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Circulating Tumor Cell Strategy Could Improve Liver Cancer Screening

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NEW YORK (GenomeWeb) – Using a microfluidic technology called the CTC-iChip to isolate circulating liver cancer cells, researchers have developed a testing method they hope can help improve screening for liver cancer in at-risk individuals and/or monitoring of patients after initial treatment.

The team, led by researchers from Massachusetts General Hospital Cancer Center, published a study in Proceedings of the National Academy of Sciences late last week, showing that by combining use of the MGH-developed CTC-iChip with digital PCR they could improve detection of cancer in patients with hepatocellular carcinoma (HCC) compared to current methods like screening serum for alpha-fetoprotein (AFP).

CTC-iChip is among a throng of new technologies aiming to enable detection of exceedingly rare cancer cells shed into circulation.

Standard techniques for isolating CTCs involve pulling these cells out of the larger mix of blood cells by targeting molecules on the tumor cell surface — epithelial cell adhesion molecule (EpCAM), for example —  and dragging them out.

But using markers like EpCAM can miss some CTCs that don't express them or don't express them highly enough, Mark Kalinch, the study's first author, said in an interview this week.

Amongst the solutions proposed to this problem are other unbiased platforms like EPIC Sciences', which spins out a cell pellet that includes all white blood cells along with any CTCs, then re-suspends this pellet and spreads it across slides in a monolayer on which imaging technology and other methods can be used to distinguish CTCs.

The CTC-iChip, in contrast, uses microfluidics to deplete hematopoietic cells from blood samples by size-based exclusion of red blood cells, platelets, and plasma, followed by magnetic deflection of white blood cells tagged with magnetic bead-conjugated CD45, CD16, and CD66b antibodies.

The remaining mix includes any CTCs along with some remaining white blood cells — fewer than 500 per milliliter of processed whole blood, according to the study authors.

In their PNAS study, the Mass General researchers described their development of a digital PCR assay for use with the CTC-iChip to identify patients with incipient HCC.

In short, the method involves encapsulating cDNA molecules within individual aqueous droplets to allow quantification of multiple transcripts of interest by in-droplet PCR amplification.

The group assayed blood samples from more than 150 patients divided into six groups: newly diagnosed HCC patients; HCC patients receiving treatment who still had evidence of disease; patients who appeared to be cured after surgical treatment; presumed healthy patients at risk for developing HCC because of other chronic liver diseases; patients with other types of cancer, including some with liver metastases; and finally healthy control subjects.

After some initial experiments, the team narrowed the potential RNA transcript targets to nine that were most significantly associated with HCC compared to normal controls.

When they applied this score to the study cohort, they were able to pick out more than half of those with untreated HCC. Meanwhile, only two of 26 age-matched controls and only one of 31 patients with at-risk nonmalignant liver disease tested positive.

In the cohort of patients currently being treated, about 28 percent of patients had positive scores. For patients with no evidence of disease after treatment the frequency of a positive score was similar to what was seen in healthy controls.

According to the authors, the results demonstrate that the CTC score can identify patients with active disease while maintaining a high degree of specificity.

Interested in whether the score could also be used to monitor disease status over time, the researchers also followed a small group of patients longitudinally. CTC scores remained high in two patients who had no therapeutic intervention or change in clinical status between blood draws, while the scores of two other patients dropped after surgical tumor removal.

Another patient's score dropped precipitously after treatment with a checkpoint inhibitor and then showed significant further reduction after a radioembolization procedure that greatly reduced the size of the tumor, the authors wrote.

Importantly, they added, for three of these five patients serum protein AFP measurements were below clinically informative values at all draw points, which is a relatively common occurrence. If the results can be further validated, the early data in these patients suggests that CTC score monitoring could serve as a complementary marker to assess disease status in such patients.

Finally, the group compared current standard AFP screening to their scoring method in 15 newly diagnosed HCC patients.

In this group, four patients were only found to be positive using the CTC score, while one was only positive by AFP. For another five, both tests picked up the presence of cancer.

Overall, the authors wrote, combining the two testing methods produced positive results for 67 percent of patients, which is a significant improvement over AFP screening alone.

According to the authors, with a cutoff of 20 ng/mL, AFP has an estimated sensitivity of 53 percent with a specificity of 87 percent. Raising the threshold to 100 ng/mL improves specificity to 99 percent but reduces the test sensitivity to 31 percent.

Though picking out a little more than 50 percent of cancers may not seem like adequate sensitivity, Kalinch said that "bootstrapping" AFP with the CTC score method is one potential was to use the approach clinically.

Alternatively, he said, if specificity can be sacrificed — for example in the context of limited screening of high-risk populations to help reduce the use of expensive follow-on imaging, the sensitivity of CTC scoring could also be raised.

HCC, which arises within well-defined at-risk populations, and in which early detection may be curative, is particularly appropriate for assessment of a CTC-based strategy like this, the study authors wrote.

The group may also be able to boost sensitivity by revisiting their transcript panel. "For these 10 … we used a bioinformatic approach to identify them, but it's entirely possible we are missing huge swaths of useful transcripts. Just because we picked these 10 doesn't mean they are the best, so we are actively pursuing single-cell RNA sequencing to identify better markers," Kalinch said.

Stymied by the challenge of isolating CTCs, and the fact that some cancers and some patients seem to shed few if any cells into their bloodstream, many groups have focused on circulating cell-free DNA as a vehicle for early cancer detection.

But Kalinch said that in HCC, genotype-based cancer detection using circulating DNA fragments would be of limited utility because of a lack of highly prevalent hallmark mutations compared to other cancer types.

"There aren't any canonical mutations in liver cancer that you could use for that, no highly penetrant mutations like there are in some other cancers," he said.

Moving forward, Kalinch and colleagues are hoping to further validate their findings in larger cohorts, but he declined to share any concrete plans.

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