Testing for biomarkers in cancer is often a routine part of medical care.1 Biomarker testing can be performed from tissue samples, or from samples obtained using less-invasive means such as the liquid biopsy. In the liquid biopsy approach, testing is performed on cancer-derived components (including CTCs) found in peripheral blood or other body fluids.2 Specifically, the testing of CTCs in liquid biopsy specimens involves cell capture, enrichment, and downstream molecular characterization, with the goal of impacting disease management in cancer.3 Current technological approaches for identifying biomarkers from CTCs include distinguishing the comparatively rare tumor cells from among the large number of normal cells by image processing and/or direct capture techniques, along with staining for the biomarker of interest.4-7
CTCs can be used to detect biomarkers important for prognosis, identifying treatment, and monitoring response to treatments in cancer. Biomarker detection from CTCs may therefore serve as a potential alternative to biomarker detection from biopsy specimens for certain cancers, including breast and prostate.4-7
In 1998 the Food and Drug Administration (FDA) approved the drug Herceptin® (trastuzumab) for the treatment of HER2 over-expressing breast cancer. Testing for HER2 has become 1 of the most important sources of information in making management decisions regarding systemic therapy in breast cancer. The NCCN guidelines on breast cancer recommend that all patients who have new primary or newly metastatic breast cancers be tested for HER2 using a methodology outlined in the ASCO/College of American Pathologists (CAP) guideline.8,9 Traditionally, testing for HER2 was performed using tissue. Testing from tissue, however, can be associated with complications. For example, breast malignancies may metastasize to the brain,10 and analyses of complications following brain biopsies have shown a wide array of complications ranging from neurologic complications to general surgical complications.11,12
Studies have demonstrated that it is feasible to collect CTCs from breast cancer and to identify the HER2 biomarker in the circulating cells, achieving results that are concordant with tissue HER2 testing.4,5 Additional studies have examined whether patients with HER2 negative tissue but HER2 positive CTCs respond to trastuzumab, and have shown minimal effect of this therapy in such patients.13,14 Conversely, research has shown that patients with HER2 positive tissue in early breast cancer may have HER2 negative CTCs, and it is posited that this may contribute to drug resistance.15 Interest persists with other studies ongoing to evaluate the use of HER2 positive CTCs to help guide treatment.16
The androgen receptor (AR) is 1 of the most important pathways in prostate cancer. It is involved in disease progression and resistance to treatment. For these reasons, it is a common target of hormone-based therapies, including androgen deprivation therapy. AR-V7 is an AR splice variant that results in constitutive activation of oncogenic signaling and cell proliferation and has been implicated in resistance to androgen receptor signaling (ARS) inhibitors.17-19 Prospective multicenter studies in men with metastatic castration-resistant prostate cancer (mCRPC) have shown that detection of AR-V7 in CTCs is associated with resistance to the ARS inhibitors enzalutamide and abiraterone, whereas in AR-V7-negative patients, the taxanes and ARS inhibitors show comparable efficacy.6,20,21 Additionally, AR-V7 status may change during therapy.22 For these reasons, NCCN guidelines recommend AR-V7 testing in CTCs to help guide selection of therapy after progression on abiraterone or enzalutamide in mCRPC.23
CTC-associated Biomarkers in Other Cancers
HER2 is overexpressed in malignancies other than breast cancer.24 Overexpression of HER2 on cancer cells has been reported in 10%–26% of gastric and esophagogastric junction cancers, particularly in cancers with intestinal-type histology.25-27 In gastric cancer, HER2 has been established as a predictive biomarker for HER2-targeted therapies, and in HER2 overexpressed metastatic gastric adenocarcinoma, trastuzumab is now recommended to be added to first-line chemotherapy.28 Additional trastuzumab-based therapies such as Enhertu® (fam-trastuzumab deruxtecan-nxki) have also been approved for the treatment of adults with locally advanced or metastatic HER2 positive gastric or gastroesophageal junction (GEJ) adenocarcinoma who have received a prior trastuzumab-based regimen.29,30 HER2 is also amplified in approximately 3% of colon cancers, and in 5%-14% of Rat sarcoma viral oncogene homolog (RAS)/ B-Raf murine sarcoma viral oncogene homolog B1 (BRAF)-wild type tumors.31,32 In colon cancer, HER2 overexpression may have a role in predicting response to HER2 targeted therapies and resistance to epidermal growth factor receptor (EGFR)-specific therapies, and anti-HER2 combination therapies have shown promise in HER2 overexpressing, RAS/RAF-wild type, metastatic colorectal cancers (mCRC).31-37 In gastric cancer, studies have shown conflicting levels of concordance of HER2 copy number between tissue and plasma samples.25,38-40 As with breast and other solid tumors, discordance is reported and thought to be, at least in part, due to tumor heterogeneity.39,40 One study reported HER2-overexpressed CTCs in up to approximately 32% of all recurrent or metastatic gastrointestinal cancer patients; this same study observed a HER2 discordance rate of 35.5% between tumor cells and CTCs.39 Though liquid biopsy holds promise, to-date NCCN guidelines recommend that methods for HER2 testing in gastric cancer remain IHC and fluorescence in situ hybridization (FISH), or another in situ hybridization (ISH) method, with next-generation sequencing (NGS) being acceptable when there is limited tissue and sequential biomarker testing is infeasible.28,41,42 Similarly, for colorectal cancer, NCCN guidelines recommend HER2 and other biomarker testing in mCRC by IHC, FISH, or NGS.43
In non-small cell lung cancer (NSCLC), NCCN guidelines state that cell-free/circulating tumor DNA (ctDNA) may be used for molecular analysis of the EGFR and anaplastic lymphoma kinase (ALK) genes, as well as other oncogenic biomarkers if tissue is insufficient for testing or if the patient is medically unfit for invasive tissue sampling.44 Detection of these biomarkers has also been achieved using CTCs; moreover, analysis at disease progression has detected new mutations in EGFR (including the T790M mutation, which confers drug resistance).45 However, studies have reported that ctDNA has shown greater sensitivity than CTCs for detecting mutations in EGFR and the Kirsten rat sarcoma virus oncogene (KRAS), when compared with mutation status in matched tumor.46,47 CTCs can also detect clinically significant genetic rearrangements in NSCLC.48,49 In 1 study using filter-adapted FISH, variations in ALK-rearranged CTC levels were observed during treatment with crizotinib.48 In another study, an increase of ROS1-rearranged CTCs was associated with resistance to crizotinib.49 Overall, biomarker testing from CTCs in lung cancer remains a promising and active area of investigation.
The detection of CTCs has been evaluated in localized as well as in metastatic cancers, and CTCs have potential utility beyond that of their molecular characteristics (biomarkers). For example, CTC enumeration and variation over time can serve as indicators of cancer detection, prognosis, recurrence, and treatment response in a variety of cancers.50,51 CTC counts have been shown to change after treatment in patients with metastatic breast (mBC), colorectal (mCRC), and prostate (mPC) cancers, and prognostic information may be obtained by the serial monitoring of patients.52 In breast cancer, numerous studies have shown enumeration of CTCs to be a good prognostic marker and measure of treatment response,53-57 though these studies do not suggest a clear effect on outcomes from a change in treatment.9,58 The STIC CTC Randomized Clinical Trial showed that CTC enumeration may be useful in guiding the choice between chemotherapy and endocrine therapy as a first-line treatment in hormone receptor–positive, HER2-negative metastatic breast cancer.59 However, in a prospective randomized trial (SWOG S0500), serial enumeration of CTC in patients with metastatic breast cancer and switching to alternate cytotoxic therapy (in patients with persistently high CTCs) did not affect progression-free survival (PFS) or overall survival (OS).58 For these reasons, the enumeration of CTCs in metastatic breast cancer is not yet included in the NCCN guidelines for assessment and monitoring.9 Moreover, in prostate cancer, a 30% decline in CTCs (approximately 1 month after treatment initiation) was shown to distinguish between patients benefiting and those not benefiting from treatment; in the latter group, those patients might be considered for a change in therapy.60 Though in metastatic prostate cancer CTC enumeration has been shown to provide good prognostic information,61-64 in localized prostate cancer, the same methodology may underestimate the actual number of CTCs.65 Limitations of CTC enumeration include low rates of detection (depending on the particular cancer type and severity), differences in the sampling and testing methods used, and the lack of standardization of cut-offs and time-points for assessment.51 CTC enumeration may be a good prognostic indicator for certain cancers, but studies do not conclusively suggest a clear effect on outcomes resulting from a change in management.