Background

Genomic profiling has revolutionized precision oncology, impacting diagnosis, prognosis, and therapy decisions. Considering the high spatiotemporal diversity and heterogeneity of breast tumor-cell genomes, small-gene panels often fail to capture rare but important genomic alterations. Conversely, comprehensive ctDNA sequencing approaches enable the identification of under-characterized 'long-tailed driver' genomic alterations and capture intra- and inter-metastatic heterogeneity. Here, we demonstrate the clinical utility of comprehensive genome profiling with higher sensitivity to predict the possibility of metastasis in early-stage breast cancer patients.

Methods

We retrospectively analyzed ctDNA and genomic DNA (gDNA) from FFPE samples, as well as circulating tumor cells (CTCs), in 10 treatment-naive, hormone-positive, and HER2-negative primary-stage breast cancer patients using the OncoIndx comprehensive 600-gene panel. The panel captures all important cancer-relevant genomic alterations, including tumor mutation burden (TMB), microsatellite instability (MSI), homologous recombination deficiency (HRD) prediction, and cfDNA tumor fraction (TF). CTCs were enumerated from 1.5 ml of blood using the OncoDiscover platform, approved by the Drug Controller General of India, using anti-EpCAM antibody-mediated immunomagnetic nanoparticles. CTCs were confirmed for cytokeratin 18+ and DAPI+ markers, and the absence of CD45.

Results

The comprehensive genomic profile obtained from ctDNA and gDNA from the FFPE of early-stage breast cancer patients predominantly exhibited the presence of alterations in PIK3CA and ESR1 signaling pathways. PIK3CA mutations were present in 77% and 44% of baseline ctDNA and gDNA samples, while ESR1 mutations were present in 44% and 22% of baseline ctDNA and gDNA, respectively. In addition, we observed about 70% additional driver mutations in ctDNA samples, suggesting the shedding of ctDNA together with CTCs (80% positive) as a likely positive biomarker of metastasis. About 50% of the patients showed higher TMB and HRD. Notably, TF representing ctDNA varied between 13% to 27% in blood samples with a corresponding ploidy range of 2.9 to 4.7. Surprisingly, ~50% of the patient population matched the mutation profile of clinically confirmed metastatic patients. All the patients harboring potential metastatic driver alterations showed the presence of CTCs in peripheral blood.

Conclusions

Comprehensive ctDNA genomic profiling showed potential metastasis-driving alterations, suggesting the role of ctDNA-based liquid biopsy to predict metastasis in early breast cancer patients. We observed enhanced TF at the time of diagnosis, possibly due to the presence of distant metastasis, high disease burden, and aggressive tumor biology. Our results suggest that ctDNA dynamics at the time of disease presentation can predict early metastasis and may demonstrate the divergent response of tumor heterogeneity to treatment in early-stage breast cancer.