A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA
Because a single cell holds only about 6 pg of DNA, mutation analysis of circulating tumor cells has usually required whole genome amplification first; this AACR 2020 poster reports an amplicon panel run directly on cell lysate and compares the mutations it found in single CTCs against matched circulating tumor DNA.
Presented by RareCyte and the University of Washington.
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- Running the amplicon panel directly on cell lysate, with no prior whole genome amplification, raised median read depth 48-fold. Across single model CTCs, 23 of 29 variants (79%) present in bulk A549 genomic DNA were recovered without WGA, against 15 of 29 (52%) after it, and the false positive error frequency was 8-fold lower and the false negative error frequency 2-fold lower than the WGA method.
- In each of two metastatic breast cancer patients, a known mutation was detected in 3 of 5 single CTCs and in 0% of the matched white blood cells. One patient’s CTCs carried PIK3CA E542K, a well-documented oncogenic mutation; the other’s carried ERBB2 L755S, which confers resistance to common anti-HER2 therapies, in a tumor that had presented as mixed invasive ductal (HER2+) and lobular (HER2-) carcinoma with differential HER2 expression.
- The same variants were present in each patient’s cell-free DNA at similar allele frequencies, whichever manufacturer’s blood collection tube the plasma came from. PIK3CA E542K read 63% in cell-free DNA from both the RareCyte tube and the Streck tube, and ERBB2 L755S read 21% from the RareCyte tube against 25% from the Streck tube, so the single-cell calls and the circulating tumor DNA agreed in both patients.
