Whole Genome Sequencing in single CTC improves clinical outcome in Her-2 negative breast cancer patients

Backgroud: Tumor tissues are usually highly heterogeneous and dicult to characterize which could mislead treatment strategy. Circulating tumor cells (CTCs) represent the most active and invasive tumor cells. This study retrospectively investigated the feasibility of individualized treatment of breast cancer patients based on genome sequencing of single cell CTC. Twenty-four CTCs were identied in three patients with breast cancer. For each patient, one polyploid CTC was captured and on which the whole genome sequencing (WGS) was performed. Based on the histopathological Her-2 status in tumor tissue and the HER2 gene status in WGS results of CTC, we adjusted treatment strategies, and monitored disease progression. Results: Patient ID1 and ID2 are with Her-2 protein overexpression in primary tumors and HER2 gene amplication in the DNA of CTCs. In patient ID3, histological examination of primary tumor and liver metastases revealed Her-2 negative, but the WGS analysis of CTC showed that the HER2 gene was amplied. After adjusting treatment by adding Her-2 inhibitors according to the results of CTC sequencing, the liver metastases and pleural effusion were signicantly reduced 2 month later, CTC number and ctDNA burden were decreased, and 18-month progression-free survival (PFS) was recorded. In addition, some potential therapeutic targets and mutations in drug-resistant genes were found. Conclusions: The results of CTC sequencing effectively guided treatment of a patient with HER2 gene amplication in CTC but with Her-2 negative on tumor tissue. CTC sequencing is useful in resolving the heterogeneity of tumors and providing precision medicine for patients.

signi cantly correlated with the prognosis and survival of multiple solid tumors, such as metastatic breast cancer [9], prostate cancer [10], and colorectal cancer [11]. Emerging studies also showed that CTC can be a potential biomarker for patient strati cation and targeted therapy selection [12]. It is di cult to obtain multiple tumor tissue samples in advanced and metastasis cancer. In this situation, CTCs could be an ideal source of cancer cells for genomic analysis. Tumor tissue sample analysis is usually unable to detect all the genetic variants present in the heterogeneous tumors such as breast cancer [13]. Evidence has pointed out that molecular pro ling of CTCs provides a better representation of tumor diversity than a single biopsy [14].
Multiple gene variations in single CTC can be investigated through whole genome sequencing (WGS) based on the next generation sequencing (NGS) technology [15,16]. With the development of CTC isolation and NGS technology, our understanding of intra-tumor heterogeneity has signi cantly improved, and the individualized treatment and precise treatment of tumors are further advanced.
In this study, CTC single cell sequencing was performed and the WGS data of CTCs was compared with tumor pathologic results of 3 patients with breast cancer. We evaluated the tumor heterogeneity, optimized the treatment decision of these patients, and achieved signi cant clinical results.

CTC enumeration of breast cancer patients
A total of 24 CTCs with chromosome aneuploidy were enriched and identi ed in all three patients (Table 1, Fig. 1). The number of CTCs for the 3 patients was 10, 7 and 7 cells/7.5 ml blood respectively.
All these cells were negative for Her-2 expression in the membrane surface. One single CTC for every patient was retrieved by micro dissection for the WGS analysis. It was pentaploid cell that collected from patient ID1 and patient ID3, and tetraploid cell from patient ID2. Paired white blood cells were used as control. of these mutations were recorded in the COSMIC database (Table 2). Besides, TP53 R248W was identi ed in patient ID3, which was identi ed by the 57-panel in the ctDNA of patient ID3 as well.
Patient ID2 with Her-2 protein expression in the tumor by IHC (Fig. 2a) showed HER2 gene ampli cation in the WGS analysis of CTC (Table 2). Nonspeci c invasive breast cancer was diagnosed in Nov 2017. After a mastectomy, she was treated with anthracycline plus cyclophosphamide (AC) every 21 days for 4 cycles followed docetaxel combined with trastuzumab (TH) for 4 cycles. Trastuzumab continued for a year. Endocrine therapy is ongoing. Disease free survival (DFS) reached 18 months after followed up to May 2019. (Fig. 3).
Very little expression (1+) of Her-2 protein was found in primary and liver metastasis tumor of Patient ID3 by IHC test, which was de ned as Her-2 negative (Fig. 2a). Subsequently, Her-2 negative was demonstrated in both tumors by FISH test (Fig. 2b). However, ampli cation in HER2 gene according to the WGS analysis of CTC was observed in this patient ( Table 2). Diagnosis of breast in ltrating ductal cancer was made in Nov 2015. Neoadjuvant chemotherapy with docetaxel, epirubicin and cyclophosphamide (TEC) was performed before the mastectomy and continued adjuvant chemotherapy and radiotherapy after the surgery. After that treatment, the patient was treated with endocrine therapy. In Oct 2017, pleural effusion, peritoneal effusion and liver multiple occupancies were observed in the patient. No cancer cell was found in the pleural effusion, but invasive adenocarcinoma was found in the biopsy of liver tissue, suggesting the liver metastasis. Her-2 negative in the metastasis tissue was con rmed by both IHC and FISH tests. After a treatment of paclitaxel combined with capecitabine, the liver metastases were reduced, and some tumor cells were identi ed in the pleural effusion. In Mar 2018, pleural effusion increased a lot and trastuzumab was added into the chemotherapy. Two month later, the pleural effusion disappeared, and tumor partial remission was much more obviously observed compared to the effect of chemotherapy only. Endocrine therapy combined trastuzumab is ongoing. Changes of CTC and ctDNA in patient ID3 before and after the treatment CTCs and mutations of ctDNA (57 gene panel) were detected at rst diagnosis and after the trastuzumab related treatment in patient ID3 (Fig. 4). At the rst diagnosis, 7 CTCs/7.5 ml blood were identi ed, a variation of TP53 R248W and ampli cation of FGFR1 were observed in the plasma. After the trastuzumab related treatment, neither CTC in blood nor gene alternation in plasma was found in this patient (

Discussion
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 di cult point of research [17]. 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 [18]. Circulating tumor cells (CTCs) have the same characteristics of primary or metastatic lesions. Some studies [17,19,20] have proved that the tumor genome heterogeneity and micrometastase can be found by whole genome sequencing of CTC, 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 results by IHC tests, and targeted therapy was in line with expectations. HER2 gene status of CTC in Patient ID3 is not consistent with tissue results by either IHC or FISH tests. Both the little expression (1+) of Her-2 protein expression and the negative result of FISH were not support the Her-2 targeted therapy.
However, Her-2 targeted therapy is effective for this patients after the failure of the rst treatment, which demonstrated that genomic charaterizations of CTC can conduct the drug guidance of tumors.
Key driver mutations such as mutations of TP53 and PIK3CA, ampli cations of MYC, CCND1, and HER2 usually take place in primary tumor cells [21,22]. Some gene alternation presented by WGS analysis of CTC, like CNV gains of HER2 gene in patient ID1 and ID2 consistent with the result of IHC detection for tissue, are potentially come from the primary tumors. Additional driver mutations or ampli cations during the tumor progression may lead to further clonal diversity in primary or metastasis tumors and treatment resistance [23]. Numerous studies have shown that discordance rates of HER2 between primary and recurrent or metastatic tumors is 8-16% respectively [24][25][26]. In our study, CNV gains of HER2 gene was identi ed in the DNA of CTC but Her-2 protein expression was not observed 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 identi ed 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 [27,28]. 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 tissues detected by IHC and FISH, membrane expression checking of CTC, or ctDNA sequencing. Only the WGS analysis of CTC revealed CNV gains 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 [29,30]. The ctDNA assay is limited by the design of the assay panel, and the tumor signal is diluted and eventually prone to false negative [31,32]. Previous studies pointed that when traditional tissue biopsies are di cult to obtain, CTCs sequencing may provide an alternative method for comprehensive genome studies to analyze tumor heterogeneity and obtain optimal targets for therapeutic [14,20], which was consistent with our results.

Conclusions
This is the rst time we have discovered an advanced breast cancer patient with a HER2 gene ampli cation in the single CTC by WGS but not histologically expressed in the tumor tissue. CTC sequencing makes us further understand the heterogeneity of breast cancer, permits the non-invasive and repeated accurate monitoring of therapeutic response and tumor progression, helps us to make therapeutic decisions and predict the outcome, which ultimately achieves personalized molecularly guided cancer treatment. Since this is a retrospective study with a small number of cases, large sample prospective control study is necessary in the future. The single cell sequencing in this study was performed two years ago, for which the quality control of CTC, single cell separation and whole genome sequencing need improving. In the past two years, the development of single cell technology has made rapid progress, which can analyze a large number of samples, increase the detection accuracy, reduce the economic cost, and improve the clinical application.

Patients and sample collection
Patients enrolled in this study were diagnosed at Shanghai Pudong Hospital. Our study was approved by the Shanghai Pudong Hospital Ethics Committee (No. W2001). Written informed consent were obtained to allow the sample collection and data analysis for research purposes. For CTC analysis, 7.5 ml peripheral blood was drawn from patients into ACD anti-coagulant tube. For ctDNA analysis, another 10 ml peripheral blood was collected into an EDTA anti-coagulant tube. Clinical characterizations of these patients were shown in supplementary table1.

Investigation of CTCs
Circulating tumor cell (CTC) detection was carried out by CTC detection kit (Majorbio, China). Brie y, this method combines subtraction enrichment, immuno uorescence staining and chromosome in situ hybridization to achieve the separation and identi cation of CTC [33]. Firstly, plasma was removed by centrifugation, and red blood cells were removed by density gradient centrifugation. White blood cells were speci cally removed by antibody-coated magnetic beads, and remaining cells including CTCs were coated on a slide for subsequent cell identi cation. Secondly, cells on the slide were stained with multitumor immuno uorescence markers CD45 and Her-2. The centromere of chromosome 8 (CEP8) was detected by uorescence in situ hybridization. Finally, CTCs were identi ed according to the criteria of CD45-, DAPI+, and CEP8 ≥ 3. The expression of Her-2 on the surface of CTC was also recorded.
Whole genome ampli cation and sequencing of single CTC For each patient, a single CTC was collected into a tube by microdissection performed on a PALM MicroBeam instrument (Zeiss) for subsequent CTC single cell whole genome ampli cation (WGA). Singlecell WGA was performed using the single-cell whole genome ampli cation kit (Yikon Genomics, China) which is based on the MALBAC method [34]. The ampli ed DNA product was assessed by a Qubit® dsDNA HS Assay kit in a Qubit 3.0 Fluorometer (Life Technologies, USA), evaluated the molecular weight on 1% agarose gel electrophoresis, and checked the genomic integrity of the ampli ed DNA product by quantitative PCR (qPCR) with eight randomly selected loci was performed. DNA samples with a total mass more than 2 µg, and DNA fragment range 300 bp to 2000 bp were considered to meet the further sequencing criteria.
Library preparation was performed according to the SureSelectXT Illumina Paired-End Sequencing Library protocol. Library quality was assessed by 2100 Bioanalyzer and qPCR with TBS380 picogreen (Invitrogen,USA). Next Seq CN500 High-throughput Sequencer (Illumina) was used for whole-genome sequencing of the captured single cells.
ctDNA analysis of the metastatic patient In this study, plasma DNA of patient ID3 with metastasis breast cancer was extracted for ctDNA analysis. A total of 67 cancer-related genes including 57 drugs related genes, 2 endocrine therapy related genes, and 9 chemotherapy related genes. Point mutations, small fragment insertions and deletions, copy number variants, and fusions of these genes were detected. Details of the gene panel are shown in Supplementary Availability of data and material: Not applicable.
Competing interest: The authors declare that they have no con icts of interest. Authors' contributions: Yongping Li, Hao Yuan, Bin Zhang were equal contribute to this article. All authors were involved in drafting and nalizing the report.
Additional le 1: Table S1. Clinical information and expression of tumor markers of the breast cancer patients.
Additional le 2: Table S2. Gene panel of ctDNA sequencing analysis. Figure 1 CTCs in patients with breast cancer. White arrows showed the identi ed CTCs. Scale bar is 10 μm.

Figure 2
Her-2/HER2 status in the tissue of the 3 patients. a. The Her-2 protein expression in the primary tissue of patient ID1, ID2, and ID3, and in the liver metastasis tissue of patient ID3 were detected by IHC test. b. The copy number of HER2 gene in the primary and liver metastasis tissue of patient ID3 were detected by FISH test.
Page 13/14 Figure 3 Clinical timelines of the 3 patients with breast cancer. The HER2 targeted drug trastuzumab were highlighted in bold type. DFS, disease-free survival; PFS, progression-free survival.