Abstract:
BACKGROUND: Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many
hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the
sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples,
although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be
used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE
Whole Genome Sequence (WGS) samples extracted from the same tumor.
METHODS: Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on
matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers,
we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity
and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally,
we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the
overlap in clonal and subclonal somatic variants.
RESULTS: We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing
that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between
callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the
sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an
average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that
part of the discrepancy between the FF and the FFPE samples can be attributed to ITH.
CONCLUSION: This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs
can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants
derived from WGS of FFPE material can be used in cohort studies
