NEW YORK (GenomeWeb) — Compared head to head, circulating tumor DNA and biopsy samples from women with metastatic breast cancer yielded similar, although not completely concordant, sequencing results in a Belgian study using the Ion Torrent AmpliSeq cancer hotspot panel.
The researchers are now following up the results, published earlier this year in Annals of Oncology, to better establish the potential utility of targeted sequencing in identifying breast cancer mutations in plasma.
Such liquid biopsy analyses, based on the group's early results, could serve as an adjunct to metastatic biopsy sequencing, which may miss relevant mutations because its specificity fails to capture tumor tissue heterogeneity, or in lieu of biopsies in cases where metastatic tissue cannot be sampled.
The use of sequencing to guide treatment in metastatic cancer is growing, according to the study authors, both in the research setting and clinical molecular screening programs. However, many metastatic cancer patients are ineligible or unable to participate in these programs because they have inaccessible or insufficient metastatic lesions or are unwilling to undergo invasive biopsy procedures.
In their initial study, the Belgian researchers set out to compare metastatic biopsy and plasma samples from a small group of 17 breast cancer patients. OncoDNA, a clinical NGS provider affiliated with the Institute of Pathology and Genetics in Gosselies, Belgium, performed the sequencing using the Ion Torrent AmpliSeq cancer hotspot panel.
Michail Ignatiadis, the study's senior author, told Clinical Sequencing News this week that other studies have looked at broader methods like exome sequencing, and others have evaluated much more targeted testing using digital PCR and bead-based methods. But he and his colleagues are the first, they believe, to apply a commercial NGS panel to plasma ctDNA in the CLIA lab setting.
Ignatiadis and his colleagues collected a total of 69 tumor samples, both primary and metastatic, and 31 plasma samples from 17 metastatic breast cancer patients to compare the two sample types.
According to the authors, the effort yielded evaluable NGS results for 60 of the tumor samples and all 31 plasma samples. All nine tumor samples that were unusable were formalin-fixed blocks from two patients' primary tumors, sampled more than 20 years prior.
Twelve of the 17 patients in the study had at least one mutation in their tumor DNA in p53, PIK3CA, PTEN, AKT1, or IDH2. Twelve of the 17 patients also had at least one mutation in their plasma in p53, PIK3CA, PTEN, AKT1, IDH2, or SMAD4.
Focusing on matched tumor and plasma samples taken at the same time point, the group compared their results and concluded that the same mutation was present in both tissue and plasma in nine patients, and no mutation was found in either sample type in another four patients.
Only two patients had a mutation in their tissue samples that was not present in the sequencing results of their plasma sample. In one, a PTEN mutation identified in both plasma and tissue was joined in the plasma analysis by a SMAD4 mutation. In the other case, a patient showed a PIK3CA mutation in plasma, but not in synchronous liver biopsy tissue.
In both cases, the allele frequency of the plasma-only mutations was high, suggesting that the mutations may represent tumor clones not captured by biopsy — and demonstrating the potential of plasma sequencing to capture tumor heterogeneity better than single biopsies.
Two other patients showed a mutation in plasma that was not detected by sequencing tumor tissue. Interestingly, when the researchers retested the plasma samples using an alternative Illumina sequencing method, they picked up the mutations. This, the authors wrote, suggests that the failure of the AmpliSeq panel to pick up the mutations is likely due to them being present at too low a frequency.
Overall, the study found that the two sample types were concordant in 13 of 17 cases, or 76 percent of the cohort. According to Ignatiadis, the results have two main implications. One is that using panels like AmpliSeq on plasma DNA could be an accurate and effective tool that would allow more metastatic breast cancer patients to be involved in molecular screening programs.
For example, "women with only bone metastases represent a substantial subset of metastatic breast cancer patients," Ignatiadis said. Tissue biopsy is not an option for them.
The other implication of the group's results is that plasma DNA might represent a more comprehensive picture of metastatic disease than single biopsies. "When you do a single biopsy it's possible you may miss a mutation that is either next to the site in the same lesion, or in another organ.
The two study cases who showed plasma-only mutations, for example, had multiple metastases in multiple organ systems, including bone, in addition to the liver metastases that were the source of their biopsy tissue.
According Ignatiatids and his coauthors, the group was able to detect mutations in plasma circulating DNA at allele frequencies as low as 0.5 percent using the AmpliSeq panel. By contrast, technologies focused on small numbers of preselected mutations — such as digital PCR and newer bead-based amplification tools — can detect DNA at allele frequencies as low as 0.01 percent.
However, these tools, because of their limited scope, look to be more useful for disease monitoring rather than molecular screening.
According to Ignatiadis and his colleagues, though their study results indicate that using commercial panels like AmpliSeq on plasma could be an important adjunct, or potentially a stand-in for metastatic tumor tissue analysis, larger studies will be needed to establish more clearly whether plasma ctDNA is truly equal to or even superior to metastatic biopsies.
Ignatiadis said his team is also involved in the ongoing Aurora effort, led by the Breast International Group, which will sequence samples from around 1,300 patients with metastatic breast cancer in collaboration with Belgium's OncoDNA, using its OncoDEEP Clinical Plus test, an adapted version of the 400-gene Ion comprehensive cancer panel on the Ion Proton.
Though ctDNA is not a main focus of Aurora, Ignatiadis said that plasma samples are being collected in the study, and his team plans to analyze them and compare the results to tissue biopsies to expand upon their recently published study.