Background: Bearing multidimensional tumor-relevant information ranging from genomic alterations to proteomic makeup, circulating tumor cells (CTCs) constitute a promising material for liquid biopsy. The clinical validity of CTCs has been most extensively studied in metastatic breast cancer (MBC). The Cellsearch assay is currently the most widely used, while alternative strategies are pursued. A filtration-based microfluidic device has been described for CTC enrichment, but its clinical relevance remains unknown. Methods: In this preliminary study, we prospectively enrolled 47 MBC patients and evaluated the performance of the abovementioned CTC assay for tumor burden monitoring and human epidermal growth factor receptor 2 (HER2) status determination. Results: At baseline, 51.1% patients (24/47) were CTC positive. CTC count and positivity were also significantly higher in samples that accompanied poorer radiographic response evaluations. Serial blood draws suggested that CTC count enabled more accurate monitoring of tumor burden than serum markers carcinoembryonic antigen and cancer antigen 15-3. Also, in contrast to previous reports, CTC-HER2 status was moderately consistent with tumor-HER2 status. CTC-HER2 status assessment was further supported by HER2 copy number measurements in select samples. Conclusion: The preliminary results from this study suggest promise for the interrogated CDC assay in several aspects, including sensitive CTC detection, accurate disease status reflection, and HER2 status determination. More studies are warranted to validate these findings and further characterize the value of CTC assay.

Circulating tumor cells (CTCs) are a rare subset of tumor cells that enter the bloodstream after shedding from primary or secondary tumors. CTCs represent an essential step in the metastatic cascade and are generally regarded as harbingers of metastasis [1]. Encouraging progress has been made over the past two decades in characterizing the clinical value of CTCs in predicting response to treatment and survival outcomes [2]. In a seminal study, Cristofanilli et al. [3] showed that CTC count before a new line of treatment was an independent predictor of progression-free survival (PFS) and overall survival (OS) in 177 patients with metastatic breast cancer (MBC). A pooled analysis of 1,944 patients further confirmed this negative correlation between CTC numbers and survival outcomes in MBC [4]. Independent prognostic significance of CTCs was also demonstrated in the setting of nonmetastatic disease. The SUCCESS trial, with >1,400 patients with primary (stage I–III) breast cancer, showed the prognostic value of CTC count both before and after adjuvant chemotherapy for disease-free survival and OS [5]. The presence of CTCs was also confirmed as a poor prognosticator for disease-free survival and OS in a subsequent meta-analysis of 3,173 patients with primary breast cancer [6]. Subsequent trials and retrospective studies further investigated the relevance of CTCs in a host of clinical scenarios [7].

In addition to CTC count and dynamics, considerable research has focused on the expression of key clinical markers, especially the human epidermal growth factor receptor 2 (HER2) status of CTCs. Two studies showed a longer PFS in patients receiving HER2-targeted therapy for those with HER2-positive CTCs [8, 9]. On the other hand, HER2-negative primary tumors are known to acquire HER2 amplification during disease progression [10]. Several large-scale trials were conducted to investigate the relevance of CTC-HER2 positivity in patients with HER2-negative MBC. The CirCe T-DM1 trial reported 14 CTC-HER2-positive patients in the 154 women who participated in the screening, although only 1 showed partial response (PR) to ado-trastuzumab emtansine treatment [11]. The DETECT III study randomized 105 MBC patients with HER2-negative tumor and HER2-positive CTCs to standard therapy alone or plus lapatinib, a dual inhibitor of HER2 and epidermal growth factor receptor. The plus lapatinib arm showed longer PFS and OS than the comparator arm [12]. These results demonstrated the value of CTC-HER2 status in making informed treatment decisions in MBC patients, even when the tumor was HER2 negative.

Despite its promise, clinical utility of CTCs is challenged by inconsistencies. Characterization of CTCs can also be confounded by the initial enrichment procedures. Studies employing the Cellsearch assay typically define CTCs as cells staining positively for the nucleus and two epithelial markers and negatively for a pan-leukocyte antigen [2]. Sensitivity of the Cellsearch assay could be impaired by the presence of CTCs undergoing epithelial-mesenchymal transition expressing no or undetectable levels of either or both of the epithelial markers [2]. Biophysics-based approaches, on the other hand, exploit the difference between CTCs and leukocytes in physical properties such as cell size [7]. One assay under this category is isolation by size of epithelial tumor cells [13]. A direct comparison between Cellsearch and isolation by size of epithelial tumor cells showed approximately 20% concordance in CTC counts, 20% in lung cancer, and 60% in MBC and prostate cancer [14]. In addition to CTC capture, low inter-approach concordance in CTC-HER2 status determination has been documented [15]. Therefore, the clinical value of CTCs can benefit from more accurate assays.

In this preliminary study, we characterize the performance of a novel filtration-based CTC assay with clinical samples from MBC patients [16]. The principles and mechanics of this test, along with demonstrations of CTC capture capability, have been reported. However, there has been no evidence regarding its value in CTC-related clinical applications. In our cohort of 47 patients, this assay resulted in a CTC positivity rate comparable to several major trials that used the Cellsearch assay but requiring less sample volume. We also found significantly higher CTC counts and positivity rate in MBC patients with poorer radiographic response than in patients with better responses and healthy volunteers. Furthermore, in patients who underwent serial biopsies, CTC dynamics may be more accurate in reflecting disease status than common serum markers. Additionally, we observed moderate inter-approach consistency in determining HER2 status with a high negative predictive value (NPV), which may be useful in facilitating treatment decisions regarding HER2-targeted therapy.

Study Design

This study enrolled 47 MBC patients and 40 healthy volunteers at Guangzhou Red Cross Hospital from December 2019 to August 2020. Inclusion criteria were as follows: the patient (1) is a woman aged 18–75 years and diagnosed with stage IV breast cancer, (2) has an ECOG score of 0–2, (3) has an OS of 3 months or longer, and (4) has at least one radiographically measurable lesion. Exclusion criteria included coagulation disorders, HIV, active chronic hepatitis B/C infection, and other severe clinical infections as defined in National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0.

Blood draws were performed at enrollment and follow-up examinations, except for the last biopsy. Results of CTC analysis corresponded to the radiologic response evaluations made at the same follow-ups, that is, by comparing the images collected at the follow-up with those acquired at the previous one. The samples were divided into two groups by type of response. The better responses group consisted of PR, complete response, and stable disease (SD) in which the tumor size did decrease but by <30%, whereas the poorer response group consisted of patients evaluated with progressive disease (PD) or SD in which the tumor size either remained the same or increased but did not reach PD.

HER2 positivity of the tumor biopsy was defined as immunohistochemical staining 3+ or IHC 2+ plus HER2/CEP17 ratio >2.2 determined by fluorescence in situ hybridization. Other clinical features, such as estrogen receptor status, progesterone receptor status, and molecular subtype, were also collected.

Blood Draw and CTC Characterization

Blood draws were performed at enrollment for MBC patients and healthy volunteers and at each follow-up for patients. CTC testing was performed within 4 h since peripheral venous blood collection at a central laboratory. Five milliliters of blood were loaded onto the RCFS-CTC platform (Anfang Biotech, Guangzhou, China) for size-based CTC enrichment. A more detailed description of the principle and specifications of the system can be found in the online supplementary File (for all online suppl. material, see www.karger.com/doi/10.1159/000520497). CTCs were defined as DAPI+, PanCK+, and CD45 cells with a diameter >5 μm, HER2+ CTC as DAPI+, PanCK+, CD45, and HER2+ cells, and CTC-HER2 positivity defined as CTC count ≥5 with the proportion of HER2+ CTCs ≥50%.

Determination of the Threshold for CTC-HER2 Positivity

Four cell lines MCF-7, ZR-75-1, MDA-MB-361, and SKBR3, which express HER2 at distinctly increasing levels, were subjected to anti-HER2 fluorescent staining and served as standards for calibration. CTC-HER2 positivity was finally defined as a ratio of average CTC-HER2 immunofluorescence intensity to background of >6. A more detailed description of the process can be found in the online supplementary File.

Tumor Cell Line Spike-In and Recovery

To assess the sensitivity and specificity of the microfilter-based CTC assay, 10 positive and 10 negative controls were subjected to the assay. The negative controls were blood collected from healthy donors, and positive controls were blood collected from healthy donors spiked with MCF-7 or SKBR3 cells. Negative samples were defined as those detected with <5 cancer cells/5 mL of blood. A detailed by-sample summary is provided in online supplementary Table 1.

Single-Cell ddPCR

CTCs were transferred with the CellTram 4r Oil hydraulic microinjector (Eppendorf, Hamburg, Germany) under a Z2 upright fluorescence microscope (Zeiss, Jena, Germany) to a PCR tube. DNA was extracted and amplified with MALBAC single-cell whole genome amplification kit (Yikon Genomics, Shanghai, China) on a RePure-A PCR thermal cycler (Bio-Gener Technology, Hangzhou, China) as per instructions. Subsequently, 14.5 μL of the droplet digital PCR mixture was subjected to PCR on a V2 droplet digital PCR chip per kit instructions (ThermoFisher, Waltham, MA, USA). Afterward, the chip was moved to QuantStudio 3D (ThermoFisher) for amplicon detection.

Statistical Analysis

Fisher’s exact test was used to assess associations between categorical variables. Cohen’s kappa was used to evaluate concordance between the HER2 status of primary tumor and of CTCs. Comparison of CTC counts was performed with Wilcoxon’s test for two groups and Kruskal-Wallis test for three groups. Statistical significance was defined as a two-sided test p < 0.05.

Patient Characteristics and CTC Detection

This study enrolled 47 MBC patients and 40 healthy volunteers. Blood was collected at enrollment for both patients and volunteers and at follow-ups for patients. Nineteen patients had at least 1 follow-up blood draw, of whom 8 had 4 or more, leading to a total of 100 samples. The patients had a median age of 51 (range 34–80) and all had previously been treated. At baseline, CTCs were detected in 40 patients (85.1%), and 51.1% patients (24/47) were considered CTC positive (≥5 CTCs/5 mL blood). CTC count in patients ranged from 0 to 253, with both a median and an interquartile range (IQR) of 6 (Fig. 1a). In contrast, CTC counts in healthy volunteers varied between 0 and 4 (median and IQR 0), with two volunteers having ≥2 CTCs and none being CTC positive. As shown in Table 1, baseline CTC positivity rate tended to be higher in patients of younger age or with histologically HER2-negative, estrogen receptor-positive, progesterone receptor-positive, or luminal B breast tumors, although this trend was statistically significant only for age.

Table 1.

Clinical features and CTC detection of the MBC patients analyzed in this study

 Clinical features and CTC detection of the MBC patients analyzed in this study
 Clinical features and CTC detection of the MBC patients analyzed in this study
Fig. 1.

CTC counts were significantly higher in MBC patients compared with healthy volunteers and in blood samples that accompanied more favorable radiologic evaluations at baseline. a Summary of key statistics of the CTC counts in healthy donors, all patient, and patients with baseline blood samples that accompanied better or poorer treatment response. Better responses were defined as radiographic evaluations of complete response, PR, or SD in which there was a decrease in tumor size by <30%. Poorer responses consisted of PD or SD in which the tumor size remained the same or increased. b CTC counts were significantly higher in MBC patients compared with healthy volunteers and in blood samples accompanying better responses at baseline. P values were calculated with Kruskal-Wallis test due to heteroscedasticity with Benjamin-Hochberg post hoc correction. IQR, interquartile range; MBC, metastatic breast cancer; CTC, circulating tumor cell; PD, progressive disease; PR, partial response; SD, stable disease.

Fig. 1.

CTC counts were significantly higher in MBC patients compared with healthy volunteers and in blood samples that accompanied more favorable radiologic evaluations at baseline. a Summary of key statistics of the CTC counts in healthy donors, all patient, and patients with baseline blood samples that accompanied better or poorer treatment response. Better responses were defined as radiographic evaluations of complete response, PR, or SD in which there was a decrease in tumor size by <30%. Poorer responses consisted of PD or SD in which the tumor size remained the same or increased. b CTC counts were significantly higher in MBC patients compared with healthy volunteers and in blood samples accompanying better responses at baseline. P values were calculated with Kruskal-Wallis test due to heteroscedasticity with Benjamin-Hochberg post hoc correction. IQR, interquartile range; MBC, metastatic breast cancer; CTC, circulating tumor cell; PD, progressive disease; PR, partial response; SD, stable disease.

Close modal

CTC Positivity Was Associated with Increased Tumor Burden

Tumor cell line spike-in and recovery was performed to assess the performance of CTC enumeration. Online supplementary Figure 2 shows representative images of the high-quality detection enabled by the assay. Blood from healthy donors was used as negative controls, and positive controls were prepared from negative controls spiked with MCF-7 or SKBR3 cells. Ten positive and 10 negative controls were subjected to the assay, which achieved 90% sensitivity and 100% specificity, suggesting high performance in CTC detection (online suppl. Table 1).

We then investigated how well CTC levels associated with radiographic evaluations of response to treatment. The blood samples from patients were divided into two groups based on the accompanying radiologic evaluations made at the time of blood draw. The better responses group consisted of samples accompanying evaluations of complete response, PR, or SD in which the tumor size decreased by <30%, whereas the poorer responses included PD and the remaining SD evaluations. At baseline 51.1% (24/47), samples were associated with poorer responses and had significantly higher CTC counts than those in the better responses group (Wilcoxon test p = 0.02), and the latter had slightly but significantly higher counts than the volunteers (averages 3.7 vs. 0.3, Wilcoxon test p < 0.001), further supporting the robustness of the assay. In terms of CTC positivity (≥5 CTCs/5 mL blood), the poorer responses group also showed a significantly higher rate than the better response group (Fisher’s exact test p < 0.001; Table 2). Consistently, radiographic responses and CTC positivity showed moderate consistency (Cohen’s kappa 0.58, 95% confidence interval 0.42–0.74). Using radiographic response as reference, CTC analysis resulted in a sensitivity of 78.0%, specificity of 80.0%, positive predictive value of 79.6%, and a NPV of 78.4%. Together, these results suggested an association between higher CTC count or CTC positivity and a greater likelihood of increased tumor burden.

Table 2.

Numbers of CTC-positive and -negative blood samples in patients with better or poor response determined by radiographic evaluation

 Numbers of CTC-positive and -negative blood samples in patients with better or poor response determined by radiographic evaluation
 Numbers of CTC-positive and -negative blood samples in patients with better or poor response determined by radiographic evaluation

CTC, CEA, and CA153 Showed Generally Consistent Dynamics, although a Few Discrepancies Suggested Higher Accuracy of CTC Count in Monitoring Disease Status

We then used the data from eight patients who underwent five or more serial blood draws to compare the dynamics of CTC count and carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA153), two common serum markers in MBC. As indicated in Figure 2, these markers showed generally consistent dynamic patterns that were also in general agreement with imaging findings. However, there were several contradictions between CTC and one or both serum markers, at which point radiography appeared to agree mostly with changes in CTC level. For example, at C4 in patients 3 and at C5 in patient 9, increase in CTC count disagreed with the drop in CEA level, when the accompanying evaluations were both PD. Also, rises in serum markers at C6 of patient 3 contrasted with a drastic fall in CTC count and an evaluation of PR. Similarly, increases in CTC count at C3 contrasted with dramatic drops in CEA in patient 6 and in CA153 in patients 8 and 9. On the other hand, discrepancies between CTC and radiography were also observed, such as at C7 in patient 6 and at C2 in patient 9, although for fewer times compared with CEA/CA153 dynamics (Fig. 2). These inconsistencies suggested the potential of greater accuracy of CTC count in reflecting the disease status, although more validation is warranted.

Fig. 2.

Dynamic changes in CTC, CEA, and cancer antigen 15-3 (CA153) levels in blood collected at enrollment (C1) and follow-up examinations (C2–C5). dSD, stable disease in which the tumor size did decrease but by <30%. iSD, SD in which the tumor size either remained the same or increased but did not reach progressive disease. PD, progressive disease; PR, partial response; CEA, carcinoembryonic antigen; SD, stable disease; CTC, circulating tumor cell.

Fig. 2.

Dynamic changes in CTC, CEA, and cancer antigen 15-3 (CA153) levels in blood collected at enrollment (C1) and follow-up examinations (C2–C5). dSD, stable disease in which the tumor size did decrease but by <30%. iSD, SD in which the tumor size either remained the same or increased but did not reach progressive disease. PD, progressive disease; PR, partial response; CEA, carcinoembryonic antigen; SD, stable disease; CTC, circulating tumor cell.

Close modal

CTC-HER2 Status Was Moderately Concordant with Tumor-HER2 Status with a High NPV

Next, we gauged the level of consistency between CTC- and tumor-HER2 status. CTC-HER2 positivity was defined as the average CTC-HER2 immunofluorescence intensity >5 times greater than the background after calibration with four cancer cell lines (online suppl. Fig. 3). After exclusion of patients with no CTC or record of tumor-HER2 status, 59 samples from 32 patients were subjected to CTC-HER2 analysis (online suppl. Fig. 3). Compared with those with HER2-negative tumors, patients with HER2-positive tumors had a significantly higher CTC-HER2 positivity rate (Fisher’s exact p < 0.001; Table 3). Using tumor as reference, CTC showed 75.0% sensitivity, 74.4% specificity, 52.2% positive predictive value, and 88.9% NPV in HER2 status determination, suggesting the potential utility of CTC-HER2 status in predicting HER2-negative samples. There was also moderate consistency between tumor- and CTC-HER2 status (Cohen’s kappa 0.43, 95% confidence interval 0.20–0.67).

Table 3.

Numbers of CTC HER2-positive and -negative blood biopsies in groups assigned based on tumor biopsy HER2 positivity

 Numbers of CTC HER2-positive and -negative blood biopsies in groups assigned based on tumor biopsy HER2 positivity
 Numbers of CTC HER2-positive and -negative blood biopsies in groups assigned based on tumor biopsy HER2 positivity

To further validate the CTC-HER2 status, CTCs from select patients were used to estimate HER2 gene copy number via single-cell PCR. Patients with different CTC-HER2 statuses were selected. As shown in online supplementary Table 2, there was promising agreement between HER2 copy number and CTC-HER2 positivity, supporting the validity of the CTC-HER2 status determined with immunofluorescence.

Blood biopsies present several promising advantages over tissue biopsies, among them noninvasiveness and amenity to dynamic monitoring. The promise of CTCs in monitoring disease status and predicting treatment efficacy has led to extensive investigation [7]. Nonetheless, CTC characterization faces several technical challenges. The rarity of CTCs typically necessitates enrichment and may, therefore, suffer from incomplete capture. For example, a comparison of biomarker- and sized-based approaches showed 20–60% inter-assay concordance in CTC counts [14]. A novel microfluidic device using microfilters with canonical-shaped holes has been reported in a proof-of-concept study [16]. The investigators demonstrated the viability of capturing CTCs with this instrument using blood samples from 6 healthy donors and 15 patients with different cancer types, which enabled CTC detection in 66.7% (10/15) patients and in none of the healthy donors. In this study, we set out to provide evidence of the clinical value of this essay in CTC-relevant applications.

We first assessed the association between CTC level and radiographic response and detected significantly more CTCs and higher positivity rate in samples that corresponded to poorer responses than those accompanying better responses (Fig. 1b; Table 2). There was also a level of agreement bordering substantial consistency between CTC positivity and radiographic evaluation (Cohen’s kappa 0.58). In addition, serial blood biopsies from eight patients showed CTC-level changes generally agreeing with tumor burden markers CEA and CA153, although the occasional discrepancies supported higher accuracy of CTC count in reflecting the disease status. These results suggested CTC positivity as a potential marker for monitoring treatment responses in MBC, which was consistent with a meta-analysis of studies that mostly used the Cellsearch assay [17]. For CTC-HER2 characterization, we developed a immunofluorescence-based method and incorporated it in the assay workflow to allow CTC enumeration and HER2 status determination in one sitting. CTC-HER2 positivity rate was significantly higher in patients with HER2-positive tumors, while there was also a moderate concordance between CTC- and tumor-HER2 status (Cohen’s kappa 0.43). CTC-HER2 status was further corroborated by HER2 copy number measurements in select samples (online suppl. Table 2).

The 51.1% (24/47) baseline CTC positivity in this study was comparable to that reported in a pooled analysis (47%) [4]. Nonetheless, less blood was used in this study (5 mL vs. 7.5 mL for Cellsearch assay), which may suggest more sensitive CTC detection. Consistently, the 39.0% (23/59) CTC-HER2 positivity rate was much higher than previously reported ones such as 20% and 22% [8, 15]. Moreover, CTC- and tumor-HER2 status appeared more consistent than previously reported. Specifically, of the patients evaluated for CTC-HER2 status, 81.8% (9/11) histologically HER2-positive patients had CTC-HER2-positive samples, and 76.2% (16/21) histologically HER2-negative patients had CTC-HER2-negative samples. In contrast, of three studies that used the Cellsearch assay, one reported a sensitivity of 62.1% (36/58) in MBC patients for HER2 status determination [9] and another reported significantly different tumor- and CTC-HER2 status [8]. The third study set a less stringent threshold for CTC-HER2 positivity (HER2-to-background immunofluorescence ratio of 3.5 instead of 6 in this study) and detected ≥1 HER2-positive CTC/7.5 mL blood in 35.9% (14/39) MBC patients [18]. Possible sources of the different CTC-HER2 positivity rates include patient cohorts and the intrinsic nature of the cells defined as CTCs by different assays, although conclusive evidence from direct comparisons is awaited.

Apart from the promising performance, there are several limitations in this study. Conclusions were drawn from 100 biopsies collected from a small cohort of 47 patients. Moreover, the cohort was not uniform in terms of treatment regimens and the time points of blood draws. These limitations suggest a preliminary nature of our results. The study was also limited by a lack of data on survival outcomes, as survival analysis would permit insights into the value of this CTC assay in predicting survival outcomes. Given the established difference tumor- and CTC-HER2 expression, one interesting question that awaits answers is whether the CTC-HER2 status determined by this assay could lead to greater accuracy in predicting the efficacy of HER2-targeted therapy. A third limitation is the requirement of cytokeratin expression for CTC positivity, which leads to the risk of not capturing CTCs that do not express cytokeratin at detectable levels. To address these limitations, future investigations will be conducted in larger cohorts documenting survival outcomes and with a modified assay that also captures the cytokeratin-negative CTCs.

In summary, we provide preliminary evidence of the clinical validity of a previously reported microfluidic device for CTC capture. CTC count and positivity rate were significantly higher in patients with worse radiographic responses. CTC-HER2 status was moderately consistent with tumor-HER2 status and showed greater sensitivity than previously reported. Larger scale studies with modified CTC definitions are warranted to further characterize the clinical value of this assay.

We thank the patients and volunteers in this study and their families for their encouraging support.

This study was approved by the Institutional Review Board of Guangzhou Red Cross Hospital (No. 2020-208-02). All patients had provided written informed consent for participating in the study.

CH is a full-time employee of Anfang Biotech Co., Ltd.

No funding was provided for this study.

X.W., C.H., and Y.W. conceived and designed the study. X.W., C.H., and J.L. collected the data. X.W., C.H., and J.Y. analyzed and interpreted the data. X.W. and C.H. wrote the manuscript. J.L., J.Y., Z.P., and Y.W. provided critical review of the manuscript. All authors approved the final version of the manuscript and are accountable for all aspects of the work.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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