The clinical usefulness of myPath® Melanoma is supported not only by a sensitivity and specificity greater than 90%, but also by the robust and reproducible nature of the assay, as demonstrated by a stringent analytical validation.
The analytical validation specifically assessed the various components of the assay by:
Measuring the precision of the assay to ensure a result was reliably reproduced.
Thirty samples were tested three separate times and the standard deviation of the myPath Melanoma score was determined to be 0.69 units. This constitutes 2.5% of the observed range of the assay, which was 27.8 units (-16.7 to +11.1) in the clinical validation cohort.
The precision of each of the assay’s 24 amplicons was measured based upon the standard deviation of each amplicon’s CT measurement. The average standard deviation for the signature and housekeeper amplicons was 0.35 and 0.21, respectively. This indicates that the amplicons within the signature are reproducible and precise.
Measuring the RNA yield from the extraction process.
None of the 464 samples used in the training of the gene signature produced an RNA concentration less than the minimum concentration requirement of 2 ng/µl. Adequate RNA was successfully extracted from melanocytic lesions as small as 0.125 mm2. This indicates that the RNA extraction is a robust process for generating sufficient material for sample analysis.
Measuring the dynamic range of RNA concentration over which the assay could reproducibly generate an accurate and consistent result.
Three samples were tested over a 33-fold range of input RNA concentrations and one was tested over a broader 2000-fold range. The standard deviation across dilution points was 0.40, indicating the test result is independent of RNA concentration.
Determining the extent to which melanin might inhibit the assay.
Melanin can inhibit PCR through competitive binding of DNA polymerase. To determine if residual melanin contamination accrued during RNA extraction could interfere with the assay, purified exogenous melanin was added to RNA previously extracted from melanocytic lesions. The inhibition point for the assay was found to be between 0.125 and 0.25 µg of purified exogenous melanin. Additionally, it was found that the amount of contaminating melanin sufficient to inhibit PCR cannot be obtained through the RNA extraction process utilized during myPath Melanoma testing.