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

Whole Genome Sequencing in single CTC improves clinical outcome in Her-2 negative breast cancer patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Whole Genome Sequencing in single CTC improves clinical outcome in Her-2 negative breast cancer patients Yongping Li, Hao Yuan, Bin Zhang, Xiaofei Jiang, Minghua Yu, Hongbo Zhu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-97189/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 8 You are reading this latest preprint version Abstract Backgroud: Tumor tissues are usually highly heterogeneous and difficult 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 identified 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 amplification 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 amplified. After adjusting treatment by adding Her-2 inhibitors according to the results of CTC sequencing, the liver metastases and pleural effusion were significantly 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 amplification 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. Epigenetics & Genomics Single Circulating Tumor Cell Breast Cancer Heterogeneity Whole Genome Sequencing (WGS) HER2 (ERBB2) Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The latest global and Chinese cancer statistics show that the incidence of breast cancer in women is still ranked the first, and the morbidity trend is increasing[ 1 – 4 ]. The median survival time of advanced breast cancer from diagnosis is 2–3 years. Only about 5–10% of patients can survive for more than 5 years[ 5 ]. According to the tumor status of the patients, various treatments are combined to formulate an effective comprehensive treatment program[ 6 ]. For the patients with strong expression (3+) of Her-2 in immunohistochemistry (IHC) report, or expression (2+) by IHC and confirmed to be positive by FISH, targeted therapy is an internationally accepted method. Currently available HER2 -targeting drugs have vastly improved outcomes in patients with HER2 /Her-2 positive status [ 7 ]. Accurate and sensitive detection of this targeted factor plays a critical role in screening patients for targeted treatment. However, the IHC/FISH technology, which is mainly used to assess the Her-2 status on tissue, cannot reveal the real gene status in tumors of the patient due to the potentially inconsistent HER2 gene status between primary and metastatic tumors. Circulating tumor cells (CTCs), a liquid biopsy specimen, are highly active tumor cells that detach from solid tumors and enter the peripheral blood. It has been confirmed that CTCs derived from highly invasive tumors and are associated with tumor metastasis[ 8 ]. Lots of previous studies have shown that CTCs are significantly 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 stratification and targeted therapy selection[ 12 ]. It is difficult 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 profiling 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 significantly 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 significant clinical results. Results CTC enumeration of breast cancer patients A total of 24 CTCs with chromosome aneuploidy were enriched and identified 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. Table 1 CTC number assessed for each patient based on chromosome aneuploidy Patient ID Number of singles for CEP8 in CTCs CTC with Her2 expression Total Triploid Tetraploid Pentaploid and above 1 7 1 2 0 10 2 6 1 0 0 7 3 bT 6 0 1 0 7 3 aT 0 0 0 0 0 bT : before treatment; aT : after treatment Somatic mutation and CNV in CTCs We identified 2,934, 497, and 2,901 mutations in the CTCs of the three patients, respectively. About 20% of these mutations were recorded in the COSMIC database (Table 2 ). Besides, TP53 R248W was identified in patient ID3, which was identified by the 57-panel in the ctDNA of patient ID3 as well. HER2 Amplification was observed in all the 3 patients (Table 2 ). Besides, CNV gains of other 2 drug-related genes (FGFR1 and FGFR2) were revealed in patient ID3. Table 2 DNA status in CTCs and ctDNA of patients with breast cancer Sample DNA of CTCs ctDNA in patient ID3 ID 1 ID2 ID3 before TM* after TM* Mutation Total recorded 2934 497 2901 Cosmic recorded 583 95 538 Breast cancer-related 30 8 39 Chromosome 17 174 35 201 TP53 mutation 0 0 R248W R248W - CNV ERBB2 3 4 8 - - FGFR1 2 2 5 3 - FGFR2 2 2 7 - - *TM: target medicine Correlation between HER2 status and patients’ treatment Patient ID1 with Her-2 protein expression in the tumor by IHC (Fig. 2 a) showed HER2 gene amplification according to the WGS analysis of CTC (Table 2 ). The first diagnosis of nonspecific invasive breast cancer was made in Oct 2017. After a mastectomy, she received chemotherapy combined with molecular targeted therapy TCH (Paclitaxel-Carboplatin-Trastuzumab) every 21 days for 6 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 ). Patient ID2 with Her-2 protein expression in the tumor by IHC (Fig. 2 a) showed HER2 gene amplification in the WGS analysis of CTC (Table 2 ). Nonspecific 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 defined as Her-2 negative (Fig. 2 a). Subsequently, Her-2 negative was demonstrated in both tumors by FISH test (Fig. 2 b). However, amplification in HER2 gene according to the WGS analysis of CTC was observed in this patient (Table 2 ). Diagnosis of breast infiltrating 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 confirmed by both IHC and FISH tests. After a treatment of paclitaxel combined with capecitabine, the liver metastases were reduced, and some tumor cells were identified 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. A 18-month progression-free survival (PFS) was recorded for Patient ID3 until the manuscript was finished (Figs. 3 and 4 ). Changes of CTC and ctDNA in patient ID3 before and after the treatment CTCs and mutations of ctDNA (57 gene panel) were detected at first diagnosis and after the trastuzumab related treatment in patient ID3 (Fig. 4 ). At the first diagnosis, 7 CTCs/7.5 ml blood were identified, a variation of TP53 R248W and amplification 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 (Table 2 , Figs. 3 and 4 ). 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 difficult 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 first 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 , amplifications 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 amplifications 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 – 26 ]. In our study, CNV gains of HER2 gene was identified 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 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 [ 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 difficult 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 first time we have discovered an advanced breast cancer patient with a HER2 gene amplification 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. Methods 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). Briefly, this method combines subtraction enrichment, immunofluorescence staining and chromosome in situ hybridization to achieve the separation and identification of CTC [ 33 ]. Firstly, plasma was removed by centrifugation, and red blood cells were removed by density gradient centrifugation. White blood cells were specifically removed by antibody-coated magnetic beads, and remaining cells including CTCs were coated on a slide for subsequent cell identification. Secondly, cells on the slide were stained with multi-tumor immunofluorescence markers CD45 and Her-2. The centromere of chromosome 8 (CEP8) was detected by fluorescence in situ hybridization. Finally, CTCs were identified 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 amplification 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 amplification (WGA). Single-cell WGA was performed using the single-cell whole genome amplification kit (Yikon Genomics, China) which is based on the MALBAC method [ 34 ]. The amplified 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 amplified 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 Table 2. Abbreviations CTCs: Circulating tumor cells; WGS:whole genome sequencing; IHC:immunohistochemistry; CEP8:centromere of chromosome 8; NGS:next generation sequencing; qPCR:quantitative PCR; DFS:Disease free survival; PFS:progression-free survival. Declarations Ethical approval and consent to participate: All procedures performed in studies involving human participants were in accordance with the ethical standards of Shanghai Pudong Hospital Ethics Committee (NO. W2001) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from individual participants. Consent for publication: Not applicable. Availability of data and material: Not applicable. Competing interest: The authors declare that they have no conflicts of interest. Funding: This work was funded by Key Specialty Construction Project of Pudong Health and Family Planning Commission of Shanghai: PWZzk2017-32, Talents Training Program of Pudong Hospital affiliated to Fudan University: RHJJ 2018-01, Subject of Advanced talents in Shanghai area YJRCJJ201701, and National Natural Science Foundation of China (81672593，81272899) . Authors' contributions: Yongping Li, Hao Yuan, Bin Zhang were equal contribute to this article. All authors were involved in drafting and finalizing the report. Acknowledgements: We thank the patients, physicians, nurses, and data managers who participated in the trial. References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424. 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Clinical information and expression of tumor markers of the breast cancer patients. Additional file 2: Table S2. Gene panel of ctDNA sequencing analysis. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major revision 06 Jan, 2021 Review # 1 received at journal 01 Dec, 2020 Reviewer # 1 agreed at journal 05 Nov, 2020 Reviewers invited by journal 01 Nov, 2020 Editor assigned by journal 27 Oct, 2020 Submission checks completed at journal 23 Oct, 2020 Editor invited by journal 16 Oct, 2020 First submitted to journal 11 Oct, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-97189","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":3895442,"identity":"534f363f-4e46-4168-8bfa-8852be5c2fde","order_by":0,"name":"Yongping Li","email":"","orcid":"","institution":"Shanghai Pudong Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongping","middleName":"","lastName":"Li","suffix":""},{"id":3895443,"identity":"5ed4b1bb-977c-488c-8bbe-2ac66ae75be0","order_by":1,"name":"Hao Yuan","email":"","orcid":"","institution":"Shanghai Pudong Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Yuan","suffix":""},{"id":3895444,"identity":"c50c2e81-d6c3-4056-b68b-ddd062a32768","order_by":2,"name":"Bin Zhang","email":"","orcid":"","institution":"Yulin City First Hospital Yulin Branch","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zhang","suffix":""},{"id":3895445,"identity":"af38bd1d-8dd2-4aef-8942-e8b13e96c937","order_by":3,"name":"Xiaofei Jiang","email":"","orcid":"","institution":"Shanghai Pudong Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaofei","middleName":"","lastName":"Jiang","suffix":""},{"id":3895446,"identity":"87fb0676-da41-427d-9d5d-14b3be9a4ed8","order_by":4,"name":"Minghua Yu","email":"","orcid":"","institution":"Shanghai Pudong Hospital","correspondingAuthor":false,"prefix":"","firstName":"Minghua","middleName":"","lastName":"Yu","suffix":""},{"id":3895447,"identity":"99c8e96c-19c1-463b-8bc5-47b75cd2de5f","order_by":5,"name":"Hongbo Zhu","email":"","orcid":"","institution":"Shanghai Pudong Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Zhu","suffix":""},{"id":3895448,"identity":"260c81d0-08c1-4217-88de-98760b3f18f7","order_by":6,"name":"Qinghua You","email":"","orcid":"","institution":"Shanghai Pudong Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"You","suffix":""},{"id":3895449,"identity":"0ef1424e-9cff-4bd7-ae95-f56251a0bf30","order_by":7,"name":"Ling Wang","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Wang","suffix":""},{"id":3895450,"identity":"f5566ab3-14ca-4b2b-8167-87c772325dcd","order_by":8,"name":"Bo Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYPCDCgk5eaIV84DJMxbGhg0kaWFsq0hkOEBApfyM3McvPu45zGAvkZ34uHKeRAJjA/PDRzfwaDG4kW5mOePZYQYeidzNhme3SeSxM7AZG+fg0yKRxmbMcwCsZZtk4zaJYsYGHjZpfFrkZwC1/IFrmSOR2HCAgBaGG2nMjxngWhqI0GJw5hkbY8+BdAaeM283GzYckzA2bCbgF/n2NOYPPw5YM7C352582FBTJyfP3vzwMV6HMTCwSTAwNNc3wPnM+JWDlXxgYKgjrGwUjIJRMApGLgAAd5FIk+5oHNwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3888-9901","institution":"Shanghai Pudong Hospital","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2020-10-23 12:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-97189/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-97189/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3291040,"identity":"9473d1bb-3e62-40a4-bb7c-b707be698d69","added_by":"auto","created_at":"2020-10-30 13:30:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":702272,"visible":true,"origin":"","legend":"CTCs in patients with breast cancer. White arrows showed the identified CTCs. Scale bar is 10 μm.","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-97189/v1/5d27e61e02244ed8ec72d508.png"},{"id":3291041,"identity":"f6a4aaa9-91b0-4178-8b6d-c224cfc42f11","added_by":"auto","created_at":"2020-10-30 13:30:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1652020,"visible":true,"origin":"","legend":"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. ","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-97189/v1/48ec837eb94bf35d5fa347c4.png"},{"id":3291042,"identity":"955c8015-ad81-4e6b-baf7-c5b3769ae55e","added_by":"auto","created_at":"2020-10-30 13:30:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29380,"visible":true,"origin":"","legend":"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.","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-97189/v1/d01fdadb48641be4fc97d78b.png"},{"id":3291043,"identity":"6023c425-10ae-4be9-81ba-9b26eeb52522","added_by":"auto","created_at":"2020-10-30 13:30:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":629398,"visible":true,"origin":"","legend":"Variations of imaging and molecular biomarker features in Patient ID3 during a series of medical treatments. Treatment process is showed in the top. Images of the lung (upper) and liver (lower) metastasis are listed in the middle. Variations of CTC (grey column) and ctDNA (black line for CNV of FGFR1; grey line for mutation of TP53 R248W) are presented in the bottom. ","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-97189/v1/0c93a0819a35421a588cccd9.png"},{"id":13607847,"identity":"73ac358b-bcdf-4a07-8d34-1e84e0320860","added_by":"auto","created_at":"2021-09-17 06:13:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3039025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-97189/v1/1b70bc7d-02f6-400b-bfaa-618503eebdff.pdf"}],"financialInterests":"","formattedTitle":"Whole Genome Sequencing in single CTC improves clinical outcome in Her-2 negative breast cancer patients","fulltext":[{"header":"Background","content":" \u003cp\u003eThe latest global and Chinese cancer statistics show that the incidence of breast cancer in women is still ranked the first, and the morbidity trend is increasing[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The median survival time of advanced breast cancer from diagnosis is 2\u0026ndash;3\u0026nbsp;years. Only about 5\u0026ndash;10% of patients can survive for more than 5 years[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to the tumor status of the patients, various treatments are combined to formulate an effective comprehensive treatment program[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For the patients with strong expression (3+) of Her-2 in immunohistochemistry (IHC) report, or expression (2+) by IHC and confirmed to be positive by FISH, targeted therapy is an internationally accepted method. Currently available \u003cem\u003eHER2\u003c/em\u003e-targeting drugs have vastly improved outcomes in patients with \u003cem\u003eHER2\u003c/em\u003e/Her-2 positive status [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Accurate and sensitive detection of this targeted factor plays a critical role in screening patients for targeted treatment. However, the IHC/FISH technology, which is mainly used to assess the Her-2 status on tissue, cannot reveal the real gene status in tumors of the patient due to the potentially inconsistent \u003cem\u003eHER2\u003c/em\u003e gene status between primary and metastatic tumors.\u003c/p\u003e \u003cp\u003eCirculating tumor cells (CTCs), a liquid biopsy specimen, are highly active tumor cells that detach from solid tumors and enter the peripheral blood. It has been confirmed that CTCs derived from highly invasive tumors and are associated with tumor metastasis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Lots of previous studies have shown that CTCs are significantly correlated with the prognosis and survival of multiple solid tumors, such as metastatic breast cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], prostate cancer[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and colorectal cancer[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Emerging studies also showed that CTC can be a potential biomarker for patient stratification and targeted therapy selection[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is difficult 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[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Evidence has pointed out that molecular profiling of CTCs provides a better representation of tumor diversity than a single biopsy[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMultiple gene variations in single CTC can be investigated through whole genome sequencing (WGS) based on the next generation sequencing (NGS) technology [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. With the development of CTC isolation and NGS technology, our understanding of intra-tumor heterogeneity has significantly improved, and the individualized treatment and precise treatment of tumors are further advanced.\u003c/p\u003e \u003cp\u003eIn 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 significant clinical results.\u003c/p\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCTC enumeration of breast cancer patients\u003c/h2\u003e \u003cp\u003eA total of 24 CTCs with chromosome aneuploidy were enriched and identified in all three patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of CTCs for the 3 patients was 10, 7 and 7 cells/7.5\u0026nbsp;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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCTC number assessed for each patient based on chromosome aneuploidy\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePatient ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eNumber of singles for CEP8 in CTCs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCTC with Her2 expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTriploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTetraploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePentaploid and above\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e \u003csup\u003e\u003cb\u003ebT\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003csup\u003e\u003cb\u003eaT\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ebT\u003c/sup\u003e: before treatment; \u003csup\u003eaT\u003c/sup\u003e: after treatment\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSomatic mutation and CNV in CTCs\u003c/h2\u003e \u003cp\u003eWe identified 2,934, 497, and 2,901 mutations in the CTCs of the three patients, respectively. About 20% of these mutations were recorded in the COSMIC database (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Besides, TP53 R248W was identified in patient ID3, which was identified by the 57-panel in the ctDNA of patient ID3 as well.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHER2\u003c/em\u003e Amplification was observed in all the 3 patients (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Besides, CNV gains of other 2 drug-related genes (FGFR1 and FGFR2) were revealed in patient ID3.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDNA status in CTCs and ctDNA of patients with breast cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eDNA of CTCs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ectDNA in patient ID3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eID 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eID2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eID3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ebefore TM*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eafter TM*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMutation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal recorded\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2934\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2901\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCosmic recorded\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBreast cancer-related\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChromosome 17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTP53 mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR248W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR248W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCNV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERBB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGFR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGFR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e*TM: target medicine\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between HER2 status and patients\u0026rsquo; treatment\u003c/h2\u003e \u003cp\u003ePatient ID1 with Her-2 protein expression in the tumor by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) showed \u003cem\u003eHER2\u003c/em\u003e gene amplification according to the WGS analysis of CTC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The first diagnosis of nonspecific invasive breast cancer was made in Oct 2017. After a mastectomy, she received chemotherapy combined with molecular targeted therapy TCH (Paclitaxel-Carboplatin-Trastuzumab) every 21\u0026nbsp;days for 6 cycles. Trastuzumab continued for a year. Endocrine therapy is ongoing. Disease free survival (DFS) reached 18\u0026nbsp;months after followed up to May 2019. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePatient ID2 with Her-2 protein expression in the tumor by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) showed \u003cem\u003eHER2\u003c/em\u003e gene amplification in the WGS analysis of CTC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nonspecific invasive breast cancer was diagnosed in Nov 2017. After a mastectomy, she was treated with anthracycline plus cyclophosphamide (AC) every 21\u0026nbsp;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\u0026nbsp;months after followed up to May 2019. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVery little expression (1+) of Her-2 protein was found in primary and liver metastasis tumor of Patient ID3 by IHC test, which was defined as Her-2 negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Subsequently, Her-2 negative was demonstrated in both tumors by FISH test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). However, amplification in \u003cem\u003eHER2\u003c/em\u003e gene according to the WGS analysis of CTC was observed in this patient (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Diagnosis of breast infiltrating 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 confirmed by both IHC and FISH tests. After a treatment of paclitaxel combined with capecitabine, the liver metastases were reduced, and some tumor cells were identified 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. A 18-month progression-free survival (PFS) was recorded for Patient ID3 until the manuscript was finished (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChanges of CTC and ctDNA in patient ID3 before and after the treatment\u003c/h2\u003e \u003cp\u003eCTCs and mutations of ctDNA (57 gene panel) were detected at first diagnosis and after the trastuzumab related treatment in patient ID3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At the first diagnosis, 7 CTCs/7.5\u0026nbsp;ml blood were identified, a variation of TP53 R248W and amplification 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 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eBreast 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 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Circulating tumor cells (CTCs) have the same characteristics of primary or metastatic lesions. Some studies [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] 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 \u003cem\u003eHER2\u003c/em\u003e 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. \u003cem\u003eHER2\u003c/em\u003e 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 first treatment, which demonstrated that genomic charaterizations of CTC can conduct the drug guidance of tumors.\u003c/p\u003e \u003cp\u003eKey driver mutations such as mutations of \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003ePIK3CA\u003c/em\u003e, amplifications of \u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eCCND1\u003c/em\u003e, and \u003cem\u003eHER2\u003c/em\u003e usually take place in primary tumor cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Some gene alternation presented by WGS analysis of CTC, like CNV gains of \u003cem\u003eHER2\u003c/em\u003e 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 amplifications during the tumor progression may lead to further clonal diversity in primary or metastasis tumors and treatment resistance [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Numerous studies have shown that discordance rates of \u003cem\u003eHER2\u003c/em\u003e between primary and recurrent or metastatic tumors is 8\u0026ndash;16% respectively [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our study, CNV gains of \u003cem\u003eHER2\u003c/em\u003e gene was identified in the DNA of CTC but Her-2 protein expression was not observed in primary and liver metastasis tumor, which implied that \u003cem\u003eHER2\u003c/em\u003e CNV gained in another metastatic tumor that is not discovered at that moment. What\u0026rsquo; s more, no \u003cem\u003eHER2\u003c/em\u003e 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 \u003cem\u003eHER2\u003c/em\u003e-positive breast cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, we speculate the existence of micrometastasis in the lung but not clinically detected. \u003cem\u003eHER2\u003c/em\u003e-positive CTCs may be associated with lung metastasis, which ultimately leads to the ineffectiveness of previous chemotherapy.\u003c/p\u003e \u003cp\u003eIn the investigation of patient ID3, no abnormal of \u003cem\u003eHER2\u003c/em\u003e 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 \u003cem\u003eHER2\u003c/em\u003e gene. \u003cem\u003eHER2\u003c/em\u003e 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The ctDNA assay is limited by the design of the assay panel, and the tumor signal is diluted and eventually prone to false negative [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which was consistent with our results.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eThis is the first time we have discovered an advanced breast cancer patient with a \u003cem\u003eHER2\u003c/em\u003e gene amplification 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.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePatients and sample collection\u003c/h2\u003e \u003cp\u003ePatients 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\u0026nbsp;ml peripheral blood was drawn from patients into ACD anti-coagulant tube. For ctDNA analysis, another 10\u0026nbsp;ml peripheral blood was collected into an EDTA anti-coagulant tube. Clinical characterizations of these patients were shown in supplementary table1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInvestigation of CTCs\u003c/h2\u003e \u003cp\u003eCirculating tumor cell (CTC) detection was carried out by CTC detection kit (Majorbio, China). Briefly, this method combines subtraction enrichment, immunofluorescence staining and chromosome in situ hybridization to achieve the separation and identification of CTC [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Firstly, plasma was removed by centrifugation, and red blood cells were removed by density gradient centrifugation. White blood cells were specifically removed by antibody-coated magnetic beads, and remaining cells including CTCs were coated on a slide for subsequent cell identification. Secondly, cells on the slide were stained with multi-tumor immunofluorescence markers CD45 and Her-2. The centromere of chromosome 8 (CEP8) was detected by fluorescence in situ hybridization. Finally, CTCs were identified according to the criteria of CD45-, DAPI+, and CEP8\u0026thinsp;\u0026ge;\u0026thinsp;3. The expression of Her-2 on the surface of CTC was also recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome amplification and sequencing of single CTC\u003c/h2\u003e \u003cp\u003eFor 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 amplification (WGA). Single-cell WGA was performed using the single-cell whole genome amplification kit (Yikon Genomics, China) which is based on the MALBAC method [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The amplified DNA product was assessed by a Qubit\u0026reg; 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 amplified DNA product by quantitative PCR (qPCR) with eight randomly selected loci was performed. DNA samples with a total mass more than 2\u0026nbsp;\u0026micro;g, and DNA fragment range 300\u0026nbsp;bp to 2000\u0026nbsp;bp were considered to meet the further sequencing criteria.\u003c/p\u003e \u003cp\u003eLibrary 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ectDNA analysis of the metastatic patient\u003c/h2\u003e \u003cp\u003eIn 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 Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCTCs: Circulating tumor cells; WGS:whole genome sequencing; IHC:immunohistochemistry; CEP8:centromere of chromosome 8; NGS:next generation sequencing; qPCR:quantitative PCR; DFS:Disease free survival; PFS:progression-free survival.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of Shanghai Pudong Hospital Ethics Committee (NO. W2001) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from individual participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Key Specialty Construction Project of Pudong Health and Family Planning Commission of Shanghai: PWZzk2017-32, Talents Training Program of Pudong Hospital affiliated to Fudan University: RHJJ 2018-01, Subject of Advanced talents in Shanghai area YJRCJJ201701, and National Natural Science Foundation of China (81672593，81272899) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYongping Li, Hao Yuan, Bin Zhang were equal contribute to this article. All authors were involved in drafting and finalizing the report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patients, physicians, nurses, and data managers who participated in the trial.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394\u0026ndash;424.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W, Zheng R, Zhang S, Zeng H, Xia C, Zuo T, Yang Z, Zou X, He J. Cancer incidence and mortality in China, 2013. 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Cell. 2012;149(5):994\u0026ndash;1007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmendro V, Kim HJ, Cheng YK, Gonen M, Itzkovitz S, Argani P, van Oudenaarden A, Sukumar S, Michor F, Polyak K. Genetic and phenotypic diversity in breast tumor metastases. Cancer Res. 2014;74(5):1338\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmir E, Miller N, Geddie W, Freedman O, Kassam F, Simmons C, Oldfield M, Dranitsaris G, Tomlinson G, Laupacis A, et al. Prospective study evaluating the impact of tissue confirmation of metastatic disease in patients with breast cancer. J Clin Oncol. 2012;30(6):587\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiikura N, Liu J, Hayashi N, Mittendorf EA, Gong Y, Palla SL, Tokuda Y, Gonzalez-Angulo AM, Hortobagyi GN, Ueno NT. Loss of human epidermal growth factor receptor 2 (HER2) expression in metastatic sites of HER2-overexpressing primary breast tumors. J Clin Oncol. 2012;30(6):593\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindstr\u0026ouml;m LS, Karlsson E, Wilking UM, Johansson U, Hartman J, Lidbrink EK, Hatschek T, Skoog L, Bergh J. Clinically Used Breast Cancer Markers Such As Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 Are Unstable Throughout Tumor Progression. J Clin Oncol. 2012;30(21):2601\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinger CF, Balma\u0026ntilde;a J, B\u0026uuml;rki N, Delaloge S, Filieri ME, Gerdes A-M, Grindedal EM, Han S, Johansson O, Kaufman B, et al. Genetic counselling and testing of susceptibility genes for therapeutic decision-making in breast cancer\u0026mdash;an European consensus statement and expert recommendations. Eur J Cancer. 2019;106:54\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHowie LJ, Scher NS, Amiri-Kordestani L, Zhang L, King-Kallimanis BL, Choudhry Y, Schroeder J, Goldberg KB, Kluetz PG, Ibrahim A, et al: FDA Approval Summary: Pertuzumab for adjuvant treatment of HER2-positive early breast cancer. Clinical Cancer Research 2018:clincanres.3003.2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan X, Ma F, Li C, Wu S, Hu S, Huang J, Sun X, Wang J, Luo Y, Cai R, et al. The prognostic and therapeutic implications of circulating tumor cell phenotype detection based on epithelial-mesenchymal transition markers in the first-line chemotherapy of HER2-negative metastatic breast cancer. Cancer Commun (Lond). 2019;39(1):1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapadaki MA, Stoupis G, Theodoropoulos PA, Mavroudis D, Georgoulias V, Agelaki S. Circulating tumor cells with stemness and epithelial-to-mesenchymal transition features are chemoresistant and predictive of poor outcome in metastatic breast cancer. Mol Cancer Ther 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClark TA, Chung JH, Kennedy M, Hughes JD, Chennagiri N, Lieber DS, Fendler B, Young L, Zhao M, Coyne M, et al. Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA. J Mol Diagn. 2018;20(5):686\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanman RB, Mortimer SA, Zill OA, Sebisanovic D, Lopez R, Blau S, Collisson EA, Divers SG, Hoon DS, Kopetz ES, et al. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA. PLoS One. 2015;10(10):e0140712.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin PP. Integrated EpCAM-independent subtraction enrichment and iFISH strategies to detect and classify disseminated and circulating tumors cells. Clin Transl Med. 2015;4(1):38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. 2012;338(6114):1622\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Information","content":"\u003cp\u003eTables S1 and S2 were not provided with this version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1.\u003c/strong\u003e Clinical information and expression of tumor markers of the breast cancer patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Table S2.\u003c/strong\u003e Gene panel of ctDNA sequencing analysis.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Single Circulating Tumor Cell, Breast Cancer, Heterogeneity, Whole Genome Sequencing (WGS), HER2 (ERBB2)","lastPublishedDoi":"10.21203/rs.3.rs-97189/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-97189/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackgroud: \u003c/strong\u003eTumor tissues are usually highly heterogeneous and difficult 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 identified 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 \u003cem\u003eHER2\u003c/em\u003e gene status in WGS results of CTC, we adjusted treatment strategies, and monitored disease progression. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003ePatient ID1 and ID2 are with Her-2 protein overexpression in primary tumors and \u003cem\u003eHER2\u003c/em\u003e gene amplification 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 \u003cem\u003eHER2\u003c/em\u003e gene was amplified. After adjusting treatment by adding Her-2 inhibitors according to the results of CTC sequencing, the liver metastases and pleural effusion were significantly 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. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e The results of CTC sequencing effectively guided treatment of a patient with \u003cem\u003eHER2\u003c/em\u003e gene amplification 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.\u003c/p\u003e","manuscriptTitle":"Whole Genome Sequencing in single CTC improves clinical outcome in Her-2 negative breast cancer patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-10-30 13:30:14","doi":"10.21203/rs.3.rs-97189/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-01-07T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-02T00:00:00+00:00","index":1,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nLi et al describe a study using whole genome sequencing (WGS) on circulating tumor cells (CTCs) of 3 breast cancer patients, one of which was found to be HER2 negative by immunohistochemistry and in-situ hybridization of the primary and metastatic tumor, but which WGS analysis of CTC showed HER2 gene amplification. This information facilitated commencement of anti-HER2 therapy, resulting in good response and tumor remission. The study thus illustrates the usefulness of CTC sequencing of one case pertaining to targeted HER2 therapy, worthy of consideration for publication. There are however there are several important points that need clarification.\n\n1. Table 1 documents 7 CTCs for patient ID3, 6 triploid and 1 pentaploid. This number is also mentioned in the main text, line 20. However, in both figure 3 and figure 4, it is written 10 CTCs detected. Kindly clarify and correct accordingly.\n2. Table 1 footnote. bT before treatment and aT after treatment. Kindly specify the treatment. Is it HER2 targeted therapy? Likewise, for patients ID1 and ID2, were the CTCs obtained before commencement of therapy? Why were CTCs not obtained after therapy for these 2 patients? This would have served the purpose of showing anti-HER2 therapy was effective in these 2 patients.\n3. Likewise, for Table 2 *TM: target medicine - should be specified - HER2 targeted therapy.\n4. Figure 2 shows patient ID1 was HER2 positive, score 3+ whilst patient ID2 was score 2+. This should not be described as positive. Moreover, the image of ID2 shows poor tissue preservation and largely cytoplasmic rather than strong circumferential membranous staining which is a requirement for positivity. As internationally recommended guidelines (J Clin Oncol. 2018 Jul 10;36(20):2105-2122.) HER2 IHC score 2+ is considered equivocal requiring ISH testing by FISH or DISH to demonstrate HER2 amplification before being considered eligible of HER2 targeted therapy. Hence also for Figure 3, patient ID2 should not be described as Her-2 positive. Why was FISH not done for patient ID2?\n5. Also, for Figure 2 patient ID3, the breast cancer would have been aggressive as seen from the clinical history as well as from the Ki-67 score which was \u003e50%, hence the histology image of the primary tumor shown in Fig 2a does not appear representative of the tumor. On the other hand, ID3 - liver tumor seems more representative, and strong membranous staining is in fact seen in some cells, suggesting score 2+, rather than score 1+.\n6. Supplementary Table 1. For Patient ID1, c-erbB2 is stated as 2+, where elsewhere is it described as 3+. Patient ID2 on the other hand is stated as 3+. Please clarify and correct accordingly. Pathological diagnosis is given \"Nonspecific invasive breast cancer\" and \"Breast infiltrating ductal cancer\". Both terms mentioned refer to Invasive breast cancer of no special type, which under the new WHO Classification 2018 is simply called Invasive Breast Cancer.\n7. Page 6, line 38-39. \"CTCs and mutations of ctDNA (57 gene panel) were detected at first diagnosis and after the trastuzumab related treatment in patient ID3\". No CTCs could be detected after trastuzumab treatment. This sentence should be corrected.\n8. Page 7, line 38-39. \"CNV gains of HER2 gene was identified in the DNA of CTC but Her-2 protein expression was not observed in the primary and liver metastasis tumor…….\" This could be better expressed as \"………but neither Her-2 protein expression nor HER2 gene amplification were observed in the primary and liver metastasis tumor…\".\n9. The discussion section could benefit greatly from better expression and English.\n10. The Material and Methods section lacked scientific details of ctDNA analysis, such as extraction of cfDNA, ddPCR assay used, its analysis and how ctDNA levels were calculated. Relevant information of HER2 immunohistochemistry and in-situ hybridization - kits, primary antibody/probes etc was also missing.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. 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