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CTC Enrichment Comparison Demonstrates Methods' Potential in Melanoma Patient Management

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NEW YORK – An international group led by researchers at Edith Cowan University in Perth, Australia have compared the abilities of Angle's Parsortix and Biolidics' ClearCell FX1 platforms for the isolation and purification of melanoma circulating tumor cells (CTCs) from patient blood samples.

While Parsortix yielded lower white blood cell (WBC) backgrounds in the samples, ClearCell detected melanoma CTCs in additional samples that Parsortix was unable to identify.

The researchers aim to integrate one of the enrichment platforms with downstream detection assays to investigate prognostic and treatment response monitoring applications for metastatic melanoma patients.

Surrey, UK-based Angle's Parsortix cell-sorting system uses microfluidic disposable cassettes to capture and harvest CTCs based on their less-deformable nature and larger size compared to other blood components. Captured cells can be fixed and stained for in-cassette identification and enumeration or can be recovered for external staining and molecular analysis with qPCR and other techniques.

Singapore-based Biolidics' ClearCell FX1 system is a label-free liquid biopsy device that the firm says automates CTC isolation and enrichment and offers two different purity protocols. After lysing red blood cells in a blood sample, the platform passes the sample through its single-use CTChip FR1 microfluidic biochip to isolate CTCs from a WBC background.

In the study, published in the British Journal of Cancer earlier this month, Elin Gray, a senior author and ECU associate professor, and her team began by collecting 8 mL of blood from a cohort of healthy controls and 37 melanoma patients.

After spiking the healthy blood samples with dyed melanoma cells, the researchers processed both sets of samples using Parsortix and ClearCell FX1. They processing spiked samples using ClearCell's low- and high-purity protocols but decided to use ClearCell's low-purity protocol to isolate CTCs from melanoma patient blood samples.

For melanoma patients, Gray and her team collected two samples per patient for each platform. The group then performed multi-marker immunostaining for phenotypic CTC detection on one sample, while using the second sample for RNA and DNA isolation for molecular detection.

The researchers then used reverse-transcriptase PCR (RT-PCR) to analyze enriched CTC fractions for five genetic transcripts. The team also performed additional genetic profiling by using a droplet digital PCR (ddPCR) assay to quantify transcript levels of 19 genes from Parsortix-enriched samples.

The team also isolated cell-free DNA from 5 mL of the melanoma patients' samples using QIAmp circulating nucleic acid kits. By quantifying circulating tumor DNA (ctDNA) using mutation-specific ddPCR, the researchers wanted to see if correlations existed between ctDNA levels and patient CTC scores.

For spiked blood samples, Gray and her team found that Parsortix yielded a higher CTC recovery at a low total WBC count than both of ClearCell's enrichment protocols.

To further compare the two enrichment platforms, Gray's team matched blood samples from 10 metastatic patients. Using immunostaining, the researchers saw that Parsortix produced, on average, a two-fold lower WBC background than produced by ClearCell. They detected CTCs in 30 percent of melanoma patient samples processed via both Parsortix (range of 1 to 13 CTCs) and ClearCell (range of 2 to 10 CTCs).

Using RT-PCR, the group successfully detected at least one melanoma transcript in 40 and 60 percent of samples enriched by Parsortix and ClearCell, respectively. The study authors also found that ClearCell detected CTCs in samples that Parsortix did not identify, noting that "ClearCell-enriched fractions also showed a slightly higher abundance of detected transcripts compared to Parsortix-derived samples."

However, Gray believes that the difference was minimal, as ClearCell only detected melanoma-specific transcripts in two additional CTC samples. Gray argued the discrepancy could either be due to sampling error or what she calls a "protective effect."

"Because you have more cells going into the RNA extraction process, you have more of a protection effect, which is kind of like putting carrier RNA into extraction," Gray explained. "Because you have a lot of RNA material, it is less sensitive to [ribonuclease]."

Because Parsortix yielded lower WBC backgrounds than ClearCell, the researchers carried out follow-up molecular analyses in Parsortix-enriched samples.

Comparing CTC fractions derived from 43 blood samples from the melanoma patients, Gray and her team detected CTCs via immunocytochemistry in 12 of the 43 samples. In addition, they saw that the CTCs varied in size, ranging from 10.2 µm to 68.3 µm.

RT-PCR analysis of five melanoma-associated genes showed that least one transcript was detected in 18 out of the 43 samples. However, only seven of these samples had detectable CTCs by immunostaining. Using ddPCR, the team then found that 23 out of the 43 CTC fractions were positive for at least one transcript.

Using the multi-marker approach, the group saw that high CTC scores strongly predicted shorter overall survival and were linked to progression-free survival in melanoma patients. CTC scores also mirrored plasma ctDNA changes and had similar pharmacodynamic changes when patients began drug treatment.  

Gray noted that her team's biggest challenge involved implementing multiple markers and different assays in order to detect melanoma CTCs.

While each approach individually produced very low and variable CTC detection rates, the researchers successfully increased the detection rate up to 72 percent in Parsortix-derived CTC fractions using all three methods. They therefore believe that applying an integrated approach may help improve melanoma CTC detection.

The study authors also believe that despite the high phenotypic and molecular heterogeneity of melanoma CTCs, "multi-marker derived CTC scores could serve as viable tools for prognostication and treatment response monitoring in patients with metastatic melanoma."

In a statement, Angle highlighted that Parsortix did not require several pre-processing steps, while competing systems needed multiple pre-processing steps to remove red blood cells. However, the firm did not respond to multiple requests for further clarification.

While Parsortix yielded the highest recovery of CTCs among the lowest WBC background, Biolidics said in an email that the "fit-for-purpose" and clinical sensitivity are the two most crucial parameters for any liquid biopsy technology.

"We are encouraged to know that with the ClearCell FX technology, the ECU researchers are able to achieve the highest CTC detection rate in melanoma clinical specimens using mRNA detection modality against all other competing technology platforms," Biolidics added.

Gray said that her team now anticipates analyzing the enriched CTC fractions on single-cell sequencing platforms offered by firms like 10X Genomics in order to visualize the "entire transcript's blueprint." She highlighted that downstream applications would benefit from a low WBC background, as sequencing all cells in the sample can be very expensive.

Biolidics added that the researchers' "interesting results further support the increasingly expanding clinical evidence that CTC-based liquid biopsy can provide insights to melanoma patient care management."

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