Evaluation of circulating tumor cells as a prognostic biomarker for early recurrence in stage II–III breast cancer patients using CytoSorter^® system: a retrospective study

Introduction

Breast cancer (BC) is the most frequent cancer among women both in China and worldwide, accounting for 17.1% of all cancer and 8.16% of cancer-related death in women in China in 2015 (Bray et al., 2018; Zheng et al., 2019). Primary treatment remedy for early BC is surgery and radiation, followed by systemic treatments, such as hormone therapy, chemotherapy, targeted therapy or combined multiple therapies (Goldhirsch et al., 2013). A precise treatment plan is determined by the molecular subtype of tumor and locoregional tumor load (Huang & Yin, 2018). As for patients with triple-negative (three receptors negative, including ER, PR and HER2) and HER2-positive (human epidermal growth factor receptor 2) early BC, a neoadjuvant therapy might be optional (Huang & Yin, 2018). Although it is believed that BC in early phase is potentially curable, still about 30% of patients develop disease recurrence (Ayala de la Peña et al., 2019). The timing of relapses fluctuates greatly, influenced by patients clinicopathological characteristics as well as received therapies (Takeuchi, Tsuji & Ueo, 2005). A relapse occurring within 5 years of initial treatment is commonly known as “early recurrence”, while appearing five or more years later is referred to as “late recurrence” (Takeuchi, Tsuji & Ueo, 2005). Late recurrence is more observed in patients with hormone receptor-positive breast cancer and is less common among patients with hormone receptor-negative diseases (Pan et al., 2017). A study with longitudinal 24 years of follow-up concerning the annual hazard rates (HR) of BC relapses has shown that the annualized HR of recurrence was highest within the first 5 years (10.4%), with a peak between years 1 and 2 (15.2%) in spite of hormone receptor status (Colleoni et al., 2016). However, beyond 5 years, patients with ER-positive (estrogen receptor) diseases had higher HR of recurrence and annualized HR remained elevated and stable, reaching a second peak between years 16 and 18 (Colleoni et al., 2016). A recent study showed how adjuvant chemotherapy would affect recurrence of BC. HR of recurrence for patients with surgery only exhibited two early sharp peaks at 9 and 33 months, a wide intermediate one spanning from about 50 to 90 months and a late peak at 115–120 months (Demicheli et al., 2020). When adjuvant chemotherapy was introduced, HR for early recurrence was reduced, leaving an early residual peak at about 18 months and two small intermediate peaks at about 50 and 80 months (Demicheli et al., 2020).

Circulating tumor cells (CTCs) refer to tumor cells detaching from the primary tumor or metastatic lesions and then entering into bloodstream (Fabisiewicz et al., 2020; Massagué & Obenauf, 2016). CTCs represent the undergoing process of metastases (Massagué & Obenauf, 2016). Therefore, in the updated TNM (tumor-node-metastasis) staging for BC, the National Comprehensive Cancer Network (NCCN) guidelines have added a new M0 (i+) category that is defined as “no clinical or radiographic evidence of distant metastases, but existence of detected tumor cells in the circulation fluids” (Gradishar et al., 2018). Clinical application of CTCs has been extensively exploited in BC. CTCs are good prognostic markers to predict patients survival outcome, and thus can be used to risk stratify patients and guide therapy (Fabisiewicz et al., 2020; Cristofanilli et al., 2005; Liu et al., 2009; Hwang, Hwang & Miyamoto, 2016). CTCs can be used as a diagnostic tool for screening and early diagnosis of BC and as a surveillance tool for real-time monitoring of disease progression and patients response to treatment (Jin et al., 2020; Yan et al., 2017). Many technologies have been developed to detect CTCs. However, for lacking a validated standard of CTC results from different platforms, CTC is not yet considered as a standard routine diagnostic tool used in clinical practice. Different CTC detection methodologies have different sensitivities, leading to varying CTC cut-off values for the same clinical application (Krawczyk et al., 2013). CellSearch^® is so far the only CTC system approved by Food and Drug Administration (FDA). But CellSearch^® had in general low CTC detection rates in BC, around 40–50% in metastatic BC and less than 30% in early BC (Eroglu, Fielder & SomLo, 2013). Based on the CellSearch^® data on BC, the 8^th edition of American Joint Committee on Cancer (AJCC) Cancer Staging Manual for BC has recognized CTC as a prognostic factor (Amin et al., 2017). Early BC patients with ≧1 CTC per 7.5 mL of blood or metastatic BC patients with ≧5 CTC per 7.5 mL of blood are considered as patients with higher risks for poor prognosis (Amin et al., 2017). With the improvement of new technologies, CTC detection has become more sensitive. As there is currently no prevailing CTC detection system, a standard must be established to compare results from different CTC detection platforms. With the ease of sample manipulation, the microfluidic-based strategies for CTC enrichment usually have a better sensitivity and specificity for CTC detection (Jin et al., 2020; Stott et al., 2010). CytoSorter^® (Hangzhou Watson Biotech, Hangzhou, China), a microfluidic-based immuno-capture CTC platform, has been used in this study for CTC detection. CytoSorter^® technology employs the positive selection utilizing a streptavidin nanoarray chip. The microfluidic chip can be coated with any biotin-labeled capture antibody to enrich desired cells. The most common CTC capture antibody is anti-epithelial cell adhesion molecules (EpCAM) CytoSorter^® CTC system has been validated in BC (Jin et al., 2020), head and neck cancer (Zheng et al., 2019), pancreatic cancer (Wei et al., 2019) and lung cancer (Xie et al., 2021). CytoSorter^® has CTC detection rates over 90% in stage II–III BC patients (Jin et al., 2020), much higher than the CellSearch^® system (Eroglu, Fielder & SomLo, 2013). With a CTC cut-off of two per four mL of blood, CytoSorter^® has a sensitivity and specificity of 76.56 % and 95.4%, respectively, for diagnosing BC (Jin et al., 2020).

It is reported that a single positive CTC result with CellSearch^® 5 years later after curative treatment could be still considered as an independent prognostic factor for late recurrence in ER-positive BC (Sparano et al., 2018). In this retrospective study, we aimed at evaluating whether preoperative (pre-op) CTCs could be used as a prognostic biomarker for early recurrence of disease in BC, and at determining the CytoSorter^® CTC cut-off value for the identification of high-risk BC patients for early recurrence.

Materials and Methods

Study design

Studies have shown that CTC enumerations prior to therapy, after initial treatment and during follow-ups could be all considered as a prognostic factor in metastatic BC (Fabisiewicz et al., 2020; Cristofanilli et al., 2005; Liu et al., 2009; Hwang, Hwang & Miyamoto, 2016). This study aimed to determine whether pre-op CTC could be used as a biomarker to predict early recurrence of disease in BC patients with resectable tumors. The first peak of early recurrence usually occurs in around 9 months after curative surgery and might be delayed to 18 months in patients with adjuvant therapies. Therefore, stage II-III BC patients who performed pre-op CTC detection prior to any treatment, and had a curative mastectomy or lumpectomy between January and May 2018 and follow-up records until December 31^st 2019 were retrospectively enrolled in this study as shown in Fig. 1. Relapses during follow-up were recorded. Correlations of relapse and survival with CTCs, patients clinicopathological characteristics, and received adjuvant therapies would be analyzed. BC patients inclusion criteria were as follows: (1) female patients aged between 18 to 75 years; (2) patients who had CTC detection prior to surgery; (3) stage II or III BC patients with a resectable tumor; (4) patients who had surgery as the primary treatment; (5) patients who had negative history of other malignancy within five years prior to CTC detection; (6) patients were treatment-naive before CTC detection. Patients of the following descriptions were excluded and rejected from the study: (1) patients who were breast-feeding or pregnant at time of CTC detection or surgery; (2) patients who lost contact during follow-up; (3) patients with ductal carcinoma in situ (DCIS), stage I tumor, or metastatic cancer; (4) patients who had other conditions which were considered not suitable for the study.

Results

Univariate analysis for RFS and D-RFS

The significance of patient clinicopathological characteristics with RFS and D-RFS are shown in Table 1. Only cancer stage and CTCs were significant for RFS (P = 0.033 and 0.013, respectively). Time to relapse ranged between 128 and 566 days. Six patients showed recurrence within 1 year after surgery. Mean (median) of follow-up was 518 (536) days. Patients with ≧4 CTCs per four mL of blood showed a significantly reduced RFS compared to patients with <4 CTCs (log-rank P = 0.013). As for D-RFS, only adjuvant radiation therapy and CTCs showed significance (log-rank P = 0.047 and 0.029, respectively). The 2-year RFS and D-RFS Kaplan-Meier survival curves are shown in Fig. 2. Patients with <4 CTCs had RFS and D-RFS of 85.2% and 88.9%, respectively, while patients with ≧4 CTCs had reduced RFS and D-RFS of 44.4% and 55.6%, respectively. When CTC cut-off was set to four, CTC had a sensitivity and specificity of 55.6% and 85.2%, respectively, for relapse. Patients with stage III tumors had a reduced RFS of 42.9% compared to stage II patients (82.8%, log-rank P = 0.033).

Discussions

Studies have shown that CTCs could be considered as an independent prognostic factor for progression-free and overall survival (PFS and OS) in metastatic BC (Cristofanilli et al., 2005; Liu et al., 2009). In the 8^th edition of AJCC Cancer Staging Manual for BC, it is written that with ≧1 and ≧5 CTCs per 7.5 mL of peripheral blood indicate poor prognosis in early and metastatic BC, respectively (Amin et al., 2017). Furthermore, a study concerning pre-op CTC enumeration and prognosis showed as well that an increased risk for distant recurrence and BC-related death is associated with the presence of pre-op CTCs in BC patients (Franken et al., 2012). Most studies investigating CTCs and recurrence in BC had follow-up time usually longer than 5 years (Cristofanilli et al., 2005; Liu et al., 2009; Stott et al., 2010; Franken et al., 2012). They were more looking at the overall relapses and late recurrence. Results of these studies indicated that CTCs could be used as a prognostic factor for RFS, D-RFS and late recurrence (Cristofanilli et al., 2005; Liu et al., 2009; Stott et al., 2010; Franken et al., 2012). The first peak of early recurrence of BC occurs within 2 years after initial treatment (Colleoni et al., 2016; Demicheli et al., 2020). The aim of this study was to evaluate the use of pre-op CTCs as prognostic marker to identify high-risk BC patients for early recurrence. Our results show that CTC with cut-off value of four with CytoSorter^® system is an independent prognostic factor for 2-year RFS and D-RFS, suggesting that CTCs can be used as a predictor for early recurrence of disease in BC.

A CTC cut-off value of four was found in our study for the identification of high-risk patients for early recurrence. As different CTC capturing systems have varying sensitivities, different CTC cut-off values for recurrence were reported. Franken et al used CellSearch^® and found a CTC cut-off value of 1 per 30 mL of blood can be used as a predictor for recurrence, distant recurrence and BC-related death (Franken et al., 2012). Sparano et al found that using CellSearch^®, CTCs with a cut-off value of 1 per 7.5 mL could be used to predict the late recurrence in ER-positive BC patients (Sparano et al., 2018). CTC cut-off values depends on patients status, clinical applications and CTC detection methods. The cut-off value would be higher in patients at advanced tumor stage, as AJCC staging manual uses CTC cut-off values of 1 and 5 per 7.5 mL of blood, respectively, in early and metastatic BC for prognosis (Stott et al., 2010). Same system may use different CTC cut-off values for different clinical applications. The previous study with CytoSorter^® system showed that CTCs with a cut-off value of two per four mL of blood could be used as a diagnostic aid for diagnosing BC with a sensitivity and specificity of 76.56% and 95.4%, respectively (Jin et al., 2020). However, results of this study show that the cut-off value of pre-op CTCs for early recurrence in BC with CytoSorter^® system should be four CTCs per four mL of blood. Different CTC systems use different strategies to detect CTCs, thus resulting in different sensitivities regarding CTC detection rates. It is reported that CellSearch^® has CTC detection rates less than 40%, while CytoSorter^® has CTC detection rates more than 80% in non-metastatic BC (Eroglu, Fielder & SomLo, 2013; Jin et al., 2020). Although CellSearch^® and CytoSorter^® EpCAM epithelial CTCs kit are both meant to capture the EpCAM-positive epithelial CTCs, they should have different CTC cut-off values for predicting recurrence. As CytoSorter^® is more sensitive than CellSearch^®, it should have a higher CTC cut-off value for poor prognosis. Patients enrolled in this study were at either stage II or stage III, in between early and metastatic BC. According to the AJCC staging manual, the CTC cut-off value for poor prognosis in enrolled patients should be in between one and five per 7.5 mL of blood. And we found a CTC cut-off value of four per four mL of blood for early recurrence. Four CTCs per four mL of blood equals to 7.5 CTCs per 7.5 mL of blood, which is in line with our hypothesis that CytoSorter^® should have a higher CTC cut-off value for poor prognosis than CellSearch^®.

Our results showed that only CTC was associated with 2-year RFS and D-RFS, while patient demographics and clinical characteristics, and adjuvant therapies were not. recurrence were surprisingly more common in patients receiving adjuvant therapy. It may be due to the small sample size of the included patients, or adjuvant therapy might be more effective in reducing late recurrence rather than early recurrence. Also, patients requiring adjuvant therapy are usually patients at higher risks for disease progression. Therefore, they might more easily develop early recurrence.

Some BC patients are considered as high-risk patients for they develop recurrence easily after conventional treatment. To improve the curative rate of BC, a precise stratification of patients that will benefit from different customized therapies is required. Recurrence of disease may originate from the minimal residue disease of a primary tumor, or from another hidden lesion site which was not discovered at diagnosis (Klein, 2009). Micrometastasis in the bone marrow is a high-risk factor for disease recurrence, and can be found in about 30% of BC patients (Braun et al., 2000; Braun et al., 2005). However, screening for occult metastatic tumor cells in the bone marrow is not yet considered as a standard procedure in clinical routine due to being too invasive (Fehm et al., 2006). Routinely used biomarkers for discriminating between low- and high-risk patients include serum tumor makers array and histological staining of biopsy samples for ER, PR, HER2 and Ki67 (Amin et al., 2017). The results are sometimes not so satisfying, for distant metastasis may cause deaths in some low-risk patients, whereas some high-risk patients would survive for decades (Fabisiewicz et al., 2020; Cristofanilli et al., 2005). An accurate method to stratify patients with different risks is still in need. Detecting tumor cells in the blood is a promising alternative for it is non-invasive and can be performed frequently. It is reported that tumor cells can be released into bloodstream even when the tumor lesion is smaller than a sesame and hardly detected by the routine used medical imaging techniques (Hu et al., 2019). Therefore, CTCs can be used as a dynamic monitoring tool to tell the presence of recurrence even before imaging methods can detect. Our results indicate that pre-op CTC detection can be used as well to predict early recurrence of disease, therefore enabling physicians to identify high-risk patients and provide a different enhanced treatment to increase their curative rate. When CTC cut-off was set to four, CTC had a sensitivity and specificity of 55.6% and 85.2%, respectively, for early recurrence. However, the sensitivity and specificity are not quite satisfying. Pre-op CTC detection should be combined with other prognostic evaluations, such as ER, PR, HER2 and Ki67, to better stratify patients with different risks.

Taken together, CTCs can be used as a prognostic factor and monitoring tool for recurrence. The major drawbacks of this study are that this is a retrospective study with limited sample size, too heterogeneous sample and short follow-up. Only 36 BC patients with diverse background were included for analysis and the median follow-up was 518 days. Patients heterogeneity led to small sample sizes in each sub-group and thus resulted in some misleading results without statistical significance. A longer follow-up may have increased the percentage of recurrence. Furthermore, most patients had only performed the CTC detection once before surgery. Since it is believed that minimal residual disease (MRD) is the key to recurrence (Lin, Orozco & Hoon, 2018), it would be more interesting to look for the minimal residual disease by postoperative CTC detection.

Conclusions

Results of this study show pre-op CTC detection could be used as an independent prognostic factor for 2-year RFS and D-RFS. A CytoSorter^® CTC cut-off value of four per four mL of blood is found to identify high-risk BC patients for early recurrence. Still a prospective study with a larger patient population with more homogeneous background and longer follow-up is needed to verify our findings.

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