Liquid Biopsy in Breast Cancer: Molecular Characterization and Support to Clinical Management

Di Cosimo, Serena (2021). Liquid Biopsy in Breast Cancer: Molecular Characterization and Support to Clinical Management. PhD thesis The Open University.



The classical paradigm of breast cancer (BC) management has been challenged by recent advances in the understanding of molecular biology, which is increasingly encouraging attempts to move away from protocol-based in favor of a personalized approach. This project aimed to test circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) to characterize the disease, and monitor patients with BC including both non-invasive and invasive cases.

For this purpose, 41 patients with newly diagnosed ductal carcinoma in situ (DCIS), and 61 triple negative BC (TNBC) patients treated with neoadjuvant chemotherapy were analyzed. Primary tumor mutations identified by targeted- gene sequencing were validated, and tracked in 182 plasma samples longitudinally collected at multiple time-points by droplet-digital Polymerase Chain Reaction. In recurrent TNBC patients, plasma DNA underwent direct targeted-gene assay, and CTCs were analyzed for Copy Number Alterations (CNAs).

In DCIS patients, ctDNA from baseline samples was evaluable in 71% of cases, with Variant Allele Frequency (VAF) values ranging between 0.001% and 5%, independently of clinico-pathological features, and likewise at initial diagnosis and at relapse. In TNBC patients, the number of primary tumor mutations showed a downward trend after treatment; proficient genes involved in the immune pathways were associated to response; and surgical samples of non responsive cases presented alterations in druggable pathways. ctDNA after treatment was associated with increased risk of relapse, and its prognostic value remained worthy even after adjusting for age, residual disease, systemic inflammatory indices, and Ki-67 (HR 1.91; 95%CI 0.51-7.08). During follow-up, ctDNA was undetectable in non-recurrent cases. Conversely, ctDNA was detected in 83% of recurrent cases, and antedated the clinical diagnosis up to 13 months. Notably, recurrent cases without ctDNA developed loco-regional, contralateral, and bone-only disease. At clinical progression, CTCs presented chromosome 10 and 21q CNAs whose network analysis showed connected modules including HER/PI3K/Ras/JAK signaling, and immune response.

These findings extend the value of liquid biopsy to further clinical scenarios by reporting - unlike previous studies - the presence of ctDNA in noninvasive BC; and substantiate the development of ctDNA in invasive BC as an exploitable tool, either alone or with CTCs, for personalized TNBC management.

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