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German Group Details Digital-Direct RT-PCR Method to Quantify Cervical Cancer CTCs

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A research team from Jena University Hospital at the Friedrich Schiller University in Germany has developed a method it calls digital direct RT-PCR (DD-RT-PCR) to quantify circulating tumor cells in cervical cancer by detecting HPV-oncogene transcripts.

The group conducted a number of experiments using both cell lines and patient samples to demonstrate the sensitivity of the method, publishing the results this month in Scientific Reports.

According to the group's corresponding author Norman Häfner, the Jena team became interested in looking for a way to quantify CTCs in cervical cancer after earlier work successfully evaluated the use of HPV oncogene transcripts as superior to epithelial-specific markers as a target for detection and prognostic assessment of cervical cancer lymph node metastases.

In the recently published study, Häfner and colleagues describe a method they developed combining Ficoll separation, ThinPrep fixation, and one-step RT-PCR in a low-throughput digital PCR format to allow the direct analysis and detection of cervical cancer CTCs without the need for RNA isolation.

In an email to PCR Insider Häfner wrote that if the team's results using this DD-RT-PCR method can be confirmed in larger cohorts, it could find use clinically, either to identify subsets of patients who might benefit from adjuvant treatment or closer follow-up than others, or to monitor the efficacy of treatment by serially tracking CTC counts.

"Additionally, this method may be transferred to other tumor entities [that express] tumor-specific transcripts and [have] a high rate of blood-based metastasis," he wrote.

In the study, Häfner and his colleagues described the DD-RT-PCR method as involving separation and fixation of mononuclear blood cells, followed by one-step PCR using Roche's Transcriptor one-step RT-PCR kit to amplify the target sequence, and then semi-nested qPCR using a Qiagen Rotor-Gene system on centrifuged products of the first reaction.

Cervical cancer, because it is virus-induced and thus expresses highly specific viral oncogene transcripts, offers an optimal subject for direct CTC enumeration without the need to estimate CTC numbers based on analysis of extracted RNA from the mononuclear cell fraction of a blood sample.

Testing the method on SiHa and HeLa cells in experimental blood samples, the group was able to detect target tumor cells among as many as 500,000 non-tumor mononuclear blood cells, but settled on a limit of 200,000 MNBCs per reaction, above which the reproducibility and sensitivity of the assay was compromised, the authors wrote.

The researchers also did spiking experiments to further validate the method, and finally tested the method in blood samples from 10 patients with HPV 16- or HPV 18-positive cervical cancer. In this cohort, the group detected CTCs in three of the 10 patients with numbers ranging from one in 540,000 to one in 1,750,000 MNBCs.

One of these three patients had local disease only, the group wrote, while the other two had systemic disease with distant metastases.

According to Häfner the Jena group has also explored analysis of intact CTCs using a continuous-flow microfluidic PCR method.

"We were not successful in establishing an in situ PCR to label HPV-positive cells [but] we could show the detection of target cells by a direct 'on-cell' PCR within microfluidic compartments. In principle, this method can be used for clinical samples but not for the detection of extremely rare CTCs [because] the high number of cells which need to be analyzed for the detection of one CTC could not be processed in the developed microfluidic system. Thus, we switched to regular PCR systems and established the described Digital-Direct RT-PCR," he said.

Newer droplet-based digital PCR methods might provide a more appropriate platform, according to Häfner. However, he said, "several aspects have to be considered," including the fact that the limited compartments per sample of current systems may require several reactions to be performed for each clinical sample if one or zero blood cells is to be present in each compartment.

Additionally, he said, "No data are available showing a stable generation of water-in-oil droplets using high numbers of intact cells. Albeit we have seen in our earlier experiments that water compartments enclosing intact cells are stable in a microfluidic system, we have no data regarding droplet suspensions as used for dPCR."

Despite these caveats, Häfner said, the group is interested in validating newer droplet-based PCR methods for cervical cancer CTC quantification. "We would like to cooperate with other research groups and companies for the evaluation of droplet-based systems," he said.

While the team focused on cervical cancer in its recent paper, Häfner said that the method is potentially directly applicable to other HPV-induced tumor types, such as head and neck cancers, or to other cancer subsets associated with tumor-specific fusion transcripts, which occur in ovarian and prostate cancers, for example.

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