Sorting Cell-Free DNA by Length May Increase Tumor Detection

There are “subtle but distinct” differences in the length between normal cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA), according to a study published July 18 in PLoS Genetics. Exploiting the consistently shorter fragment length of ctDNA may improve the sensitivity of liquid biopsy testing, the authors say.

“This development has the potential to enable earlier detection of solid tumors through a simple blood draw by substantially improving our ability to detect very low quantities of circulating DNA derived from tumor cells,” says lead author Hunter Underhill, M.D., Ph.D., in a statement. “It’s possible that jump in sensitivity could make the difference between being able to detect a cancer, and not.”

While the prospect for noninvasively detecting and monitoring cancer is exciting, the clinical utility of liquid biopsies has been limited by its sensitivity, particularly in detecting ctDNA from nonmetasticized solid tumors.

Detecting ctDNA against the abundant backdrop of normally occurring cfDNA derived from healthy cells has been likened to detecting a needle in a haystack. However, researchers are working on developing novel approaches to improve detection of ctDNA, including assessing differences in fragment length between healthy cfDNA and ctDNA.

Underhill and colleagues utilized massively parallel sequencing to define these fragment length differences. First, animal models of glioblastoma and hepatocellular carcinoma showed that the most common fragment lengths of ctDNA were 134 and 144 bp, compared to the 167 bp fragment length most seen in normal cfDNA.

As a next step, the researchers found similar differences in the fragment length of ctDNA in humans with melanoma and lung cancer, compared to healthy controls. The most common fragment length for the BRAF V600E mutant allele in the melanoma patient was shorter than the most common fragment length of the wild-type allele (132–145 bp versus 165 bp, respectively). Similarly, size selecting for shorter cfDNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. Overall, the authors say that fractional selection of cfDNA that is 20 to 50 bp shorter than the size of normal healthy cfDNA may “substantially enrich” samples for ctDNA in human cancer testing.

“Size-selection for shorter cfDNA fragments increased the proportion of ctDNA within a sample,” writes Underhill and colleagues. “These results provide compelling evidence that development of techniques to isolate a subset of cfDNA consistent with the ctDNA fragment lengths described in our study may substantially improve detection of non-metastatic solid tumors. As such, our findings may have a direct impact on the clinical utility of ctDNA for the non-invasive detection and diagnosis of solid tumors (i.e., the “liquid biopsy”), monitoring tumor recurrence, and evaluating tumor response to therapy.”

Takeaway: Using ctDNA tumor length to segregate cfDNA may improve the clinical utility of liquid biopsy technology for the detection of non-metastatic, solid tumors.


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