Disease characterization in liquid biopsy from HER2-mutated, non-amplified metastatic breast cancer patients treated with neratinib

Abstract

Metastatic breast cancer (mBC) patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving five years or more. In rare instances (2-4% of cases) patients with mBC have ERBB2 (HER2) activating mutations but are ERBB2 non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell assay (HDSCA) workflow to investigate the clinical significance of the liquid biopsy in ERBB2 mutant, non-amplified, post-menopausal mBC patients starting neratinib and fulvestrant combination therapy. Characterization with a comprehensive liquid biopsy methodology (HDSCA) included genomic analysis of both the cell-free DNA (cfDNA) and single circulating tumor cells (CTCs) to monitor tumor evolution and identify potential mutational variants unique to the patient's clinical response. A limited series of five sequentially enrolled patients presented here were from the MutHER ( https://www.clinicaltrials.gov , NCT01670877) or SUMMIT ( https://www.clinicaltrials.gov , NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. CTC enumeration alone was not sufficient to predict clinical response. Treatment pressure was shown to lead to an observable change in CTC morphology and genomic instability (GI), suggesting these parameters may inform prognosis. Single cell copy number alteration (CNA) analysis indicated that the persistence or development of a clonal population of CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally, the genomic analysis of the cfDNA, identified new mutations in ERBB2, PIK3CA, and TP53 that arose likely due to treatment pressure in a patient with poor response, further providing insights on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HDSCA methodology. Additional studies are necessary to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology.

Conflict of interest statement

The HDSCA technology described here is licensed to Epic Sciences. J.H. and P.K. have ownership in Epic Sciences. All other authors declare no conflicts of interest.

© 2022. The Author(s).

Figures

Fig. 1. Patient specific ERBB2 mutations identified in the tumor at the time of enrollment.

Patients 1 and 2 had pathological assessments conducted by Foundation One, while Patients 3–5 had pathological assessment conducted by CARIS. Purple: Excellent responder, Pink: Average responder, Yellow: Poor responder.

Fig. 2. Comprehensive analysis of the liquid biopsy for Patient 1.

a Similar clonal architecture identified in the CTCs was also detectable in the cfDNA, with a greater tumor fraction present in follow-up sampling. b Heatmap and phylogenic tree of single cell CNAs across the entire population of cells isolated. Draw number and clones are identified using color key. c Longitudinal analysis of CTCs and tumor antigen burden in Patient 1. Clinical features include CEA and CA27.29 measurements taken periodically over the course of treatment. SD stable disease, PD progressive disease. Representative images of CTCs taken at 400X. Scale bar = 10 µm. White: ER, Red: CK, Green: CD45, Blue: DAPI.

Fig. 3. Comprehensive analysis of the liquid biopsy for Patient 2.

a Similar clonal architecture identified in the CTCs was also detectable in the cfDNA, with a greater tumor fraction present in follow-up sampling. b Heatmap and phylogenic tree of single cell CNAs across the entire population of cells isolated. Draw number and clones are identified using color key. c Longitudinal analysis of CTCs and tumor antigen burden in Patient Clinical features include CEA measurements taken periodically over the course of treatment. SD stable disease, PD progressive disease. Representative images of CTCs taken at 400X. Scale bar = 10 µm. White: ER, Red: CK, Green: CD45, Blue: DAPI.

Fig. 4. Comprehensive analysis of the liquid biopsy for Patient 3.

a Similar clonal architecture identified in the CTCs was also detectable in the cfDNA, with a greater tumor fraction present in follow-up sampling. b Heatmap and phylogenic tree of single cell CNAs across the entire population of cells isolated. Draw number and clones are identified using color key. c Longitudinal analysis of CTCs and tumor antigen burden in Patient 3. Clinical features include CEA and CA27.29 measurements taken periodically over the course of treatment. PD progressive disease. Representative images of CTCs taken at 400X. Scale bar = 10 µm. White: ER, Red: CK, Green: CD45, Blue: DAPI.

Fig. 5. Comprehensive analysis of the liquid biopsy for Patient 4.

a Similar clonal architecture identified in the CTCs was also detectable in the cfDNA, with a greater tumor fraction present in follow-up sampling. b Heatmap and phylogenic tree of single cell CNAs across the entire population of cells isolated. Draw number and clones are identified using color key. c Longitudinal analysis of CTCs and tumor antigen burden in Patient 4. Clinical features include CEA and CA15-3 measurements taken periodically over the course of treatment. SD stable disease, PD progressive disease. Representative images of CTCs taken at 400X. Scale bar = 10 µm. White: ER, Red: CK, Green: CD45, Blue: DAPI.

Fig. 6. Comprehensive analysis of the liquid biopsy for Patient 5.

a Similar clonal architecture identified in the CTCs was also detectable in the cfDNA, with a greater tumor fraction present in follow-up sampling. b Heatmap and phylogenic tree of single cell CNAs across the entire population of cells isolated. Draw number and clones are identified using color key. c Longitudinal analysis of CTCs in Patient 5. SD stable disease, PD progressive disease. Representative images of CTCs taken at 400X. Scale bar = 10 µm. White: ER, Red: CK, Green: CD45, Blue: DAPI.

Fig. 7. Genomic analysis of single cells isolated from the liquid biopsy from mBC patients receiving neratinib treatment prior to progression.

Heatmap and phylogenic tree of CNVs across the entire population of cells from patient PB samples collected prior to progression. Patient number is identified using color key.