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Circulating Biomarkers in Breast Cancer

  • Katelyn N Seale
    Affiliations
    University of Maryland, School of Medicine, Marlene and Stewart Greenebaum Comprehensive Cancer Center, 22 South Greene Street, S9D12, Baltimore, MD 21201
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  • Katherine H R Tkaczuk
    Correspondence
    Address for correspondence: Katherine H R Tkaczuk, MD, FACP, University of Maryland, 22 South Greene Street, S9D12, Baltimore, MD 21201
    Affiliations
    University of Maryland, School of Medicine, Marlene and Stewart Greenebaum Comprehensive Cancer Center, 22 South Greene Street, S9D12, Baltimore, MD 21201
    Search for articles by this author
Open AccessPublished:September 22, 2021DOI:https://doi.org/10.1016/j.clbc.2021.09.006

      Abstract

      Breast cancer management has progressed immensely over the decades, but the disease is still a major source of morbidity and mortality worldwide. Even with enhanced imaging detection and tissue biopsy capabilities, disease can progress on an ineffective treatment before additional information is obtained through standard methods of response evaluation, including the RECIST 1.1 criteria, widely used for assessment of treatment response and benefit from therapy.
      • Eisenhauer EA
      • Therasse P
      • Bogaerts J
      • et al.
      New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
      Circulating biomarkers have the potential to provide valuable insight into disease progression and response to therapy, and they can serve to identify actionable mutations and tumor characteristics that can direct therapy. These biomarkers can be collected at higher frequencies than imaging or tissue sampling, potentially allowing for more informed management. This review will evaluate the roles of circulating biomarkers in breast cancer, including the serum markers Carcinoembryonic antigen CA15-3, CA27-29, HER2 ECD, and investigatory markers such as GP88; and the components of the liquid biopsy, including circulating tumor cells, cell free DNA/DNA methylation, circulating tumor DNA, and circulating microRNA.

      Introduction

      While increasingly treatable when discovered in early stages, breast cancer is still a major cause of death worldwide Breast cancer was the most commonly diagnosed female cancer in the world in 2020 with an incidence of over 2.26 million, accounting for 11.7% of total cancer cases worldwide; it was responsible for over 680,000 deaths.

      Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clinn/a(n/a). doi:10.3322/caac.21660

      The American Cancer Society's 2019 update estimated that over 250,000 new cases of invasive breast cancer were diagnosed in US women, and over 41,000 women died from the disease.
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      • et al.
      Breast cancer statistics, 2019.
      Furthermore, since 2004, the overall invasive breast cancer incidence increased by 0.3% per year, which is likely contributable to increases in body mass index and declines in fertility rate.
      • Pfeiffer RM
      • Webb-Vargas Y
      • Wheeler W
      • Gail MH.
      Proportion of U.S. trends in breast cancer incidence attributable to long-term changes in risk factor distributions.
      Breast imaging is crucial in the screening, diagnosis, and preoperative work-up of breast cancer. Due to high cost and low likelihood of positive findings, screening for recurrence outside of routine mammography is not recommended for patients with early stage breast cancer.

      National Comprehensive Cancer Network. Breast Cancer (Version 4.2021). National Comprehensive Cancer Network.

      ,
      • Khatcheressian JL
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      Breast cancer follow-up and management after primary treatment: american society of clinical oncology clinical practice guideline update.
      However, for patients with metastatic breast cancer (MBC), recurrent diagnostic imaging as often as every 6 weeks is standard. The Response Evaluation Criteria in Solid Tumors (RECIST 1.1), in which lesions are characterized with imaging, has been widely accepted as a standardized measure of tumor response to treatment, particularly in clinical trials.
      • Eisenhauer EA
      • Therasse P
      • Bogaerts J
      • et al.
      New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
      However, imaging is expensive and cumbersome, and the use of iodine-based or gadolinium contrast can cause toxicity. Moreover, disease may progress on an ineffective treatment for a significant time before additional imaging is obtained.
      Biomarkers can provide additional insight into a patient's prognosis and response to treatment. Numerous biomarkers are currently used in breast cancer management, notably tissue expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Serum biomarkers are especially appealing for the potential to detect disease progression or response to treatment before imaging can. However, serum biomarkers are not as established in breast cancer management. Here we will review the circulating markers CEA, CA15-3, and CA27-29, along with HER2 ECD and the experimental marker GP88. We will also discuss the biomarkers encompassed by the term “liquid biopsy”, including circulating tumor cells (CTCs), circulating cell-free DNA (ccfDNA), circulating tumor DNA (ctDNA), and circulating miRNA.

      Carcinoembryonic Antigen (CEA), CA15-3, CA27-29 in Early Breast Cancer

      CEA is a glycoprotein involved in cell adhesion and can be elevated in numerous cancers.
      • Grunnet M
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      Carcinoembryonic antigen (CEA) as tumor marker in lung cancer.
      CA15-3 and CA27-29 are carbohydrate-containing protein antigens of the transmembrane glycoprotein MUC-1, which appears to inhibit tumor cell lysis and reduce cell-cell interactions.
      • David JM
      • Hamilton DH
      • Palena C.
      MUC1 upregulation promotes immune resistance in tumor cells undergoing brachyury-mediated epithelial-mesenchymal transition.
      In primary breast cancer, several studies have found that elevated CEA levels at diagnosis portend a negative prognosis.
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      Prognostic value of CEA and ferritin assay in breast cancer: A multivariate analysis.
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      c-erbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value.
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      Serum levels of CEA and CA15-3 in different molecular subtypes and prognostic value in Chinese breast cancer.
      • Uehara M
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      Long-term prognostic study of carcinoembryonic antigen (CEA) and carbohydrate antigen 15-3 (CA 15-3) in breast cancer.
      • Ebeling FG
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      Serum CEA and CA 15-3 as prognostic factors in primary breast cancer.
      Similarly, various studies have demonstrated that elevated serum CA15-3 values at diagnosis are associated with higher breast cancer stage, tumor size, positive axillary lymph nodes, and worse OS and DFS.
      • Molina R
      • Jo J
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      c-erbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value.
      ,
      • Uehara M
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      • Hojo T
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      • Iwamoto E
      • Fukutomi T.
      Long-term prognostic study of carcinoembryonic antigen (CEA) and carbohydrate antigen 15-3 (CA 15-3) in breast cancer.
      ,
      • Shering SG
      • Sherry F
      • McDermott EW
      • O'Higgins NJ
      • Duffy MJ
      Preoperative CA 15-3 concentrations predict outcome of patients with breast carcinoma.
      • Shao Y
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      • Liu C
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      Elevated levels of serum tumor markers CEA and CA15-3 are prognostic parameters for different molecular subtypes of breast cancer.
      • Fu Y
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      Assessing Clinical Significance of serum CA15-3 and carcinoembryonic antigen (CEA) levels in breast cancer patients: a meta-analysis.
      • Di Gioia D
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      • Mayr D
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      Serum HER2 in combination with CA 15-3 as a parameter for prognosis in patients with early breast cancer.
      Of the two MUC-1 biomarkers, most studies focused on CA15-3 than on CA27-29, although comparative studies have established similar correlations with stage and tumor size.
      • Gion M
      • Mione R
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      Comparison of the diagnostic accuracy of CA27.29 and CA15.3 in primary breast cancer.
      ,
      • Gion M
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      • Leon AE
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      CA27.29: a valuable marker for breast cancer management. A confirmatory multicentric study on 603 cases.
      These biomarkers clearly have prognostic significance in early breast cancer; but they demonstrate poor sensitivity (<7%
      • Hou MF
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      • Tseng TF
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      Evaluation of serum CA27.29, CA15-3 and CEA in patients with breast cancer.
      ) and lack prospective randomized control trial evidence establishing their use in early disease.

      CEA, CA15-3, CA27-29 in Metastatic Disease

      In MBC, all three biomarkers have increased sensitivity,
      • Hou MF
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      • Tseng TF
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      Evaluation of serum CA27.29, CA15-3 and CEA in patients with breast cancer.
      • Pathak KA
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      Carcinoembryonic antigen: an invaluable marker for advanced breast cancer.
      • Guadagni F
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      A re-evaluation of carcinoembryonic antigen (CEA) as a serum marker for breast cancer: a prospective longitudinal study.
      • Safi F
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      Comparison of CA 15-3 and CEA in diagnosis and monitoring of breast cancer.
      • Kallioniemi OP
      • Oksa H
      • Aaran RK
      • Hietanen T
      • Lehtinen M
      • Koivula T.
      Serum CA 15-3 assay in the diagnosis and follow-up of breast cancer.
      • Beveridge RA.
      Review of clinical studies of CA 27.29 in breast cancer management.
      with Hou et al demonstrating sensitivities of 62.8%, 82.8%, and 85.7% for CEA, CA15-3, and CA27-29, respectively.
      • Hou MF
      • Chen YL
      • Tseng TF
      • et al.
      Evaluation of serum CA27.29, CA15-3 and CEA in patients with breast cancer.
      The serum markers were measured serially in a number of studies assessing their applications in early detection of disease progression and monitoring therapy response.
      • Pathak KA
      • Khanna RR
      • Khanna HD
      • Khanna SS
      • Gupta SS
      • Khanna NN.
      Carcinoembryonic antigen: an invaluable marker for advanced breast cancer.
      ,
      • Guadagni F
      • Ferroni P
      • Carlini S
      • et al.
      A re-evaluation of carcinoembryonic antigen (CEA) as a serum marker for breast cancer: a prospective longitudinal study.
      ,
      • Lokich JJ
      • Zamcheck N
      • Lowenstein M.
      Sequential carcinoembryonic antigen levels in the therapy of metastatic breast cancer.
      • Wojtacki J
      • Kruszewski WJ
      • Sliwińska M
      • et al.
      [Elevation of serum Ca 15-3 antigen: an early indicator of distant metastasis from breast cancer. Retrospective analysis of 733 cases].

      Tampellini M, A B, R B, et al. Prognostic significance of changes in CA 15-3 serum levels during chemotherapy in metastatic breast cancer patients. Breast Cancer Res Treat doi:10.1007/s10549-005-9155-y

      • Chourin S
      • Veyret C
      • Chevrier A
      • Loeb A
      • Gray C
      • Basuyau J
      [Routine use of serial plasmatic CA 15-3 determinations during the follow-up of patients treated for breast cancer. Evaluation as factor of early diagnosis of recurrence].
      • Yang Y
      • Zhang H
      • Zhang M
      • Meng Q
      • Cai L
      • Zhang Q
      Elevation of serum CEA and CA153 levels during antitumor therapy predicts poor therapeutic response in advanced breast cancer patients.
      • Kim HS
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      • Park MJ
      • et al.
      Clinical significance of a serum CA15-3 surge and the usefulness of CA15-3 kinetics in monitoring chemotherapy response in patients with metastatic breast cancer.
      • Jensen JL
      • Maclean GD
      • Suresh MR
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      Possible utility of serum determinations of CA 125 and CA 27.29 in breast cancer management.
      Of note, Wojtacki et al demonstrated that an increase of >30 u/mL in CA15-3 levels preceded the diagnosis of relapse with a median lead time of 9 months in MBC patients.
      • Wojtacki J
      • Kruszewski WJ
      • Sliwińska M
      • et al.
      [Elevation of serum Ca 15-3 antigen: an early indicator of distant metastasis from breast cancer. Retrospective analysis of 733 cases].
      Further analyzing CA15-3’s kinetics, Tampellini et al conducted a prospective trial of 526 MBC patients during initiation of anthracycline-based chemotherapy. Median time to progression decreased from patients with normal levels of CA15-3 (15.3 months), patients with initially elevated levels and a 25% decrease (11.7 months), patients with elevated levels throughout (9.6 months), to patients with increasing levels (8.6 months).

      Tampellini M, A B, R B, et al. Prognostic significance of changes in CA 15-3 serum levels during chemotherapy in metastatic breast cancer patients. Breast Cancer Res Treat doi:10.1007/s10549-005-9155-y

      CEA, CA15-3, and CA27-29 have prognostic and predictive significance in MBC and have shown value in serial monitoring for response to therapy.
      Two randomized controlled trials will compare disease monitoring with CEA, CA15-3/CA27-29 markers to traditional image-based monitoring. A multicenter European trial will follow asymptomatic early breast cancer patients during and after adjuvant therapy and randomize them to one of two groups. In the experimental arm, serum CEA and CA15-3 will be collected every 3 months, with no imaging studies performed unless there is a critical increase of the biomarkers. This arm will be compared to the control arm that monitors patients with imaging studies and serum markers performed according to local practice (NCT02261389). The SWOG S1703 trial also plans to randomize patients with HR-positive HER2-negative metastatic breast cancer to two arms; arm I (control) will monitor patients with imaging studies at a minimum frequency of every 12 weeks alone or in conjunction with serum tumor markers CEA, CA15-3, and/or CA27-29, and arm II will monitor patients with serum tumor marker (STM) evaluation every 4-8 weeks without imaging until an elevation of at least one STM. In the event of STM elevation, the patient will have imaging within 4 weeks for evaluation of disease. The primary outcome assesses whether patients monitored with the serum biomarkers have non-inferior overall survival compared to those monitored with usual methods (NCT03723928). By establishing non-inferiority with the current standard image-based monitoring, the results of these trials can potentially increase the clinical significance of CEA, CA15-3, and CA27-29.

      Serum HER2 Extracellular Domain (ECD)

      The human epidermal growth factor receptor 2 (HER2) gene express a 185 kDA glycoprotein receptor that has intrinsic tyrosine kinase activity.
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      • Sudo C
      • Ogawara H
      • Toyoshima K
      • Yamamoto T
      The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity.
      HER2 has been shown to be amplified in 20% to 30% of breast tumors,
      • Slamon DJ
      • Clark GM
      • Wong SG
      • Levin WJ
      • Ullrich A
      • McGuire WL
      Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
      ,
      • King CR
      • Kraus MH
      • Aaronson SA
      Amplification of a novel v-erbB-related gene in a human mammary carcinoma.
      and gene amplification is associated with decreased DFS and OS.
      • Seshadri R
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      • Setlur V
      • Kitchen P
      Clinical significance of HER-2/neu oncogene amplification in primary breast cancer. The South Australian Breast Cancer Study Group.
      ,
      • Tandon AK
      • Clark GM
      • Chamness GC
      • Ullrich A
      • McGuire WL
      HER-2/neu oncogene protein and prognosis in breast cancer.
      Tissue HER2 has been established as a prognostic and predictive biomarker for response to anti-HER2 therapies.
      • Joensuu H
      • Kellokumpu-Lehtinen P-L
      • Bono P
      • et al.
      Adjuvant docetaxel or vinorelbine with or without trastuzumab for breast cancer.
      • Romond EH
      • Perez EA
      • Bryant J
      • et al.
      Trastuzumab plus Adjuvant chemotherapy for operable HER2-positive breast cancer.
      • Piccart-Gebhart MJ
      • Procter M
      • Leyland-Jones B
      • et al.
      Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.
      • Cameron D
      • Piccart-Gebhart MJ
      • Gelber RD
      • et al.
      11 years’ follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive early breast cancer: final analysis of the HERceptin Adjuvant (HERA) trial.
      NCCN and ASCO guidelines recommend assessing immunohistochemistry expression levels for HER2 at diagnosis of breast cancer to guide therapy

      National Comprehensive Cancer Network. Breast Cancer (Version 4.2021). National Comprehensive Cancer Network.

      ,
      • Hammond MEH
      • Hayes DF
      • Dowsett M
      • et al.
      American society of clinical oncology/college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version).
      • Wolff AC
      • Hammond MEH
      • Hicks DG
      • et al.
      Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.
      • Harris L
      • Fritsche H
      • Mennel R
      • et al.
      American society of clinical oncology 2007 update of recommendations for the use of tumor markers in breast cancer.
      and in newly diagnosed metastases that are easily biopsied.

      National Comprehensive Cancer Network. Breast Cancer (Version 4.2021). National Comprehensive Cancer Network.

      ,
      • Van Poznak C
      • Somerfield MR
      • Bast RC
      • et al.
      Use of biomarkers to guide decisions on systemic therapy for women with metastatic breast cancer: american society of clinical oncology clinical practice guideline.
      HER2/neu protein has three domains, including a 105 kDa ECD, which is shed from tumor cells and can be quantified in the serum.
      • Zabrecky JR
      • Lam T
      • McKenzie SJ
      • Carney W.
      The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.
      ,
      • Lin YZ
      • Clinton GM.
      A soluble protein related to the HER-2 proto-oncogene product is released from human breast carcinoma cells.
      Several studies have established a correlation between serum HER2 and tissue HER2 expression
      • Molina R
      • Jo J
      • Filella X
      • et al.
      c-erbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value.
      ,
      • Ludovini V
      • Gori S
      • Colozza M
      • et al.
      Evaluation of serum HER2 extracellular domain in early breast cancer patients: correlation with clinicopathological parameters and survival.
      • Müller V
      • Witzel I
      • Lück HJ
      • et al.
      Prognostic and predictive impact of the HER-2/neu extracellular domain (ECD) in the serum of patients treated with chemotherapy for metastatic breast cancer.
      • Garoufali A
      • Kyriakou F
      • Kountourakis P
      • et al.
      Extracellular domain of HER2: a useful marker for the initial workup and follow-up of HER2-positive breast cancer.
      • Narita T
      • Funahashi H
      • Satoh Y
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      C-erbB-2 protein in the sera of breast cancer patients.
      , but others failed to show such a relationship.
      • Willsher PC
      • Beaver J
      • Pinder S
      • et al.
      Prognostic significance of serum c-erbB-2 protein in breast cancer patients.
      • Quaranta M
      • Daniele A
      • Coviello M
      • et al.
      c-erbB-2 protein level in tissue and sera of breast cancer patients: a possibly useful clinical correlation.

      Pd S, Eh J, St L, et al. Serum HER-2 concentrations for monitoring women with breast cancer in a routine oncology setting. clinical chemistry and laboratory medicine. doi:10.1515/CCLM.2009.241

      • Harris LN
      • Liotcheva V
      • Broadwater G
      • et al.
      Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy.
      Considering these results, serum HER2 ECD is not a substitute for tissue HER2 status determination.

      HER2 ECD as a Prognostic and Predictive Marker to HER2-Targeted Therapies

      Serum HER2 ECD has demonstrated prognostic significance, with elevated HER2 ECD at diagnosis being associated with tumor burden, metastatic disease, shorter OS.
      • Ludovini V
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      Evaluation of serum HER2 extracellular domain in early breast cancer patients: correlation with clinicopathological parameters and survival.
      ,
      • Müller V
      • Witzel I
      • Lück HJ
      • et al.
      Prognostic and predictive impact of the HER-2/neu extracellular domain (ECD) in the serum of patients treated with chemotherapy for metastatic breast cancer.
      ,
      • Willsher PC
      • Beaver J
      • Pinder S
      • et al.
      Prognostic significance of serum c-erbB-2 protein in breast cancer patients.
      ,
      • Harris LN
      • Liotcheva V
      • Broadwater G
      • et al.
      Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy.
      • Hayes DF
      • Yamauchi H
      • Broadwater G
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      Circulating HER-2/erbB-2/c-neu (HER-2) extracellular domain as a prognostic factor in patients with metastatic breast cancer: Cancer and Leukemia Group B Study 8662.
      • Reix N
      • Malina C
      • Chenard M-P
      • et al.
      A prospective study to assess the clinical utility of serum HER2 extracellular domain in breast cancer with HER2 overexpression.
      • Moreno-Aspitia A
      • Hillman DW
      • Dyar SH
      • et al.
      Soluble human epidermal growth factor receptor 2 (HER2) levels in patients with HER2-positive breast cancer receiving chemotherapy with or without trastuzumab: Results from North Central Cancer Treatment Group adjuvant trial N9831.
      • Carney WP
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      • Leitzel K
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      Potential clinical utility of serum HER-2/neu oncoprotein concentrations in patients with breast cancer.
      • Gasol Cudós A
      • Morales Murillo S
      • Veas Rodriguez J
      • Canosa Morales C
      • Olivé JM
      • Salud A
      Clinical utility of SERUM extracelullar domain of HER2 receptor (ECD) in HER2-positive breast cancer.
      In the neoadjuvant setting, Witzel et al measured serum HER2 before initiation and after treatment with trastuzumab and demonstrated a significant positive association between high baseline serum HER2 and pathologic complete response at time of surgery. They also found a similar correlation between a decrease in serum HER2 ECD levels and pathologic complete response.
      • Witzel I
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      • von Minckwitz G
      • et al.
      Monitoring serum HER2 levels during neoadjuvant trastuzumab treatment within the GeparQuattro trial.
      In metastatic patients, a reduction of serum HER2 ECD of at least 20% after receiving trastuzumab had a higher response rate, longer time to progression, and longer DFS and OS.
      • Reix N
      • Malina C
      • Chenard M-P
      • et al.
      A prospective study to assess the clinical utility of serum HER2 extracellular domain in breast cancer with HER2 overexpression.
      ,
      • Ali SM
      • Esteva FJ
      • Fornier M
      • et al.
      Serum HER-2/neu change predicts clinical outcome to trastuzumab-based therapy.
      Similar results were seen with lapatinib monotherapy.
      • Lipton A
      • Leitzel K
      • Ali SM
      • et al.
      Human epidermal growth factor receptor 2 (HER2) extracellular domain levels are associated with progression-free survival in patients with HER2-positive metastatic breast cancer receiving lapatinib monotherapy.
      Additionally, when measuring serial levels, rising serum HER2 ECD may predict disease progression in metastatic disease.
      • Molina R
      • Jo J
      • Filella X
      • et al.
      c-erbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value.
      ,
      • Reix N
      • Malina C
      • Chenard M-P
      • et al.
      A prospective study to assess the clinical utility of serum HER2 extracellular domain in breast cancer with HER2 overexpression.
      ,

      Pd S, Eh J, St L, et al. Serum HER-2 concentrations for monitoring women with breast cancer in a routine oncology setting. clinical chemistry and laboratory medicine. doi:10.1515/CCLM.2009.241

      ,
      • Schöndorf T
      • Hoopmann M
      • Warm M
      • et al.
      Serologic concentrations of HER-2/neu in breast cancer patients with visceral metastases receiving trastuzumab therapy predict the clinical course.
      However, studies have shown inconsistent results regarding the correlation of serum HER2 ECD levels with treatment response
      • Lennon S
      • Barton C
      • Banken L
      • et al.
      Utility of serum HER2 extracellular domain assessment in clinical decision making: pooled analysis of four trials of trastuzumab in metastatic breast cancer.
      , and HER2-targeted therapies possibly interfere with the release of HER2 ECD from tumor cells.
      • Molina MA
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      • Albanell J
      • Rojo F
      • Arribas J
      • Baselga J.
      Trastuzumab (herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast cancer cells.
      ,
      • Vazquez-Martin A
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      • Cufí S
      • Del Barco S
      • Martin-Castillo B
      • Menendez JA.
      Lapatinib, a dual HER1/HER2 tyrosine kinase inhibitor, augments basal cleavage of HER2 extracellular domain (ECD) to inhibit HER2-driven cancer cell growth.
      Leyland-Jones et al performed a comprehensive review of 63 studies, demonstrating an inconsistent association of serum HER2 ECD with poor prognostic and predictive results. The authors therefore concluded that there is insufficient evidence to support the biomarker's clinical use.
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      Serum HER2 testing in patients with HER2-positive breast cancer: the death knell tolls.

      GP88, a Driver of Tumorigenesis

      The clinical use of current serum biomarkers alone in identifying disease progression and response to therapy is limited. Thus, there is a need to identify circulating biomarkers that are drivers of disease and tumorigenesis and can potentially monitor real-time biological processes. An example of this is GP88, also known as progranulin, an 88 kda glycoprotein and the largest member of a family of cysteine-rich polypeptide growth modulators
      • Bhandari V
      • Palfree RG
      • Bateman A.
      Isolation and sequence of the granulin precursor cDNA from human bone marrow reveals tandem cysteine-rich granulin domains.
      ,
      • Plowman GD
      • Green JM
      • Neubauer MG
      • et al.
      The epithelin precursor encodes two proteins with opposing activities on epithelial cell growth.
      isolated from the PC-cell line.
      • Zhou J
      • Gao G
      • Crabb JW
      • Serrero G.
      Purification of an autocrine growth factor homologous with mouse epithelin precursor from a highly tumorigenic cell line.
      Preclinical studies found GP88 to be a biological driver of cell proliferation, survival, and invasiveness,
      • He Z
      • Bateman A.
      Progranulin (granulin-epithelin precursor, PC-cell-derived growth factor, acrogranin) mediates tissue repair and tumorigenesis.
      • Pizarro GO
      • Zhou XC
      • Koch A
      • et al.
      Prosurvival function of the granulin-epithelin precursor is important in tumor progression and chemoresponse.
      • Serrero G.
      Autocrine growth factor revisited: PC-cell-derived growth factor (progranulin), a critical player in breast cancer tumorigenesis.
      and it is associated with drug resistance.
      • Abrhale T
      • Brodie A
      • Sabnis G
      • et al.
      GP88 (PC-Cell Derived Growth Factor, progranulin) stimulates proliferation and confers letrozole resistance to aromatase overexpressing breast cancer cells.
      • Lu R
      • Serrero G.
      Mediation of estrogen mitogenic effect in human breast cancer MCF-7 cells by PC-cell-derived growth factor (PCDGF/granulin precursor).
      • Tangkeangsirisin W
      • Serrero G.
      GP88 (Progranulin) confers fulvestrant (Faslodex, ICI 182,780) resistance to human breast cancer cells.
      • Kim WE
      • Serrero G.
      PC Cell–derived growth factor stimulates proliferation and confers trastuzumab resistance to her-2-overexpressing breast cancer cells.
      Furthermore, inhibition of GP88 expression in PC-cells and human breast cancer cell lines resulted in the dramatic inhibition of tumorigenesis.
      • Zhang H
      • Serrero G.
      Inhibition of tumorigenicity of the teratoma PC cell line by transfection with antisense cDNA for PC cell-derived growth factor (PCDGF, epithelin/granulin precursor).
      ,
      • Lu R
      • Serrero G.
      Inhibition of PC cell-derived growth factor (PCDGF, epithelin/granulin precursor) expression by antisense PCDGF cDNA transfection inhibits tumorigenicity of the human breast carcinoma cell line MDA-MB-468.
      Considering these effects, GP88 is an attractive marker to study in breast cancer.

      Serum GP88 as a Prognostic Marker in Early Disease and Metastatic Disease

      GP88 (PC-Cell Derived Growth Factor, progranulin) is a glycoprotein overexpressed in breast and other tumors and secreted into crirculation. GP88 is involved in tumot cell proliferation and survival. GP88 tumor tissue expression was evaluated and found to be prognostic in several studies, correlating with tumor histological grade, DFS, OS, nodal status, and tumor size.
      • Serrero G
      • Ioffe OB.
      Expression of PC-cell-derived growth factor in benign and malignant human breast epithelium.
      • Serrero G
      • Hawkins DM
      • Yue B
      • et al.
      Progranulin (GP88) tumor tissue expression is associated with increased risk of recurrence in breast cancer patients diagnosed with estrogen receptor positive invasive ductal carcinoma.
      • Serrero G
      • Hawkins DM
      • Bejarano PA
      • et al.
      Determination of GP88 (progranulin) expression in breast tumor biopsies improves the risk predictive value of the Nottingham Prognostic Index.
      Subsequently, a method to quantify circulating GP88 sandwich enzyme-linked immunosorbent assay was developed.
      • Serrero G
      • Hicks D.
      Measurement of circulating progranulin (PGRN/GP88/GEP) by enzyme-linked immunosorbent assay and application in human diseases.
      In breast cancer patients of all stages, circulating levels of GP88 were higher compared to healthy volunteers.
      • Tkaczuk KR
      • Yue B
      • Zhan M
      • et al.
      Increased circulating level of the survival factor gp88 (Progranulin) in the serum of breast cancer patients when compared to healthy subjects.
      GP88 was further investigated in cohort study of 697 patients who underwent curative surgery for primary breast cancer; elevated preoperative serum GP88 values were associated with recurrence.
      • Koo DH
      • Park C-Y
      • Lee ES
      • Ro J
      • Oh SW.
      Progranulin as a prognostic biomarker for breast cancer recurrence in patients who had hormone receptor-positive tumors: a cohort study.
      Current serum markers in clinical practice have proven to be more useful in monitoring treatment response in metastatic disease. Tkaczuk et al measured both GP88 and CA15-3 serially in 101 MBC patients; patients with GP88 <55 ng/mL had a four-fold increased OS compared with patients with GP88 >55 ng/mL. Furthermore, CA15-3 was significantly associated with disease progression but not treatment response, while GP88 was significantly associated with both.
      • Tkaczuk KHR
      • Hawkins D
      • Yue B
      • Hicks D
      • Tait N
      • Serrero G.
      Association of serum progranulin levels with disease progression, therapy response and survival in patients with metastatic breast cancer.
      Prognostically, these are promising results that provide additional information to standard biomarkers. A prospective trial by researchers from the University of Maryland Greenebaum Comprehensive Cancer Center is currently enrolling MBC patients starting a new line of standard-of-care chemotherapy; they will be monitored by routine imaging every 2 to 3 months and routine bloodwork with the addition of serum GP88 levels drawn at routine MD visits every 3 to 8 weeks. The primary objective is to identify if there is a statistically significant change in GP88 levels associated with time to progression as determined by the RECIST 1.1 criteria, with secondary objectives assessing for treatment response and use in combination with CA15-3 and/or CA27-29. Research concerning GP88 as a therapeutic target in cancer is also being pursued; a first-in-human trial with a chimeric anti-GP88 monoclonal antibody is planned at the University of Maryland in both breast cancer
      • Serrero G
      • Dong J
      • Marquez J
      • Yue B
      • Hayashi J.
      Abstract 4514A: antibody to progranulin (Anti-GP88) potentiates tamoxifen and letrozole effect in estrogen receptor positive breast cancer cells.
      ,
      • Guha R
      • Dong J
      • Yue B
      • Serrero G.
      Abstract 1934: Anti-Progranulin (GP88) antibody AG01 therapeutic effect in triple negative breast cancer invasiveness.
      and lung cancer.
      • Edelman MJ
      • Feliciano J
      • Yue B
      • et al.
      GP88 (progranulin): a novel tissue and circulating biomarker for non-small cell lung carcinoma.
      ,
      • Serrero G
      • Xia G
      • Dong J
      • et al.
      Abstract 2054: GP88 (Progranulin) is a therapeutic and prognostic target for non-small cell lung carcinoma.
      Novel targets like GP88 that serve as drivers of tumorigenesis are exciting avenues to explore in the treatment of all cancers.

      Liquid Biopsy – Circulating Tumor Cells (CTCs), Cell-Free DNA/DNA Methylation (ccfDNA), Circulating Tumor DNA (ctDNA), and Circulating microRNA (miRNA)

      Circulating Tumor Cells

      CTCs were first described in the late 1800s as carcinoma associated circulating epithelial cells.
      • ASHWORTH TR
      A case of cancer in which cells similar to those in the tumours were seen in the blood after death.
      They comprise of a heterogeneous population of viable and nonviable tumor cells that are shed from the primary tumor into the circulation, and they have been detected in both early and metastatic breast cancer. CTCs can evaluate the biology of cancer cells using a peripheral blood test, but they are rare in number and greatly diluted by surrounding blood cells, making their detection technologically challenging. Over the past decade, several techniques have been developed to detect CTCs in blood, including size-based, PCR-based, and epithelial-based techniques; most commercial systems, including FDA-approved Cell Search System, use the latter.
      • Sarangi S
      • Mosulpuria K
      • Higgins MJ
      • Bardia A.
      The evolving role of circulating tumor cells in the personalized management of breast cancer: from enumeration to molecular characterization.

      CTCs in Early Breast Cancer

      CTC enumeration has been evaluated for prognostication in early breast cancer. Tkaczuk and colleagues conducted a prospective study of 487 breast cancer patient blood samples; CTCs were detected in 56% of all patients, with 83% of those having stage IV disease. The presence of more than 10 CTCs per sample was associated with decreased survival.
      • Tkaczuk KHR
      • Goloubeva O
      • Tait NS
      • et al.
      The significance of circulating epithelial cells in Breast Cancer patients by a novel negative selection method.
      Other large multicenter trials have established that elevated CTCs at primary diagnosis are associated with shorter DFS and OS,
      • Janni WJ
      • Rack B
      • Terstappen LWMM
      • et al.
      Pooled analysis of the prognostic relevance of circulating tumor cells in primary breast cancer.
      • Rack B
      • Schindlbeck C
      • Jückstock J
      • et al.
      Circulating tumor cells predict survival in early average-to-high risk breast cancer patients.
      • Janni W
      • Rack BK
      • Fasching PA
      • et al.
      Persistence of circulating tumor cells in high risk early breast cancer patients five years after adjuvant chemotherapy and late recurrence: Results from the adjuvant SUCCESS A trial.
      and the presence of CTCs 5 years after completion of chemotherapy was prognostic for poor RFS.
      • Janni W
      • Rack BK
      • Fasching PA
      • et al.
      Persistence of circulating tumor cells in high risk early breast cancer patients five years after adjuvant chemotherapy and late recurrence: Results from the adjuvant SUCCESS A trial.
      ,
      • Sparano J
      • O’Neill A
      • Alpaugh K
      • et al.
      Association of circulating tumor cells with late recurrence of estrogen receptor–positive Breast Cancer.
      Interestingly, a retrospective study by Goodman et al suggested that CTC detection after surgery in early breast cancer patients predicts benefit of adjuvant radiotherapy. These results need to be validated in prospective interventional trials, but they illustrate CTC's potential in detecting minimal residual disease (MRD), or occult micrometastases, in breast cancer.
      • Goodman CR
      • Seagle B-LL
      • Friedl TWP
      • et al.
      Association of circulating tumor cell status with benefit of radiotherapy and survival in early-stage breast cancer.

      CTCs in Metastatic Disease

      In metastatic disease, CTCs were also established as an independent prognostic factor.

      Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. http://dx.doi.org/10.1056/NEJMoa040766. doi:10.1056/NEJMoa040766

      • Wallwiener M
      • Hartkopf AD
      • Baccelli I
      • et al.
      The prognostic impact of circulating tumor cells in subtypes of metastatic breast cancer.
      • Bidard F-C
      • Peeters DJ
      • Fehm T
      • et al.
      Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data.
      In a pooled analysis of 2436 MBC patients, Cristofallini et al used CTCs to group patients into stage IV aggressive (greater than or equal to 5 cells/7.5 mL) and stage IV indolent (less than 5 cells/7.5 mL). Stage IV indolent patients had longer OS than stage IV aggressive (36.3 months versus 16.0 months) independent of clinical and molecular variables.
      • Cristofanilli M
      • Pierga J-Y
      • Reuben J
      • et al.
      The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.
      Budd et al demonstrated that CTCs are a more reproducible indicator of disease status than imaging studies, with the OS of MBC patients with radiologic non-progression and ≥5 CTCs being shorter than those with radiologic non-progression and <5 CTCs. The median OS trends were similar in patients with evidence of radiologic progression.
      • Budd GT
      • Cristofanilli M
      • Ellis MJ
      • et al.
      Circulating tumor cells versus imaging—predicting overall survival in metastatic breast cancer.
      These trials establish CTCs as a prognostic biomarker in MBC and illustrate their advantage over traditional imaging methods.
      One promising application of CTCs is the detection of treatment response or failure in MBC. Smerage et al published the results of the SWOG S0500 trial, investigating whether changing chemotherapy improves OS in patients with persistently elevated CTCs. Specifically, 595 patients were placed into three arms prior to starting a new line of chemotherapy based on CTC levels – arm C had patients with persistently elevated CTCs. These patients were then randomly assigned to continue initial therapy (arm C1) or change to an alternative therapy (arm C2); while CTCs were strongly prognostic between the three arms, there was no significant difference in median OS between C1 and C2.
      • Smerage JB
      • Barlow WE
      • Hortobagyi GN
      • et al.
      Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500.
      The authors concluded that the results may reflect the need for more effective treatment in this population rather than a failure of CTCs to detect ineffective treatment. The STIC CTC trial, a multicenter prospective trial randomized 761 MBC patients into either an a priori clinical group, where the decision to administer hormone therapy or chemotherapy was made clinically before CTC results were disclosed; or a CTC-driven group, where hormone therapy was given if CTC <5/7.5 mL and cytotoxic chemotherapy given if CTC ≥5/7.5 mL. PFS was not inferior in the CTC-driven group. Interestingly, in patients with the clinical a priori choice of hormone therapy with elevated CTC count, PFS was significantly longer in the CTC-driven arm (where chemotherapy was administered) than in the clinically-driven arm.
      • Bidard F-C
      • Jacot W
      • Dureau S
      • et al.
      Abstract GS3-07: Clinical utility of circulating tumor cell count as a tool to chose between first line hormone therapy and chemotherapy for ER+ HER2- metastatic breast cancer: Results of the phase III STIC CTC trial.
      This trial illustrates the potential of CTCs to direct therapy, highlighting the need for additional studies.

      HER2 Discordance

      The HER2 status of the CTCs can be obtained and compared to the primary tumor tissue HER2 status. In some cases, patients can have CTCs positive for HER2 mRNA with negative tissue HER2 expression, known as HER2 discordance. For example, the GeparQuattro trial studied early breast cancer patients receiving neoadjuvant therapy and found that 19% of patients with HER2-negative breast cancer had discordant CTC HER2 positivity.
      • Riethdorf S
      • Müller V
      • Zhang L
      • et al.
      Detection and HER2 expression of circulating tumor cells: prospective monitoring in breast cancer patients treated in the neoadjuvant geparquattro trial.
      Similar findings have been observed in metastatic disease.
      • De Gregorio A
      • Friedl TWP
      • Huober J
      • et al.
      Discordance in human epidermal growth factor receptor 2 (HER2) phenotype between primary tumor and circulating tumor cells in women with her2-negative metastatic breast cancer.
      ,
      • Shah AN
      • Gerratana L
      • Zhang Q
      • et al.
      Abstract P3-01-08: HER2-negative metastatic breast cancer with HER2-positive circulating tumor cells (CTCs): A new CTC-defined HER2-positive subgroup.
      Wang et al measured CTC HER2 expression in 105 MBC patients with negative tissue HER2 status. The participants were deemed low-risk cHER2 (CTC HER2 <2) or high-risk cHER2 (CTC HER2 > or equal to 2), and those with high-risk cHER2 had worse survival and increased risk for disease progression. Furthermore, among the high-risk cHER2 patients, those who received HER2-targeted therapies had improved PFS compared to those who did not. Alternatively, targeted therapies did not affect PFS among the low-risk cHER2 patients.
      • Wang C
      • Mu Z
      • Ye Z
      • et al.
      Prognostic value of HER2 status on circulating tumor cells in advanced-stage breast cancer patients with HER2-negative tumors.
      These results illustrate CTCs’ potential utility in detecting HER2 discordance and predicting response to HER2 targeted therapies.
      In ongoing clinical trials, CTCs are being investigated in breast cancer detection, early disease, and metastatic disease. A large prospective cohort trial out of Walter Reed Army Medical Center will attempt to validate CTC technology as a breast cancer screening test to identify subclinical disease in subjects at risk of progression to clinically apparent disease (NCT01322750). Another non-inferiority randomized trial will randomly assign early HR-positive breast cancer patients into two groups; the trial group will follow patients after definitive surgery with regular CTC evaluation, with elevated levels prompting immediate PET-CT evaluation. The control group will be reevaluated after surgery with regular PET-CT evaluation. The outcome measures include DFS, OS, total length of hospital stay, and total cost of reexaminations (NCT04065321). Ongoing trials in MBC include the DETECT trials. DETECT III (NCT01619111) is a multicenter phase III trial that compares standard therapy alone versus standard therapy plus lapatinib in MBC patients with HER2 discordance. The primary endpoint is CTC clearance, and the secondary endpoints are PFS, overall, all response rate, and dynamic of CTCs.
      • Krause S
      • Friedl T
      • Romashova T
      • et al.
      Abstract OT1-10-01: DETECT III/IV study trial – The multicenter study program in patients with HER2-negative metastatic breast cancer and circulating tumor cells.
      DETECT IV (NCT02035813) is a phase II trial with cohort A having HR-positive MBC patients treated with endocrine therapy combined with everolimus or ribociclib, and with cohort B having triple negative or higher risk HR-positive MBC patients treated with mitotic inhibitor eribulin. The primary endpoints are CTC clearance rate in cohort A and PFS in cohort B.
      • Krause S
      • Friedl T
      • Romashova T
      • et al.
      Abstract OT1-10-01: DETECT III/IV study trial – The multicenter study program in patients with HER2-negative metastatic breast cancer and circulating tumor cells.
      In all trials of DETECT, CTCs are measured serially to obtain data on CTCs dynamics. There is also the DETECT-CTC project to apply innovative biomarkers and assays focusing on molecular characteristics of CTCs to study their potential function as a liquid biopsy tool.
      • Schochter F
      • Friedl TWP
      • deGregorio A
      • et al.
      Are circulating tumor cells (CTCs) ready for clinical use in breast cancer? an overview of completed and ongoing trials using ctcs for clinical treatment decisions.
      These trials can potentially increase CTC's role in breast cancer screening, surveillance monitoring after primary treatment, and disease monitoring in MBC.

      Circulating Cell-Free DNA (ccfDNA)

      The term “liquid biopsy” not only refers to CTCs, but also circulating ccfDNA, including ctDNA. Specifically, circulating DNA is released from both normal and tumor cells into the bloodstream
      • Stroun M
      • Maurice P
      • Vasioukhin V
      • et al.
      The origin and mechanism of circulating DNA.
      and thought to be due to lysis of circulating cancer cells, tumor necrosis, apoptosis, and/or the release of DNA from rapidly dividing cells.
      • Stroun M
      • Maurice P
      • Vasioukhin V
      • et al.
      The origin and mechanism of circulating DNA.
      ,
      • Diehl F
      • Li M
      • Dressman D
      • et al.
      Detection and quantification of mutations in the plasma of patients with colorectal tumors.
      It should be noted that ctDNA describes circulating DNA derived from tumor cells, while ccfDNA describes any free DNA circulating in the bloodstream, regardless of origin. While ccfDNA is present in healthy controls, the quantity of ccfDNA is higher in cancer patients compared to healthy subjects
      • Schwarzenbach H
      • Hoon DSB
      • Pantel K.
      Cell-free nucleic acids as biomarkers in cancer patients.
      , including breast cancer.
      • Catarino R
      • Ferreira MM
      • Rodrigues H
      • et al.
      Quantification of free circulating tumor DNA as a diagnostic marker for breast cancer.
      • Leon SA
      • Shapiro B
      • Sklaroff DM
      • Yaros MJ.
      Free DNA in the serum of cancer patients and the effect of therapy.
      • Panagopoulou M
      • Karaglani M
      • Balgkouranidou I
      • et al.
      Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers.
      Correlations between ccfDNA levels and cancer stage, tumor size, and nodal involvement were established.
      • Panagopoulou M
      • Karaglani M
      • Balgkouranidou I
      • et al.
      Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers.
      • Tangvarasittichai O
      • Jaiwang W
      • Tangvarasittichai S.
      The plasma DNA concentration as a potential breast cancer screening marker.
      • Agostini M
      • Enzo MV
      • Bedin C
      • et al.
      Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients.
      In metastatic disease specifically, Panagopoulou et al demonstrated that ccfDNA quantification serves as a prognostic marker for PFS, and their machine-learning-driven analysis identified ccfDNA quantification as a potent predictive marker for response to first-line chemotherapy.
      • Panagopoulou M
      • Karaglani M
      • Balgkouranidou I
      • et al.
      Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers.
      These results are in accord with a meta-analysis by Tan et al, which found ccfDNA quantification to have prognostic value in both early breast cancer and metastatic disease; however, the authors recognized that their statistical analysis could have overestimated ccfDNA's prognostic role, and there was marked heterogeneity between studies due to differences in analysis type, sampling time, and sampling method.
      • Tan G
      • Chu C
      • Gui X
      • Li J
      • Chen Q
      The prognostic value of circulating cell-free DNA in breast cancer.

      ccfDNA Methylation in Early Breast Cancer

      Like tissue-bound and cell-bound DNA, ccfDNA can be further analyzed for epigenetic alterations, including DNA methylation. When promoter sites of genes are methylated, transcription is typically repressed. In tissue samples, DNA methylation of promoter sites of tumor suppressor genes has been indicated in breast cancer.
      • Widschwendter M
      • Jones PA
      DNA methylation and breast carcinogenesis.
      DNA methylation has also been evaluated in ccfDNA, particularly as a diagnostic tool; both single and panels of genes have been shown to discriminate healthy patients from breast cancer patients with varying sensitivities and specificities.
      • Qian X
      • Ruan L.
      APC gene promoter aberrant methylation in serum as a biomarker for breast cancer diagnosis: A meta-analysis.
      • Shan M
      • Yin H
      • Li J
      • et al.
      Detection of aberrant methylation of a six-gene panel in serum DNA for diagnosis of breast cancer.
      • Salta S
      • P. Nunes S
      • Fontes-Sousa M
      • et al.
      A DNA methylation-based test for breast cancer detection in circulating cell-free DNA.
      • Kim J-H
      • Shin M-H
      • Kweon S-S
      • et al.
      Evaluation of promoter hypermethylation detection in serum as a diagnostic tool for breast carcinoma in Korean women.
      • Radpour R
      • Barekati Z
      • Kohler C
      • et al.
      Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer. Lyko F.
      • Yamamoto N
      • Nakayama T
      • Kajita M
      • et al.
      Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay.
      A meta-analysis of 12 studies evaluating the methylation of the APC gene in ccfDNA yielded a low sensitivity (20%) but high specificity (96%)
      • Qian X
      • Ruan L.
      APC gene promoter aberrant methylation in serum as a biomarker for breast cancer diagnosis: A meta-analysis.
      for detecting breast cancer, while other studies studying methylation panels of at least 2 genes yielded higher sensitivities, with Kim et al and Radpour et al showing 94.1% and 90% for their 2 and 8-gene methylation panel, respectively.
      • Kim J-H
      • Shin M-H
      • Kweon S-S
      • et al.
      Evaluation of promoter hypermethylation detection in serum as a diagnostic tool for breast carcinoma in Korean women.
      ,
      • Radpour R
      • Barekati Z
      • Kohler C
      • et al.
      Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer. Lyko F.
      Notably, the 3-gene methylation panel that Yamamoto et al developed was found to have greater sensitivity than the serum markers CEA and/or CA15-3 in early breast cancer.
      • Yamamoto N
      • Nakayama T
      • Kajita M
      • et al.
      Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay.
      These aforementioned panels included the promoter for gene RASS1FA
      • Shan M
      • Yin H
      • Li J
      • et al.
      Detection of aberrant methylation of a six-gene panel in serum DNA for diagnosis of breast cancer.
      • Salta S
      • P. Nunes S
      • Fontes-Sousa M
      • et al.
      A DNA methylation-based test for breast cancer detection in circulating cell-free DNA.
      • Kim J-H
      • Shin M-H
      • Kweon S-S
      • et al.
      Evaluation of promoter hypermethylation detection in serum as a diagnostic tool for breast carcinoma in Korean women.
      • Radpour R
      • Barekati Z
      • Kohler C
      • et al.
      Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer. Lyko F.
      • Yamamoto N
      • Nakayama T
      • Kajita M
      • et al.
      Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay.
      , and it is one of the most commonly studied methylation markers.
      • de Ruijter TC
      • van der Heide F
      • Smits KM
      • Aarts MJ
      • van Engeland M
      • Heijnen VCG
      Prognostic DNA methylation markers for hormone receptor breast cancer: a systematic review.
      However, Cao et al failed to establish a significant difference between methylation levels of RASSF1A in breast cancer patients and healthy controls, highlighting the heterogeneity inherent to methylation of ccfDNA.
      • Cao X
      • Tang Q
      • Holland-Letz T
      • et al.
      Evaluation of promoter methylation of RASSF1A and ATM in peripheral blood of breast cancer patients and healthy control individuals.
      Methylation of ccfDNA holds promise for diagnostic purposes, necessitating further research to reach a consensus on gene panels and collection methods.
      Prognostically, several studies demonstrated that ccfDNA methylation of various promoter sites correlated with worse survival.
      • Widschwendter M
      • Evans I
      • Jones A
      • et al.
      Methylation patterns in serum DNA for early identification of disseminated breast cancer.
      • Göbel G
      • Auer D
      • Gaugg I
      • et al.
      Prognostic significance of methylated RASSF1A and PITX2 genes in blood- and bone marrow plasma of breast cancer patients.
      • Fujita N
      • Nakayama T
      • Yamamoto N
      • et al.
      Methylated DNA and total DNA in serum detected by one-step methylation-specific pcr is predictive of poor prognosis for breast cancer patients.
      • Fujita N
      • Kagara N
      • Yamamoto N
      • et al.
      Methylated DNA and high total DNA levels in the serum of patients with breast cancer following neoadjuvant chemotherapy are predictive of a poor prognosis.
      Fujita et al established a ccfDNA methylation assay of three genes (GSTP1, RASSF1A, RARB2) in primary breast cancer patients undergoing neoadjuvant chemotherapy; those that were positive showed a worse OS compared to negative patients, independent of pathological complete response.
      • Fujita N
      • Nakayama T
      • Yamamoto N
      • et al.
      Methylated DNA and total DNA in serum detected by one-step methylation-specific pcr is predictive of poor prognosis for breast cancer patients.
      ,
      • Fujita N
      • Kagara N
      • Yamamoto N
      • et al.
      Methylated DNA and high total DNA levels in the serum of patients with breast cancer following neoadjuvant chemotherapy are predictive of a poor prognosis.
      Comprehensively, de Ruijter et al conducted a systematic review on the prognostic potential of ccfDNA methylation in breast cancer. They identified the hypermethylation of 7 genes, including RASSF1A, and the aforementioned panel of GSTP1, RASSF1A, and RARB as potential markers of poor outcome; the authors also performed an analysis of study reporting and found high heterogeneity, concluding that future prognostic DNA methylation marker research would benefit from standardized assessment methods and endpoint definitions.
      • de Ruijter TC
      • van der Heide F
      • Smits KM
      • Aarts MJ
      • van Engeland M
      • Heijnen VCG
      Prognostic DNA methylation markers for hormone receptor breast cancer: a systematic review.
      For monitoring and predicting treatment response in primary breast cancer, there are few trials to evaluate. Liggett et al measured ccfDNA methylation levels at baseline, after surgery, and after surgery on tamoxifen; baseline patterns that were elevated initially became similar to the levels of healthy controls, suggesting ccfDNA methylation can be used to monitor treatment.
      • Liggett TE
      • Melnikov AA
      • Marks JR
      • Levenson VV
      Methylation patterns in cell-free plasma DNA reflect removal of the primary tumor and drug treatment of breast cancer patients.
      To assess the clinical efficacy of neoadjuvant chemotherapy in primary breast cancer patients, Han et al evaluated serum RASSF1A methylation in patients receiving TAC (docetaxel, pirarubicin/epirubicin, and cyclophosphamide), and it was 85.0% sensitive and 50.0% specific for clinical outcome prediction.
      • Han Z-H
      • Xu C-S
      • Han H
      • Wang C
      • Lin S-G
      Value of the level of methylation of RASSF1A and WIF-1 in tissue and serum in neoadjuvant chemotherapeutic assessment for advanced breast cancer.
      On the other hand, Connolly et al also looked at serum DNA methylation levels to predict pathological complete response in early breast cancer patients undergoing neoadjuvant therapy and found that positive serum methylation of a 10-gene panel did not correlate with pCR.
      • Connolly RM
      • Fackler MJ
      • Zhang Z
      • et al.
      Tumor and serum DNA methylation in women receiving preoperative chemotherapy with or without vorinostat in TBCRC008.
      This necessitates larger prospective trials with consistent methylation methods and endpoints to fully assess ccfDNA methylation's predictive potential.

      ccfDNA Methylation in Metastatic Disease

      ccfDNA methylation has been studied more extensively as a diagnostic biomarker in primary breast cancer as opposed to a prognostic or therapy monitoring marker in metastatic disease. However, there are several recent trials that show promise in advanced breast cancer. Fackler et al and Visvanathan et al developed a serum DNA methylation assay as a prognostic marker in MBC, and an elevated cumulative methylation index at baseline and at week 4 had worse PFS and OS. Furthermore, an increasing cumulative methylation index from baseline to week 4 was associated with worse PFS and progressive disease at first restaging.
      • Fackler MJ
      • Bujanda ZL
      • Umbricht C
      • et al.
      Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer.
      ,
      • Visvanathan K
      • Fackler MS
      • Zhang Z
      • et al.
      Monitoring of serum DNA methylation as an early independent marker of response and survival in metastatic breast cancer: TBCRC 005 prospective biomarker study.
      The same authors have recently developed a 9-gene methylation panel that utilizes automated cartridge-based technology, the GX Breast Cancer Monitoring Assay, which preliminarily demonstrates a sensitivity of 85% and specificity of 92%.
      • Fackler MJ
      • Tulac S
      • Venkatesan N
      • et al.
      Abstract PS4-03: An automated DNA methylation assay for monitoring treatment response in patients with metastatic breast cancer.
      Additionally, Panagopoulou et al established a ccfDNA methylation panel of 5 genes, and methylation of 3 or more and 4 or more genes correlated to OS and no pharmacotherapy response, respectively. Using machine-learning and statistical analysis, they developed models for prediction of treatment response to chemotherapy and for OS in metastatic disease that used ccfDNA levels and ccfDNA methylation as parameters.
      • Panagopoulou M
      • Karaglani M
      • Balgkouranidou I
      • et al.
      Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers.
      This demonstrates the application of new technology like automated machine-learning in developing and selecting the panels for prospective study, which is becoming more commonplace in the field. In both primary breast cancer and metastatic breast cancer, ccfDNA methylation of promoter sites is a promising tool with potential application in diagnosis, prognosis, and monitoring treatment. Detecting and quantifying a methylated fraction of ccfDNA is a particular challenge given the low yield, and there are numerous methods.
      • Stastny I
      • Zubor P
      • Kajo K
      • Kubatka P
      • Golubnitschaja O
      • Dankova Z
      Aberrantly Methylated cfDNA in body fluids as a promising diagnostic tool for early detection of breast cancer.
      With newer technologies like automated machine-learning developing and selecting panels for prospective study, we may be closer to reaching the consensus needed to progress ccfDNA methylation research.

      Circulating Tumor DNA (ctDNA) in Early Breast Cancer

      As mentioned previously, ccfDNA includes ctDNA, which is DNA derived from the tumor itself. To distinguish ctDNA from cfDNA, specific somatic DNA mutations or epigenetic alterations are identified in the extracted DNA, meaning individualized panels with tumor-specific mutations can be developed for each patient. ctDNA can be analyzed using next generation sequencing and newer digital PCR techniques, including droplet digital PCR
      • Hindson BJ
      • Ness KD
      • Masquelier DA
      • et al.
      High-throughput droplet digital pcr system for absolute quantitation of DNA copy number.
      , and there are numerous commercial kits available (Table 2). Because breast cancer is heterogeneous with multiple mutations involved in tumorigenesis, ctDNA analysis is not as useful in the screening and diagnosis of breast cancer; however, the detection of ctDNA has shown direct correlation with tumor burden.
      • Catarino R
      • Ferreira MM
      • Rodrigues H
      • et al.
      Quantification of free circulating tumor DNA as a diagnostic marker for breast cancer.
      ,
      • Agostini M
      • Enzo MV
      • Bedin C
      • et al.
      Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients.
      ,
      • Bettegowda C
      • Sausen M
      • Leary RJ
      • et al.
      Detection of circulating tumor DNA in early- and late-stage human malignancies.
      • Huang ZH
      • Li LH
      • Hua D
      Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients.
      • Dawson S-J
      • Tsui DWY
      • Murtaza M
      • et al.
      Analysis of circulating tumor DNA to monitor metastatic breast cancer.
      Prognostically, the presence of ctDNA correlates with worse outcomes in early breast cancer.
      • Liang DH
      • Ensor JE
      • Liu Z-B
      • et al.
      Cell-free DNA as a molecular tool for monitoring disease progression and response to therapy in breast cancer patients.
      • Silva JM
      • Silva J
      • Sanchez A
      • et al.
      Tumor DNA in plasma at diagnosis of breast cancer patients is a valuable predictor of disease-free survival.
      • Olsson E
      • Winter C
      • George A
      • et al.
      Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease.
      • Cavallone L
      • Aguilar-Mahecha A
      • Lafleur J
      • et al.
      Prognostic and predictive value of circulating tumor DNA during neoadjuvant chemotherapy for triple negative breast cancer.
      • Radovich M
      • Jiang G
      • Hancock BA
      • et al.
      Association of circulating tumor DNA and circulating tumor cells after neoadjuvant chemotherapy with disease recurrence in patients with triple-negative breast cancer: preplanned secondary analysis of the BRE12-158 randomized clinical trial.
      • Magbanua MJM
      • Swigart LB
      • Wu H-T
      • et al.
      Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.
      • Guan X
      • Liu B
      • Niu Y
      • et al.
      Longitudinal HER2 amplification tracked in circulating tumor DNA for therapeutic effect monitoring and prognostic evaluation in patients with breast cancer.
      Using data from 196 early-stage TNBC patients, Radovich et al compared ctDNA and CTCs in predicting disease recurrence; detection of ctDNA was significantly associated with inferior DDFS, DFS, and OS at 24 months, and the combination of ctDNA and CTCs increased sensitivity and discriminatory capacity.
      • Radovich M
      • Jiang G
      • Hancock BA
      • et al.
      Association of circulating tumor DNA and circulating tumor cells after neoadjuvant chemotherapy with disease recurrence in patients with triple-negative breast cancer: preplanned secondary analysis of the BRE12-158 randomized clinical trial.
      Because of the lack of prospective trials and consensus in detection methods, ctDNA's prognostic potential for widespread use in early disease is currently limited.
      • Rohanizadegan M
      Analysis of circulating tumor DNA in breast cancer as a diagnostic and prognostic biomarker.
      Notably, studies have assessed ctDNA levels in patients receiving neoadjuvant chemotherapy (NAC).
      • Magbanua MJM
      • Swigart LB
      • Wu H-T
      • et al.
      Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.
      ,
      • Guan X
      • Liu B
      • Niu Y
      • et al.
      Longitudinal HER2 amplification tracked in circulating tumor DNA for therapeutic effect monitoring and prognostic evaluation in patients with breast cancer.
      ,
      • Riva F
      • Bidard F-C
      • Houy A
      • et al.
      Patient-specific circulating tumor DNA detection during neoadjuvant chemotherapy in triple-negative breast cancer.
      • Garcia-Murillas I
      • Schiavon G
      • Weigelt B
      • et al.
      Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.
      • Parsons HA
      • Rhoades J
      • Reed SC
      • et al.
      Sensitive detection of minimal residual disease in patients treated for early-stage breast cancer.
      • McDonald BR
      • Contente-Cuomo T
      • Sammut S-J
      • et al.
      Personalized circulating tumor DNA analysis to detect residual disease after neoadjuvant therapy in breast cancer.
      Magbanua et al tested a personalized ctDNA test in early breast cancer patients undergoing NAC at several different time points - pretreatment, after therapy initiation, between regimens, or prior to surgery. Patients who remained ctDNA-positive after initiation were more likely to have residual disease after NAC compared to those who were negative. After NAC, all patients who achieved pathological complete response (pCR) were ctDNA negative. Of the patients who did not achieve pCR, those that were ctDNA positive had a significantly higher risk of metastatic recurrence compared to those with ctDNA clearance.
      • Magbanua MJM
      • Swigart LB
      • Wu H-T
      • et al.
      Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.
      Others also demonstrated patients with pCR had a larger decrease in ctDNA concentrations during NAC
      • McDonald BR
      • Contente-Cuomo T
      • Sammut S-J
      • et al.
      Personalized circulating tumor DNA analysis to detect residual disease after neoadjuvant therapy in breast cancer.
      , illustrating that individualized ctDNA panels have disease monitoring potential in the neoadjuvant setting.
      Several trials specifically assessed ctDNA's ability to detect MRD in the post-neoadjuvant and post-surgery setting with mutation tracking in personalized ctDNA assays
      • Garcia-Murillas I
      • Schiavon G
      • Weigelt B
      • et al.
      Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.
      ,
      • Parsons HA
      • Rhoades J
      • Reed SC
      • et al.
      Sensitive detection of minimal residual disease in patients treated for early-stage breast cancer.
      ; detection of patient-specific mutations in serial samples of ctDNA can predict early relapse, with Garcia-Murillas et al reporting a median lead time of 7.9 months over clinical relapse.
      • Garcia-Murillas I
      • Schiavon G
      • Weigelt B
      • et al.
      Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.
      Parsons et al developed a ctDNA panel that can potentially track up to 488 mutations with 100-fold more sensitivity than droplet digital PCR; depending on the specific patient number of tumor mutations available to track, its clinical sensitivity was 19% in detecting MRD at 1-year postoperatively, which was positively associated with distant recurrence. Median lead time from first positive test to recurrence was 18.9 months.
      • Parsons HA
      • Rhoades J
      • Reed SC
      • et al.
      Sensitive detection of minimal residual disease in patients treated for early-stage breast cancer.
      These patient-focused ctDNA panels are an attractive biomarker to explore in the field of precision medicine, and development of new panels and methods of sensitive detection are underway.

      ctDNA in Metastatic Disease

      As in early breast cancer, the quantity of ctDNA correlates with poor survival in metastatic disease
      • Dawson S-J
      • Tsui DWY
      • Murtaza M
      • et al.
      Analysis of circulating tumor DNA to monitor metastatic breast cancer.
      ,
      • Hrebien S
      • Citi V
      • Garcia-Murillas I
      • et al.
      Early ctDNA dynamics as a surrogate for progression-free survival in advanced breast cancer in the BEECH trial.
      • Kruger DT
      • Jansen MPHM
      • Konings IRHM
      • et al.
      High ctDNA molecule numbers relate with poor outcome in advanced ER+, HER2− postmenopausal breast cancer patients treated with everolimus and exemestane.
      • O'Leary B
      • Cutts RJ
      • Huang X
      • et al.
      Circulating tumor DNA markers for early progression on fulvestrant with or without palbociclib in er+ advanced breast cancer.
      • Jacob S
      • Davis AA
      • Gerratana L
      • et al.
      The use of serial circulating tumor DNA to detect resistance alterations in progressive metastatic breast cancer.
      • Aguilar-Mahecha A
      • Lafleur J
      • Brousse S
      • et al.
      Early, on-treatment levels and dynamic changes of genomic instability in circulating tumor DNA predict response to treatment and outcome in metastatic breast cancer patients.
      • Darrigues L
      • Pierga J-Y
      • Bernard-Tessier A
      • et al.
      Circulating tumor DNA as a dynamic biomarker of response to palbociclib and fulvestrant in metastatic breast cancer patients.
      , and serial measurement of ctDNA has the potential to monitor and predict treatment response.
      • Dawson S-J
      • Tsui DWY
      • Murtaza M
      • et al.
      Analysis of circulating tumor DNA to monitor metastatic breast cancer.
      ,
      • Jacob S
      • Davis AA
      • Gerratana L
      • et al.
      The use of serial circulating tumor DNA to detect resistance alterations in progressive metastatic breast cancer.
      • Aguilar-Mahecha A
      • Lafleur J
      • Brousse S
      • et al.
      Early, on-treatment levels and dynamic changes of genomic instability in circulating tumor DNA predict response to treatment and outcome in metastatic breast cancer patients.
      • Darrigues L
      • Pierga J-Y
      • Bernard-Tessier A
      • et al.
      Circulating tumor DNA as a dynamic biomarker of response to palbociclib and fulvestrant in metastatic breast cancer patients.
      In a proof-of-concept analysis, Dawson et al showed that increasing ctDNA levels were more sensitive in predicting progressive disease than CA15-3 and CTCs.
      • Dawson S-J
      • Tsui DWY
      • Murtaza M
      • et al.
      Analysis of circulating tumor DNA to monitor metastatic breast cancer.
      Furthermore, Darrigues et al demonstrated that treatment with palbociclib and fulvestrant can be successfully monitored with serial ctDNA measurements.
      • Darrigues L
      • Pierga J-Y
      • Bernard-Tessier A
      • et al.
      Circulating tumor DNA as a dynamic biomarker of response to palbociclib and fulvestrant in metastatic breast cancer patients.
      Serial sampling of ctDNA is very promising, and large-cohort prospective randomized trials are needed. One such trial, PADA-1, is currently investigating the utility of serial ESR1 ctDNA measurements in HER2-negative MBC patients treated with palbociclib and aromatase inhibitor; if patients have a rising ESR1 ctDNA level, they are then randomized to two groups, one with a change in therapy to palbociclib plus fulvestrant and one with no change in therapy. The authors plan to assess whether the change in therapy will benefit patients
      • Bidard FC
      • Sabatier R
      • Berger F
      • et al.
      PADA-1: A randomized, open label, multicentric phase III trial to evaluate the safety and efficacy of palbociclib in combination with hormone therapy driven by circulating DNA ESR1 mutation monitoring in ER-positive, HER2-negative metastatic breast cancer patients.
      (NCT03079011).
      In addition to quantification, ctDNA can determine the mutation status of specific genes, including HER2
      • Guan X
      • Liu B
      • Niu Y
      • et al.
      Longitudinal HER2 amplification tracked in circulating tumor DNA for therapeutic effect monitoring and prognostic evaluation in patients with breast cancer.
      , ESR1
      • Li X
      • Lu J
      • Zhang L
      • Luo Y
      • Zhao Z
      • Li M
      Clinical implications of monitoring ESR1 mutations by circulating tumor DNA in estrogen receptor positive metastatic breast cancer: a pilot study.
      , and TP53
      • Yi Z
      • Ma F
      • Rong G
      • Guan Y
      • Li C
      • Xu B
      Clinical spectrum and prognostic value of TP53 mutations in circulating tumor DNA from breast cancer patients in China.
      , providing insight into prognosis and therapy response. These mutations are actionable, meaning patients can benefit from targeted therapies. The ongoing plasmaMATCH trial, a multicenter, multicohort, phase IIa trial of ctDNA testing in MBC patients from 18 UK hospitals, has published preliminary findings. Over 1000 patients were placed into four treatment cohorts based on mutations identified in ctDNA – cohort A with mutations in ESR1 mutations, cohort B in HER2, cohort C in AKT1 in ER-positive cancer, and corhort D in AKT1 in ER-negative disease/PTEN. Long-term follow-up of these patients is pending, but preliminary findings have shown positive response in cohorts B and C, demonstrating the ctDNA's ability to identify targetable mutations in metastatic patients
      • Turner NC
      • Kingston B
      • Kilburn LS
      • et al.
      Circulating tumour DNA analysis to direct therapy in advanced breast cancer (plasmaMATCH): a multicentre, multicohort, phase 2a, platform trial.
      (NCT03182634). Using ctDNA to identify potential treatments is still being investigated, but the results from these trials serve to strengthen the liquid biopsy's role in breast cancer management.
      In identifying PIK3CA mutations, ctDNA is already an established predictive biomarker.
      • Kodahl AR
      • Ehmsen S
      • Pallisgaard N
      • et al.
      Correlation between circulating cell-free PIK3CA tumor DNA levels and treatment response in patients with PIK3CA-mutated metastatic breast cancer.
      ,
      • Tzanikou E
      • Markou A
      • Politaki E
      • et al.
      PIK3CA hotspot mutations in circulating tumor cells and paired circulating tumor DNA in breast cancer: a direct comparison study.
      Phosphatidylinositol 3-kinases, or PI3K, are part of an intracellular signaling pathway involved in cell cycle regulation.
      • Mukohara T
      PI3K mutations in breast cancer: prognostic and therapeutic implications.
      PI3K has a subunit encoded by the PIK3CA gene, and mutations in this oncogene were identified in human cancers.
      • Samuels Y
      • Wang Z
      • Bardelli A
      • et al.
      High frequency of mutations of the PIK3CA gene in human cancers.
      ,
      • Anderson EJ
      • Mollon LE
      • Dean JL
      • et al.
      A systematic review of the prevalence and diagnostic workup of PIK3CA mutations in HR+/HER2– metastatic breast cancer.
      These PIK3CA mutations are attractive therapy targets, and in May 2019, the FDA approved the drug alpelisib in combination with fulvestrant for postmenopausal patients with HR-positive HER2-negative and PIK3CA-mutated MBC as a second-line therapy.

      FDA approves alpelisib for metastatic breast cancer. FDA. Published December 20, 2019. Accessed July 26, 2020. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-alpelisib-metastatic-breast-cancer

      The SOLAR-1 trial determined PIK3CA mutation-status using both tissue samples and ctDNA. For those patients with PIK3CA mutations identified by ctDNA who received alpelisib plus fulvestrant, there was a 45% risk reduction in PFS.
      • Juric D
      • Ciruelos E
      • Rubovszky G
      • et al.
      Abstract GS3-08: Alpelisib + fulvestrant for advanced breast cancer: Subgroup analyses from the phase III SOLAR-1 trial.
      As a result, the FDA approved tests to detect PIK3CA mutations not only in tumor tissue specimens but also in ctDNA samples. The NCCN's most recent guidelines reflect this, recommending PIK3CA mutation testing on tumor tissue samples or ctDNA in HR-positive/HER2-negative MBC patients.

      National Comprehensive Cancer Network. Breast Cancer (Version 4.2021). National Comprehensive Cancer Network.

      ctDNA is a promising and useful application of personalized medicine in breast cancer, and several trials are testing this application in disease monitoring, particularly in early disease. A prospective observational study from China is monitoring ctDNA levels at post-operative timepoints in early breast cancer patients, comparing the time to recurrence between the detectable ctDNA and nondetectable ctDNA subgroups (NCT04353557). ctDNA-driven disease monitoring may also benefit patients with higher-risk disease. A multi-center randomized control trial in the United Kingdom aims to regularly monitor ctDNA levels following primary treatment in TNBC patients; those who have a positive ctDNA result are randomized to receive either pembrolizumab or no intervention. The primary outcomes of this trial are to assess both ctDNA's ability to detect residual disease and the efficacy of pembrolizumab in preventing disease recurrence (NCT03145961). A similarly designed multicenter trial in the United States screens ctDNA in clinically high-risk stage II-III ER-positive HER2-negative patients receiving adjuvant hormonal therapy; those with detectable ctDNA in plasma and no other evidence of metastasis are then randomized to receive palbociclib plus fulvestrant or continue with adjuvant therapy (NCT04567420).
      Despite the increasing role of liquid biopsy and personalized medicine in metastatic disease, disease monitoring still includes radiological evaluation every 3-4 months. A Swedish prospective observational study aims to develop a biomarker-based prediction model that can potentially substitute for frequent imaging for disease monitoring. ER-positive HER2-negative MBC patients receiving standard therapy will be assessed with ctDNA, CA15-3, and thymidine kinase 1 (TK1) at all imaging timepoints. The primary aim is to develop an algorithm in which these biomarkers, alone or in conjunction, can predict whether a scan is necessary with sufficient sensitivity and specificity; the overall goal with this personalized medicine panel is to accurately assess disease status in these patients so that routine imaging can be delayed until biomarker elevation (NCT04597580).

      Circulating miRNAs: Emerging Prognostic and Predictive Biomarkers

      Another emerging class of serum biomarkers is circulating microRNAs (miRNAs), which are a class of non-coding RNA 18 to 25 nucleotides long that bind and inhibit the translation of their target mRNAs.
      • Bushati N
      • Cohen SM
      microRNA Functions.
      MiRNAs play a critical role in post-transcriptional regulation of gene expression and are involved in proliferation, differentiation, apoptosis, and metabolism of cells. Therefore, dysregulation of miRNAs is associated with cancer.
      • Croce CM
      Causes and consequences of microRNA dysregulation in cancer.
      MiRNAs can serve as a circulating biomarker that can give valuable insight into disease process.
      • Mitchell PS
      • Parkin RK
      • Kroh EM
      • et al.
      Circulating microRNAs as stable blood-based markers for cancer detection.
      Numerous studies have been done with various miRNA types and panels to determine if they have prognostic significance in breast cancer.
      • Mangolini A
      • Ferracin M
      • Zanzi MV
      • et al.
      Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR.
      • Kleivi Sahlberg K
      • Bottai G
      • Naume B
      • et al.
      A serum microRNA signature predicts tumor relapse and survival in triple-negative breast cancer patients.
      • Roth C
      • Rack B
      • Müller V
      • Janni W
      • Pantel K
      • Schwarzenbach H
      Circulating microRNAs as blood-based markers for patients with primary and metastatic breast cancer.
      • Shaker O
      • Maher M
      • Nassar Y
      • Morcos G
      • Gad Z
      Role of microRNAs -29b-2, -155, -197 and -205 as diagnostic biomarkers in serum of breast cancer females.
      • Eichelser C
      • Flesch-Janys D
      • Chang-Claude J
      • Pantel K
      • Schwarzenbach H
      Deregulated serum concentrations of circulating cell-free microRNAs miR-17, miR-34a, miR-155, and miR-373 in human breast cancer development and progression.
      For example, Kleivi Sahlberg et al identified a four-miRNA signature (miR-18b, miR-103, miR-107, and miR-652) associated with relapse and OS in patients with primary TNBC.
      • Kleivi Sahlberg K
      • Bottai G
      • Naume B
      • et al.
      A serum microRNA signature predicts tumor relapse and survival in triple-negative breast cancer patients.
      Another study demonstrated that increased expression of circulating miR-34a correlated with tumor stage, and upregulation of miR-10b, miR-34a, and miR-155 was associated with metastatic disease.
      • Roth C
      • Rack B
      • Müller V
      • Janni W
      • Pantel K
      • Schwarzenbach H
      Circulating microRNAs as blood-based markers for patients with primary and metastatic breast cancer.
      Excitingly, circulating miRNA panels selected by machine-learning models have been developed for the early detection of numerous solid tumor malignancies, demonstrating a 91% sensitivity and 90% specificity in breast cancer, specifically. This further demonstrates the exciting potential of liquid biopsy combined with machine-learning.
      • Matsuzaki J
      • Yamamoto Y
      • Yi O
      • et al.
      Machine learning-based multiple cancer detections with circulating miRNA profiles in the blood.
      Few studies have also investigated circulating miRNAs as a predictive biomarker in breast cancer.
      • Wang H
      • Tan G
      • Dong L
      • et al.
      Circulating MiR-125b as a marker predicting chemoresistance in breast cancer.
      ,
      • Sun Y
      • Wang M
      • Lin G
      • et al.
      Serum microRNA-155 as a potential biomarker to track disease in breast cancer.
      In one study, the authors demonstrated a possible correlation between miR-125b and chemotherapeutic resistance.
      • Wang H
      • Tan G
      • Dong L
      • et al.
      Circulating MiR-125b as a marker predicting chemoresistance in breast cancer.
      Sun et al found that miR-155 serum levels decreased after surgery and four cycles of chemotherapy in breast cancer patients, suggesting miR-155 can possibly indicate treatment response.
      • Sun Y
      • Wang M
      • Lin G
      • et al.
      Serum microRNA-155 as a potential biomarker to track disease in breast cancer.
      While these studies are very promising, there are challenges in establishing an accurate and reliable panel of circulating miRNA, including their low abundance, differences in processing, and current inability to detect novel miRNAs.
      • Hamam R
      • Hamam D
      • Alsaleh KA
      • et al.
      Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers.
      There is minimal overlap in the miRNA panels across the different studies, reflecting the complex biology of miRNA expression the serum of breast cancer patients. While very promising, multi-center profiling studies are needed to provide more valuable information on circulating miRNA panels. One such trial taking place in Nigeria aims to evaluate the potential of using circulating miRNA and CTCs as a surrogate marker of chemotherapy resistance in TNBC patients in the neoadjuvant setting. Blood samples will be collected before and after chemotherapy, and the percentage of participants achieving pathological complete response (pCR) at surgery will be determined. An outcome measure of the trial is the change in circulating levels of miRNA during and after treatment, quantifying the dynamics and ability of miRNA in monitoring treatment response (NCT04771871).

      Clinical Practice Points

      Expert panels have published recommendations for clinical use for some of the biomarkers in this review, outlined in Table 1. NCCN and ASCO guidelines recommend serial monitoring of CEA, CA 15-3, and/or CA 27-29 in metastatic disease; particularly, an elevation of 20% to 30% in any of these markers in addition to clinical exam can indicate treatment failure.
      • Harris L
      • Fritsche H
      • Mennel R
      • et al.
      American society of clinical oncology 2007 update of recommendations for the use of tumor markers in breast cancer.
      ,
      • Van Poznak C
      • Somerfield MR
      • Bast RC
      • et al.
      Use of biomarkers to guide decisions on systemic therapy for women with metastatic breast cancer: american society of clinical oncology clinical practice guideline.
      The European Society of Medical Oncology, National Academy of Clinical Biochemistry (NACB), European Society of Mastology, and European Group on Tumor Markers (EGTM) all concur, recommending serial measurement in metastatic disease.
      • Cardoso F
      • Kyriakides S
      • Ohno S
      • et al.
      Early breast cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up†.
      • Sturgeon CM
      • Duffy MJ
      • Stenman U-H
      • et al.
      National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers.
      • Blamey RW
      EUSOMA
      Guidelines on endocrine therapy of breast cancer EUSOMA.
      • Molina R
      • Barak V
      • van Dalen A
      • et al.
      Tumor markers in breast cancer- European Group on Tumor Markers recommendations.
      These groups do not recommend measuring these markers for screening and staging in primary disease. Differing from the other groups, the NACB and EGTM recommend measuring CEA, CA 15-3, and CA 27-29 for postoperative surveillance, even without evidence of progression.
      • Sturgeon CM
      • Duffy MJ
      • Stenman U-H
      • et al.
      National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers.
      ,
      • Molina R
      • Barak V
      • van Dalen A
      • et al.
      Tumor markers in breast cancer- European Group on Tumor Markers recommendations.
      The EGTM alone recommends preoperative measurement of CEA or CA 15-3/CA 27-29 in combination with established factors to aid in determining prognosis.
      • Molina R
      • Barak V
      • van Dalen A
      • et al.
      Tumor markers in breast cancer- European Group on Tumor Markers recommendations.
      Regarding serum HER2 ECD, EGTM and ASCO recommend against use to predict benefit from therapy
      • Duffy MJ
      • Harbeck N
      • Nap M
      • et al.
      Clinical use of biomarkers in breast cancer: Updated guidelines from the European Group on Tumor Markers (EGTM).
      ,
      • Harris LN
      • Ismaila N
      • McShane LM
      • et al.
      Use of biomarkers to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer: American society of clinical oncology clinical practice guideline.
      , specifically citing the Leyland-Jones et al review.
      • Leyland-Jones B
      • Smith BR.
      Serum HER2 testing in patients with HER2-positive breast cancer: the death knell tolls.
      The newer player GP88 is still in the early phases of biomarker investigation and therefore is not included in the expert panel recommendations.
      Table 1Serum Biomarkers in Breast Cancer Management
      Table 2Selected Commercial ctDNA Tests Available for Solid Tumor Cancers

      Pathway Genomics Launches First Liquid Biopsy Test To Detect Cancer-Associated Mutations In High-Risk Patients. BioSpace. https://www.biospace.com/article/releases/pathway-genomics-launches-first-liquid-biopsy-test-to-detect-cancer-associated-mutations-in-high-risk-patients-/. Published 2015. Accessed January 7, 2021.

      CellMax-Lbx Liquid Biopsy Cancer Blood test. 73 Gene Profile. CellMax Life. https://cellmaxlife.in/liquid-biopsy-lbx/. Accessed January 7, 2021.

      Circulogene. Circulogene. https://circulogene.com/. Accessed January 7, 2021.

      FoundationOne Liquid CDx | Foundation Medicine. Foundationmedicine.com. https://www.foundationmedicine.com/test/foundationone-liquid-cdx. Accessed January 7, 2021.

      Guardant360 CDx - Guardant360. Guardant360. https://guardant360cdx.com/guardant360-cdx/. Accessed January 7, 2021.

      liquidgx | AdmeraHealth. Admera Health. https://www.admerahealth.com/liquidgx-2/. Accessed January 7, 2021.

      Signatera Advanced Cancer Detection. Natera.com. https://www.natera.com/oncology/signatera-advanced-cancer-detection. Accessed January 7, 2021.

      Target Selector™ NGS Breast Panel – Biocept. Biocept.com. https://biocept.com/ngs-breast-panel/. Accessed January 7, 2021.

      In their 2007 recommendation, ASCO stated that CTCs demonstrated insufficient evidence to support routine use.
      • Harris L
      • Fritsche H
      • Mennel R
      • et al.
      American society of clinical oncology 2007 update of recommendations for the use of tumor markers in breast cancer.
      Although CTCs are prognostic, they are not predictive when used to guide or influence decisions on systemic therapy.
      • Van Poznak C
      • Somerfield MR
      • Bast RC
      • et al.
      Use of biomarkers to guide decisions on systemic therapy for women with metastatic breast cancer: american society of clinical oncology clinical practice guideline.
      NCCN released guidelines that do not yet include the clinical use of CTCs in MBC; again, they recognized the prognostic value of CTCs, but at the time of their recommendations, CTCs failed to show a predictive value.

      National Comprehensive Cancer Network. Breast Cancer (Version 4.2021). National Comprehensive Cancer Network.

      NCCN specifically cited the SWOG S0500 trial in their decision.
      • Smerage JB
      • Barlow WE
      • Hortobagyi GN
      • et al.
      Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500.
      The NACB also reports that CTC use in assessing prognosis and monitoring therapy in metastatic disease is under evaluation pending prospective randomized trial results.
      • Sturgeon CM
      • Duffy MJ
      • Stenman U-H
      • et al.
      National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers.
      Considering the detection of HER2 discordance and upcoming DETECT trials, expert panel recommendations on clinical use of CTCs may change in the coming years. Many of these guidelines do not discuss ccfDNA and ctDNA. However, specifically in testing for actionable mutations, the NCCN's most recent guidelines recommend measuring ctDNA to test for PIK3CA, reflecting the recent FDA approval of a PIK3CA ctDNA assay in HR-positive-HER2-negative MBC patients.

      National Comprehensive Cancer Network. Breast Cancer (Version 4.2021). National Comprehensive Cancer Network.

      Finally, miRNA needs to be studied further to establish the consistency and prognostic potential to be included in the current guidelines.

      Regarding the Clinical Practice Points

      This is covered under Expert Panel Recommendations, so I revised this section heading, please let me know if you agree.

      Conclusion

      Biomarkers provide valuable insight into a breast cancer patient's prognosis and response to treatment. Serum biomarkers offer an attractive alternative to tissue biomarkers, especially in the setting of monitoring for disease recurrence or progression, following response to treatment, and even determining targetable mutations to direct therapy. CA15-3, CA27-29, and CEA have low sensitivity in early breast cancer, but they have proved useful adjuncts in the management of metastatic disease, particularly in monitoring response to treatment with serial measurement. While not a substitute for tissue HER2 expression determination, serum HER2 ECD has potential, particularly in cases of HER2 discordance; although not currently recommended for use by expert panels, HER2 ECD potentially provides additional information in the decision to initiate anti-HER2 therapies, necessitating further study. GP88 is directly involved in tumorigenesis and can provide meaningful prognostic and predictive information, especially in metastatic disease. It needs to be studied more in depth, both as a biomarker and as a therapeutic target; but it demonstrates how novel biomarkers involved in the various stages of tumor formation are exciting players in oncology. Another exciting avenue for biomarker research lies in the liquid biopsy. CTCs are the most established and are promising prognostic and predictive tools, but current evidence is limited to establish their widespread use. However, upcoming trials such as the DETECT trials will attempt to address these limitations, especially in monitoring therapy in metastatic disease. ccfDNA and ctDNA have many applications, from aiding in diagnosis with ccfDNA methylation to determining the mutational status of actionable genes with ctDNA. The latter application, specifically with PIK3CA mutations in MBC, has achieved FDA approval and guideline recommendations. Finally, circulating miRNA panels have prognostic and predictive potential in all cancers, including breast cancer, but requires further investigation before widespread clinical use. Many of the ongoing trials studying these biomarkers, from the established markers CEA, CA15-3, CA27-29 to the components of the liquid biopsy (CTCs, ccfDNA/ctDNA, miRNA), will compare disease monitoring guided by serial biomarker evaluation to the traditional monitoring methods. These trials are necessary to define the roles of these circulating biomarkers and may potentially change the imaging-driven response assessment for all types of solid tumor malignancies. Furthermore, with the combination of liquid biopsy and machine learning, as demonstrated in the selection of panels in ccfDNA methylation and miRNA, personalized medicine can potentially be integrated more easily into the standard-of-care. The field of biomarker-related investigations in breast cancer continues to grow, and few biomarkers eventually make it to the final finish line and are considered standard-of-care for clinical management of breast cancer.

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