With the development of oncology in recent years, diagnosis and treatment of breast cancer have been continuously improved, and the mortality rate shows a downward trend. However, breast cancer is a heterogeneous disease with high incidence, easy to recur and metastasis, which leads to that accurate diagnosis and treatment is still a hot and difficult point of research . The intra-tumor heterogeneity of breast cancer is manifested in spatial and temporal, and individual tumors in one patient have different subpopulations of cancer cells in distant regions . Circulating tumor cells (CTCs) have the same characteristics of primary or metastatic lesions. Some studies [20, 22, 23] have proved that the tumor genome heterogeneity and micrometastase can be found by CTC whole genome sequencing, and it is feasible to conduct tumor drug guidance according to the characteristics of CTC genome. In our study, genomic HER2 characteristics of CTC in Patient ID1 and Patient ID2 were consistent with tissue test results, and targeted therapy was in line with expectations. However, HER2 status of CTC in Patient ID3 is not consistent with tissue, but targeted therapy is effective, which shows that our results are consistent with the reports.
Key driver mutations such as mutations of TP53 and PIK3CA, amplifications of MYC, CCND1, and HER2 usually take place in primary tumor cells [24, 25]. Some gene alternation presented by WGS analysis of CTC, like CNV gains of HER2 gene in patient ID1 and ID2 which were consistent with the result of IHC detection for tissue, and TP53 R248W in patient ID3 which was consistent with the result of ctDNA detection, are potentially come from the primary tumors. Additional driver mutations or amplifications during the tumor progression may lead to further clonal diversity in primary or metastasis tumors and treatment resistance. Numerous studies have shown that discordance rates of HER2 between primary and recurrent or metastatic tumors is 8–16% respectively [27–29]. In our study, CNV gains of HER2 gene was identified in the DNA of CTC but in primary and liver metastasis tumor, which implied that HER2 CNV gained in another metastatic tumor that is not discovered at that moment. What’s more, no HER2 alternation was presented in ctDNA, which implied that the potential metastasis was too tiny to be identified clinically. Although increased pleural effusion suggested pulmonary metastasis, no tumor cell was found in pleural effusion sample. Heterogeneity in tumors poses a severe challenge to the diagnosis and prognosis of diseases. The heterogeneity of breast cancer is the main cause of many treatment failures. For the patient ID3, pleural effusion was still in the state of disease progression after chemotherapy. However, this pleural abnormality was released, enumeration of CTC and the burden of ctDNA in patient ID3 were decreased after the original regimen combined with trastuzumab treatment. Trastuzumab is an effective target drug for HER2-positive breast cancer [30, 31]. Therefore, we speculate the existence of micrometastasis in the lung but not clinically detected. HER2-positive CTCs may be associated with lung metastasis, which ultimately leads to the ineffectiveness of previous chemotherapy.
In the investigation of patient ID3, no abnormal of HER2 was observed in the tumor tissue detected by IHC and FISH, membrane expression checking of CTC, or ctDNA sequencing. Only the WGS analysis of CTC revealed both the CNV gains and mutations of the HER2 gene. HER2 detection on tissues is commonly used as a criterion for targeting therapy in the clinic. However, due to the heterogeneity of tumors and the defects of detection techniques, the detection results are inaccurate. Likewise, the detection of Her-2 protein expression on CTC cell membranes also has problems considering the epithelial-mesenchymal transition [32, 33]. The ctDNA assay is limited by the design of the assay panel, and the tumor signal is diluted and eventually prone to false negative [34, 35]. Previous studies pointed that when traditional tissue biopsies are difficult to obtain, CTCs sequencing may provide an alternative method for comprehensive genome studies to analyze tumor heterogeneity and obtain optimal targets for therapeutic [15, 23], which was consistent with our results.