Liquid biopsy profiling for the detection of therapy evasion and tumor evolution in breast cancer

In a large cohort of patients with breast cancer who received tissue and liquid biopsy-based genomic profiling, we portray the utility of liquid biopsies in better understanding the tumor evolution, monitoring progression and resistance, and identifying rare targets for treatment.

Liquid biopsy-based profiling in breast cancer

Personalized medicine is shaping the future of cancer treatment. In recent years, several advances have been made in the development of precision therapies for patients with breast cancer including PI3K inhibition, PARP inhibition, antibody drug conjugates and immune checkpoint inhibition1–6 (Figure 1). Next generation sequencing-based genomic profiling can aid in personalized medicine by identifying actionable gene alterations and complex biomarkers. To date, most genomic profiling is performed on tumor tissue biopsies. More recently, genomic profiling of the circulating tumor DNA has emerged with utility across a patient’s journey, from cancer diagnosis and treatment selection to monitoring response and relapse (Figure 1). The FDA has approved gene panel tests that profile cancer-related genes using deep targeted sequencing, from a simple blood draw, as a companion diagnostic. With FDA-approval and associated payor reimbursement, liquid biopsies have the potential for more widespread uptake.

            With the growing interest in using liquid biopsy profiling to inform a patient’s journey, we sought to investigate a large real-world cohort comprising >30,000 patients who underwent tissue biopsy (TBx) or liquid biopsy (LBx)-based genomic profiling as part of routine clinical care, including 712 patients who received testing on both platforms, to better understand the mutational landscape associated with tumor evolution and characterize the diversity of acquired mutations and their associations with therapeutic interventions.

Figure 1. Clinical utility of liquid biopsies during the course of care in patients with breast cancer

Liquid biopsies identify acquired resistance mechanisms and rare actionable targets

The mutational patterns in known breast cancer initiating events (e.g., PIK3CA, BRCA1/2) derived from liquid biopsies (LBx) were largely similar to that of tissue biopsies (TBx), in our study cohort. Notably, there was a higher prevalence of alterations typically associated with therapeutic resistance in LBx (e.g., ESR1, NF1, RB1). Consistent with LBx typically being used in a late-stage setting, we observed a higher concordance between LBx and metastatic TBx compared to breast-biopsied TBx. To further investigate acquired resistance mechanisms and tumor evolution, we examined 712 patients with LBx biopsy collected at the time of or as a follow-up to TBx. Using TBx as a baseline, positive percent agreement (PPA) was 72% for LBx, heavily influenced by estimated tumor fraction in the LBx (>90% PPA for driver gene alterations where the LBx tumor fraction was >10%) and predicted germline status (PPA: 100% for common germline vs. 70% for predicted somatic alterations).

            Acquired alterations in the follow-up LBx were detected in a majority of patients, with TP53, ESR1, NF1, PIK3CA, PTEN, RB1 and ERBB2 being the most common. Acquired alterations were associated with intervening therapies. Patients treated with endocrine therapy showed frequent acquisition of ESR1, NF1, and PIK3CA alterations, while those receiving CDK4/6 inhibitors exhibited RB1 alterations as well. Potential resistance alterations in ERBB2, FGFR2, KRAS, and PIK3CA were observed in patients receiving HER2 targeted therapies, while BRCA1/2 reversions were predominantly detected following PARP inhibitor therapy. Acquired gene amplifications and complex rearrangements were also detected in LBx. Additionally, there were several rare, yet actionable, acquired alterations detected in the follow-up LBx, including those in MAPK-Ras-Fgfr signaling pathway (e.g., EGFR, BRAF, HRAS, NRAS, KRAS, FGFR1/2/3) and PI3K pathway (e.g., AKT1, MTOR). Acquired alterations in FGFR1-3 were observed in 3% of cases, predominantly in ER+ disease, suggesting that patients with relapsed ER+ disease may benefit from targeted enrollment for the various FGFR inhibitors in development. Acquired KRAS alterations, including those in codon 12 (e.g., KRAS G12C) were also detected in follow-up LBx; with solid-tumor basket trials underway, this may serve as an emerging treatment option for these patients. A RET fusion was identified in a single patient, with possible response to RET inhibitors that may benefit the treatment course for this patient. These rare, yet actionable alterations that are observed in follow-up liquid biopsies, may offer improved treatment strategies, perhaps through combinatorial mechanisms, for patients with advanced breast cancer.

Tumor evolution is a rule rather than an exception during disease progression

Tumor heterogeneity plays a critical role in tumor evolution, from the initiation and development of pre-malignant lesions to invasive tumors, tumor progression and metastasis, as well as late-stage relapse. Despite treatment advances, tumors often develop resistance that ultimately leads to recurrence/relapse7,8. Subclonal populations may respond differently to therapy and independently acquire resistance mutations. Since liquid biopsies can capture circulating tumor DNA from different metastatic sites and subclones, this platform may provide a more comprehensive picture of the tumor, including mechanisms of treatment selection and/or relapse. Consistent with this hypothesis, acquired LBx alterations in our cohort were frequently found at lower allelic fractions (subclonal) and often detected multiple alterations within the same gene (polyclonal). Of note, acquired BRCA1/2 reversions potentially conferring resistance to PARPi or platinum therapy, were found in 21% of patients with baseline BRCA mutations and were almost universally subclonal and polyclonal in nature. Similar findings were seen for ESR1, PIK3CA, and ERBB2, with patients exhibiting multiple resistance-associated alterations in one or more of these genes from a single LBx. These polyclonal alterations allude to convergent evolution mechanisms in acquiring resistance. While therapies provide one source of evolutionary pressure, selection burden through mutations that promote oncogenicity, prevent cell death, or provide a clonal advantage for tumor expansion may also occur over time.

            Even in the absence of longitudinal data, alterations frequently observed to be subclonal and/or polyclonal in a large patient population have the potential to aid in the discovery of rare resistance mechanisms and the identification of new targets for drug discovery. Taken together, our findings showcase the diversity of the mutational landscape derived from liquid biopsy-based genomic profiling and provide valuable insights on the dynamics of tumor evolution that may better guide therapy selection for patients with advanced breast cancer.


  1. André, F. et al. Alpelisib for PIK3CA-mutated, hormone receptor–positive advanced breast cancer. N. Engl. J. Med. 380, 1929–1940 (2019).
  2. Robson, M. E. et al. OlympiAD final overall survival and tolerability results: Olaparib versus chemotherapy treatment of physician’s choice in patients with a germline BRCA mutation and HER2-negative metastatic breast cancer. Ann. Oncol. 30, 558–566 (2019).
  3. von Minckwitz, G. et al. Trastuzumab emtansine for residual invasive HER2-positive breast cancer. N. Engl. J. Med. 380, 617–628 (2019).
  4. Modi, S. et al. Trastuzumab deruxtecan in previously treated HER2-positive breast cancer. N. Engl. J. Med. 382, 610–621 (2020).
  5. Marabelle, A. et al. Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study. Lancet Oncol. 21, 1353–1365 (2020).
  6. Schmid, P. et al. Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer. N. Engl. J. Med. 379, 2108–2121 (2018).
  7. Pasha, N. & Turner, N. C. Understanding and overcoming tumor heterogeneity in metastatic breast cancer treatment. Nat Cancer 2, 680–692 (2021).
  8. Fisher, R., Pusztai, L. & Swanton, C. Cancer heterogeneity: implications for targeted therapeutics. Br. J. Cancer 108, 479–485 (2013).

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