Harmonization trial on testing strategies in ER+/HER2- breast cancer patients: an Italian experience.

Aims: To date, activating mutations acts as key player to clinically stratify estrogen receptor (ER)+/HER2-advanced breast cancer (BC) patients eligible to novel new generation oral Selective Estrogen Receptor Degraders (SERD) relapsing after first line aromatase inhibitors. Liquid biopsy represents the most useful biological source to detect activating mutations in clinical setting, but the lack of standardized pre-analytical and analytical procedures drastically impacts on detection rate of mutations in diagnostic specimens. Here, we sought to harmonize technical procedures comparing technical performance of diagnostically available testing strategies on a series of three reference specimens (sample A, B, C) harboring p.D538G mutation at different mutant allele fraction (MAF) (5.0 %, 1.0 %, 0.5 %) shared with n = 10 Italian referral institutions.

Methods: A total of 10 μl of gDNA from each reference sample built to mimic clinically detectable molecular alteration (5.0 %, 1.0 %, 0.5 % VAF) was shipped by coordinator institution to each participating group to test p.D538G alteration leveraging own routinely available testing strategy. Artificial reference sample was previously validated by the University of Naples Federico II before arranging the shipment.

Results: exon 10 p.(D538G) hotspot mutation was successfully identified in 90.0 % of samples A, B whereas 8 out of 10 (80.0 %) participating institutions detected sample referenced alteration in sample C. No statistically significant variations were observed between dPCR and NGS based workflows in terms of detectability rate on standard reference samples. A single participating institution (ID#5) failed to detect p.(D538G) alteration but supervised procedures by coordinator institution enabled to detect referenced mutation in engineered reference samples set adopting an orthogonal technology (dPCR). In addition, NGS and dPCR platforms displayed a similar technical performance in detecting across samples A-C.

Conclusions: NGS and dPCR systems may be considered valid technical solutions to target low frequency alterations in diagnostic routine samples. Harmonized ring trials are key weapons to standardize analytical and post-analytical procedures optimizing clinical stratification of BC patients.