FDA Validation of Companion Diagnostic (PCR test)

I’ve been helping get a companion diagnostic get approved as a FDA test. This blog series will describe the statistics (R) used for the FDA validation of the companion diagnostic.

The companion diagnostic test is a PCR test to check for a fusion / rearrangement on the customer’s DNA. Normally, people have gene A and gene B. If the patient’s DNA shows a fusion between gene A and gene B, then she should be administered a drug that is tailored to the A+B fusion.

The lab did the experiments and I performed the statistical analysis in R. This series of posts will describe the analysis.

Note: This isn’t production-level code, but it’ll do the job.

Experimental Setup

The PCR test measures FAM and Texas Red values. There are 42 PCR cycles, and so if a fluorescent signal is observed, the Ct signal must be below 42.

The FAM is the signal for the rearrangement — if it’s detected, then the Ct value (the PCR cycle that it’s detected in) should be less than 42. If the fusion is not present, then the signal is never detected and the FAM Ct value is NaN.

The Texas Red value is the signal for wild-type or normal gene A. It is the internal control — to make sure that DNA is in the test sample, we expect some normal DNA in every test because the fusion is heterozygous and a cancer sample may not be pure. We will figure out the reportable range for Texas Red in the Reportable Range Section .

The Cy5 channel measures the presence for wild-type or normal gene B. it is another internal control.

The method we used to decide whether the fusion is present is delta Ct = FAM-Texas Red.

(You could also use FAM signal only if you didn’t want an internal control of wild-type DNA, it depends on how your validation plan was written.)

FDA validation is a long process that takes a few hundred experiments to examine:
Part 2. Pre-processing
Part 3. Analytical Specificity
Part 4. Accuracy
Part 5. Run Control Specification
Part 6. Reportable Range
Part 7. Limit of Detection (LoD)
Part 8. Precision (repeatability & reproducibility)
Part 9. Checking Controls