Prevalence of BRCA homopolymeric indels in an ION Torrent-based tumour-to-germline testing workflow in high-grade ovarian carcinoma

Abstract

Tumour DNA sequencing is essential for precision medicine since it guides therapeutic decisions but also fosters the identification of patients who may benefit from germline testing. Notwithstanding, the tumour-to-germline testing workflow presents a few caveats. The low sensitivity for indels at loci with sequences of identical bases (homopolymers) of ion semiconductor-based sequencing techniques represents a well-known limitation, but the prevalence of indels overlooked by these techniques in high-risk populations has not been investigated. In our study, we addressed this issue at the homopolymeric regions of BRCA1/2 in a retrospectively selected cohort of 157 patients affected with high-grade ovarian cancer and negative at tumour testing by ION Torrent sequencing. Variant allele frequency (VAF) of indels at each of the 29 investigated homopolymers was systematically revised with the IGV software. Thresholds to discriminate putative germline variants were defined by scaling the VAF to a normal distribution and calculating the outliers that exceeded the mean + 3 median-adjusted deviations of a control population. Sanger sequencing of the outliers confirmed the occurrence of only one of the five putative indels in both tumour and blood from a patient with a family history of breast cancer. Our results indicated that the prevalence of homopolymeric indels overlooked by ion semiconductor techniques is seemingly low. A careful evaluation of clinical and family history data would further help minimise this technique-bound limitation, highlighting cases in which a deeper look at these regions would be recommended.

Figure 1

BRCA1/2 testing results in our tumour-to-germline analysis workflow in a cohort of patients with high-grade ovarian carcinoma (HGOC). PVs pathogenic/likely pathogenic variants, MLPA multiplex ligation-dependent probe amplification, WT wild type.

Figure 2

Boxplots showing the allele frequencies at tumour testing of duplications (ins) or deletions (del) at homopolymeric regions in patients with extra-homopolymer pathogenic/likely pathogenic variants (PVs, red boxes) compared with patients from the study cohort (green boxes); panels from 1 to 4 show outlier variants identified in the four patients from the study cohort (red dots), panels I and II show the two homopolymeric variants previously identified through germline testing (green dots).

Figure 3

BRCA2 homopolymeric region c.4279_4284 from patient 3 visualised with the IGV software (ver. 2.3.97); the count of the total, inserted (INS) and deleted (DEL) reads at the first base of the thymine stretch (highlighted with a red rectangle) are shown in the box.