Manarisi said, noting that the Menarini techinique is approximately an order of magnitude lower in cost with respect to array CGH, a method widely adopted in the clinical setting.

Menarini is also working on expanding the CellSearch cell isolation capabilities beyond epithelial cells by developing additional markers and capture agents to pick out other cancer cells types from blood, Manaresi said. The company will be presenting at the upcoming American Association of Cancer Research conference on an application for multiple myeloma cells that uses the entire workflow, with data showing subclonal evolution. That application is currently research use only and also available in the firm's clinical service lab.