NEW YORK (GenomeWeb News) – A proof-of-principle study appearing online last night in the New England Journal of Medicine suggests that circulating, cell-free DNA can serve as an effective blood-based marker for monitoring metastatic breast cancer in women undergoing treatment for the disease.
Researchers from the UK and Australia started by using targeted and/or whole-genome sequencing to find characteristic somatic mutations in metastatic breast tumors from 30 women. They then tested for the presence or absence of these alterations in cell-free DNA in each woman's blood over the course of her treatment, looking at how this technique fared both for finding metastatic disease and for tracking treatment response and progression.
The team found that the technique compared favorably with conventional, imaging-based tests for metastatic breast cancer and with other blood-based methods for finding the disease, such as cancer antigen testing and analyses aimed at nabbing intact tumor cells in blood samples.
"We were pleasantly surprised," co-senior author Nitzan Rosenfeld, a researcher with the University of Cambridge and the Cancer Research UK Cambridge Institute, told GenomeWeb Daily News. "We were expecting that this would be informative, but we didn't know to what extent that would be."
Not every tumor carried mutations that could be picked up with the targeted sequencing method that investigators used for this study, which focused on two breast cancer-related genes. But Rosenfeld said he is confident that with additional genetic analyses — be it whole-genome sequencing or more extensive targeted testing — mutation markers could be ascertained for those tumors, too.
For the new study, researchers started with tumor and normal samples from 52 women with metastatic breast cancer.
All of the matched tumor-normal pairs were first tested using a targeted sequencing approach aimed at finding somatic point mutations in two oft-altered breast cancer genes — PIK3CA and TP53.
The tagged-amplicon deep sequencing analyses, which relied on Fluidigm's access array paired with the Illumina GAIIx and HiSeq 2000 sequencing instruments, uncovered mutations affecting at least one of the two genes in 25 of the 52 cases.
For nine of the women — including several individuals whose tumors did not contain PIK3CA or TP53 mutations — the team went on to do whole-genome paired-end sequencing on tumors and matched normal samples. From that data, the group picked up tumor-specific structural changes in eight of the cases.
From there, researchers explored the possibility of using the somatic mutations or structural variations in each woman's tumor to detect metastatic disease via circulating cell-free tumor DNA.
To do this, they tested 141 blood samples collected from the 30 women over the course of their treatment, using tagged-amplicon deep sequencing and/or digital PCR to track down appropriate, tumor-specific somatic mutations and/or structural variants for each individual.
The team was able to exploit the tumor-specific mutation profiles to detect tumor DNA in the cell-free portion of more than 80 percent of the blood samples. This approach proved useful for finding tumor DNA in the blood of all but one of the 30 women —97 percent of the cases for which telltale mutations were originally identified.
Tests for circulating tumor cells in the blood picked up existing metastatic disease in 87 percent of cases, and blood tests targeting an antigen called CA 15-3 correctly uncovered 78 percent of cases.
Over the time tested, meanwhile, levels of the tumor cell-free DNA tended to rise or fall in a manner consistent with treatment response, as assessed by imaging.
Moreover, for 20 women who went on to have recurrent disease, the cell-free sequencing method was the first to find this progression in just over half of cases. "Among the measures tested," study authors noted, "circulating tumor DNA provided the earliest measure of treatment response in 10 of 19 women (53 percent)."
Somatic mutations were not detected in tumors from 22 of the original 52 women, though, meaning there were no specific genetic markers to test for in their circulating cell-free DNA.
Rosenfeld explained that these tumors almost certainly contain characteristic structural changes and/or somatic mutations that could be found through more detailed genetic analyses.
"I would be very surprised if there was a case where we would sequence the tumor's whole genome and not find any mutations to track," he said.
As sequencing costs continue to decline, ever more tumors are expected to undergo the whole-genome sequencing analyses needed to get a look at the complete suite of structural and sequence changes in a given cancer.
In the meantime, those involved in the current study are expanding their analyses of circulating cell-free DNA to include other types of cancer, cancer at different stages, and in individuals undergoing various treatment regimens, Rosenfeld noted. They're also considering additional candidate genes for targeted testing during the mutation marker-finding step.