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.
Highlights
Breast cancer (BC) is the most common cancer in women worldwide, accounting for 24.5% of all newly diagnosed cancer cases[1]
The importance of genomic sequencing is apparent with the identification of rare genetic subtypes of cancer, such as the patients presented here with ERBB2 mutant, non-amplified Metastatic BC (mBC)
We observed an average response to neratinib treatment for Patients 2, 4, and 5, all of whom harbored mutations in the tyrosine kinase domain, confirming sensitivity previously reported in other studies[13,45,46,47,48]
Summary
Breast cancer (BC) is the most common cancer in women worldwide, accounting for 24.5% of all newly diagnosed cancer cases[1]. At draw 2, Patient 3 presented with a new mutation in ERBB2 (L755S) located in the tyrosine kinase intracellular domain, as well as new mutations in PIK3CA (E726K) and TP53 (G245D) At baseline, this patient presented with 37.08 CTCs/ml with a subsequent increase to 52.49 CTCs/ml after the start of therapy with confirmed disease progression (Fig. 4c). Hierarchical clustering of CNA profiles of CTCs isolated from the last draw collected prior to progression indicates that CTCs from Patients 1, 2, and 5 are more similar based on single cell genomics, than Patient 3. By aligning the CNA profiles to the COSMIC catalogue of somatic mutations in cancer we identified 30 genes that are altered in CTCs prior to progression (Supplemental Table 2) Further exploration of these alterations may provide insight into treatment sensitivity or mutation driven resistance mechanisms. All four of the cases where a CNA profile was detected showed at least five of these early luminal characteristic CNAs, along with other less frequent events, indicating a luminal, as opposed to basal-like, origin
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