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The prognostic value of chemotherapy or endocrine therapy choice according to circulating tumor cell count in HR+HER2− metastatic breast cancer: a retrospective study

Background Circulating tumor cell (CTC) count have prognostic role for metastatic breast cancer (MBC). No clear biomarkers can guide selection of chemotherapy (CT) or endocrine therapy (ET) in 1st-line setting of hormone-receptor positive and human epidermal growth factor receptor 2 negative (HR+HER2−) MBC. The present study investigated the prognostic role CT or ET according to the CTC count in HR+HER2− MBC. Methods We consecutively collected the data of 53 HR+HER2− MBC patients who received 1st-line CT or ET, who had CTC count detected by our peptide-based nanomagnetic CTC isolation system (Pep@MNPs) from January 2014 to December 2015. The clinicopathological characteristics according the CTC count and 1st-line ET vs. CT were compared. Follow-up was conducted every 6 months. The primary endpoint was progression-free survival (PFS) and overall survival (OS). A Cox regression analysis was conducted to determine the prognostic roles of CTC and 1st-line therapy of ET vs. CT for PFS and OS. Results The median CTC count of the 53 patients was 2 (range, 0–18). The clinicopathological characteristics of the patients in the CTC count <2 group and the CTC count ≥2 group were similar. The patients with a CTC count <2 had a significantly longer PFS than those with a CTC count ≥2 (P=0.005, hazard ratio =4.138, 12.1 vs. 7.1 months). The patients who received CT had a significantly longer PFS than those who received ET (P=0.041, hazard ratio =2.721, 9.9 vs. 7.2 months). In the CTC count ≥2 group, the patients who received CT had a significantly longer PFS than those who received ET (P=0.048, hazard ratio =2.475, 8.7 vs. 6.3 months). In the CTC count <2 group, there was no significant difference in PFS between the CT and ET groups (P=0.071). Additionally, the CTC count had no significant effect on OS (P=0.116, hazard ratio =2.391, 54.2 vs. 34.2 months). Conclusions The present study showed that CTC count determined by the Pep@MNP system confirmed the prognostic value in the HR+HER2− MBC patients. And it might be helpful in choosing a 1st-line treatment of CT or ET for HR+HER2− MBC patients.


Introduction
Circulating tumor cells (CTCs) are cancer cells that circulate in the peripheral blood, originate from the primary tumor or metastasis, and are responsible for distant metastasis (1). The prognostic value of CTC counts as assessed by the Cell Search System for metastatic breast cancer (MBC) patients was first demonstrated in 2004 (2). Since then, several studies had found similar results (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). A pooled analysis in 2014 confirmed that a CTC count of ≥5 cells per 7.5 mL blood is an independent prognostic factor for worse progression-free survival (PFS) and overall survival (OS) in MBC patients compared with patients with a CTC count of less than 5 per 7.5 mL at baseline (13).
Our research group developed a novel nanotechnologybased CTC detecting platform, called Pep@MNPs, which has a strong CTC capture efficiency because of the high affinity between the self-designed EpCAM recognition peptide and the EPCAM molecule (KD/ 1.98×10 −9 mol/L) (14). In a previous study, we found that the CTC enumerations at the baseline and the 1st clinical evaluation had the best prognostic value for MBC patients. Further, we found that the CTC count had prognostic value for PFS in the patients with stable disease (SD) (15).
The primary goal of MBC treatment is to prolong patient survival and improve their quality of life. A variety of factors are taken into account to produce the most effective results with minimal side effects (16). For patients with hormone-receptor positive and human epidermal growth factor receptor 2 negative (HR + HER2 − ) MBC, endocrine therapy (ET) and chemotherapy (CT) are both 1st-line treatment options. ET has the similar a survival benefit and response rate to CT; however, ET requires more time to reduce the tumor. Thus, in the absence of any clinical evidence of rapid invasive disease or visceral crisis, ET is usually the preferred option (17,18). However, to date, no clear biomarkers have been identified that can guide clinicians in the section of treatment; thus, different doctors may make different recommendation for the same patient. Therefore, the prognostic factors of ET and CT are highly needed. The outcome of the treatment is associated with molecular characteristics of tumor cells, which is determined on the primary tumor. However, the characteristics of metastatic tumor will change over time in a certain proportion of patients (19). However, tissue biopsy from metastatic lesions is invasive and impossible because of inaccessible lesions. CTC is an attractive alternative noninvasive method to test the characteristics of metastatic tumor. Printz et al. reported that if the treatment choice of HR + HER2 − MBC patients was based on the CTC count rather than clinical experience, the risk of death was significantly reduced by 35% (20).
The present study sought to assess the prognostic value of 1st-line treatment of CT or ET for HR + HER2 − MBC patients in the CTC count high and low group determined by our Pep@MNP system. We present the following article in accordance with the REMARK reporting checklist (available at https://atm.amegroups.com/article/ view/10.21037/atm-22-3797/rc).

Patients and study design
We retrospectively collected the data of 53 HR + HER2 − MBC patients at the Peking University Cancer Hospital and Institute from January 2014 to December 2015 in continuous method. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Medical Ethics Committee of Peking University Cancer Hospital and Institute (approval No. 2013KT29) and informed consent was taken from all the patients.
To be eligible for inclusion in this study, the patients had to meet the following inclusion criteria: (I) have been diagnosed with HR + HER2 − MBC; (II) be undergoing 1stline therapy (CT or ET); (III) have an expected survival time of >3 months; (IV) have measurable lesions; and (V) have provided written informed consent. The patients lost follow-up before progression were excluded. In this study, estrogen receptor (ER) and/or progesterone receptor (PR) positivity was defined as ≥1%. value ≥20%. HER2 was defined as negative with an immunohistochemical score of 0, 1+ or 2+ with fluorescence in situ hybridization (FISH) (−). PFS was defined as the time interval from the start of 1st-line treatment to cancer progression, mortality from any cause. OS was defined from the start of 1st-line treatment to mortality from any cause. All patients were followed up every 6 months by telephone or electronic medical records.
The 53 patients' CTCs were detected and their clinicopathological information was recorded before the start of treatment. We collected 8.0 mL of peripheral blood from each patient in the CellSave ® (Immunivest Corporation, Wilmington, DE, USA) collection tube. The samples were processed within 96 hours at room temperature, and 2.0 mL blood was used for the CTC enumeration using the Pep@MNP technique. The Pep@MNP technique has been described previously (14). The previous report demonstrated that the Pep@MNP technique has a comparable capture efficiency (reaching above 90%) and purity (reaching above 93%) for breast, prostate and liver cancers from spiked human blood. Further, the captured cells maintain their viability for further molecular biological analyses.

CTC isolation and enumeration
The CTCs were counted using the Pep@MNP method as previously described (14). In brief, iron oxide magnetic nanoparticles (MNPs) was coated with EPCAM recognition peptide via a biotineavidin interaction. For ease of detection, 2.0-mL peripheral blood samples were mixed with 5.0 mL of pre-vortexed Pep@MNPs (10 mg Fe/mL). Then the samples were incubated with gentle shaking at 37 ℃ for 30 min. The Pep@MNPseCTC complexes were subsequently isolated and washed with phosphate buffered solution at least 3 times under a magnetic field. The captured CTCs were then stained with 4',6-diamidino-2phenylindole (DAPI), CD45-phycoerythrin and cytokeratin (CK)19-fluorescein isothiocyanate. Then the ZeissVert A1 fluorescent microscope (Carl Zeiss Microscopy GmbH, München, Germany) was used for identification and enumeration of the CTCs.

Statistical analysis
The continuous variables are presented as the median and range, while the categorical variables are presented as the number and frequency. The chi-square test or Fisher's exact test was used to compare the categorical variables. The PFS and OS were calculated using the Kaplan-Meier method and compared using the log-rank test. The multivariate Cox proportional risk regression model was used to analyze the potential confounding factors for PFS and OS. The interactive effect between CTC and treatment method was also analyzed in the multivariate Cox proportional risk regression model. All the statistical data were examined using SPSS 20.0 software package. A two-sided P value <0.05 was considered statistically significant.

Patient characteristics
A total of 53 patients were included in the study. The patients had a median CTC count of 2 (range, 0-18). The baseline characteristics of the patients with a CTC count <2 (18 cases, 34%) or a CTC count ≥2 (35 cases, 66%) are summarized in Table 1. The patients in the 2 groups had similar clinicopathological characteristics [in terms of age, pathological type, grade, stage at surgery, ER expression, PR expression, Ki-67 expression, adjuvant ET, diseasefree survival (DFS), and metastatic sites]. The patients who received neoadjuvant or adjuvant CT had a higher CTC count at the baseline than those who did not (P=0.015).

The relevance of the baseline CTC count and PFS or OS
T h e m e d i a n f o l l o w -u p t i m e w a s 3 8 . 5 ( r a n g e , 4.2-87.8) months. All the patients had disease progression or died during the follow-up period. In total, 6 patients were lost to follow-up of OS and 33 patients died. The median PFS and OS of all the patients was 9.0 [95% confidence interval (CI): 6.4-11.7] months and 38.8 (95% CI: 33.3-44.3) months. Next, we analyzed the influence of age, pathological grade, subtype (luminal A-like or luminal B-like breast cancer), visceral metastasis, the number of metastasis sites, 1st-line therapy (CT or ET), and the CTC count on PFS and OS (see Tables 2,3). The patients who received CT (38 patients, 71.7%) had a significantly longer median PFS than those who received ET (15 patients, 28.3%) in the univariate and multivariate analyses [P=0.041, hazard ratio =2.721, 9.9 vs. 7.2 months; see Figure 1A]. The median PFS of the patients with a CTC count <2 was significantly longer than that of the patients with a CTC count ≥2 (P=0.005,  Figure 1B). In a further analysis of OS, we found that the patients with no visceral metastasis had a significantly longer OS than those with visceral metastasis (P=0.011, hazard ratio =4.300, 64.3 vs. 34.2 months; see Figure 1C). However, the CTC count had no significant effect on OS (P=0.116, hazard ratio =2.391, 54.2 vs. 34.2 months; see Figure 1D).

The CTC count predicted the 1st-line choice of CT or ET
To explore the predictive value of the CTC count on the choice of CT or ET, we analyzed the PFS of CT and ET in different CTC count groups. The clinicopathological characteristics did not differ significantly between the CT and ET groups (P>0.05; see Table 4). The median PFS of patients who received CT or ET did not differ significantly in the CTC count <2 group (P=0.071, 16.0 vs. 11.3 months; see Figure 2A). However, the patients who received CT had a significantly longer PFS than those who received ET (P=0.035, 8.7 vs. 6.3 months) in the CTC count ≥2 group ( Figure 2B). Next, we analyzed the effects of the other potential prognostic factors (i.e., age, pathological grade, subtype, visceral metastasis, and number of metastatic sites) on PFS in the CTC count ≥2 group with a multivariate Cox proportional risk regression model. The 1st-line CT or ET significantly affected PFS in the CTC count ≥2 group (hazard ratio =2.475, P=0.048; see Table 5).
We further analyzed the prognostic value of the CTC count in the patients with or without visceral metastasis. In the group with visceral metastasis, the median OS of the patients with a CTC count <2 was significantly longer than that of patients with a CTC count ≥2 (P=0.008, 38.8 vs. 25.3 months; see Figure 3A). However, in the group without visceral metastasis, the CTC count had no significant effect on OS (P=0.106, -vs. 63.1 months; see Figure 3B). The median OS of the CTC count <2 group in the patients without visceral metastasis has not reached (only 3 patients of the 8 patients died).

Discussion
In this study, we found that CT led to a significantly longer PFS than ET for patients with HR + HER2 − MBC who had a baseline CTC count ≥2. Our results support those of a phase 3 randomized controlled trial (STIC CTC) that were presented at the 2018 San Antonio Breast Cancer Symposium in 2018 under the direction of Dr. Bidard (21). In a previous study, we showed the robust prognostic value of the CTC count using our Pep@MNP system both at the baseline and in combination with the 1st clinical evaluation time point for PFS. In the current study, we further explored whether the CTC count could be used to guide the treatment choice for patients with HR + HER2 − MBC.
In the pooled analysis, the prognostic value of the CTC count for MBC patients has been confirmed by the highest level of evidence (13). Previous findings have consistently indicated that a baseline CTC count higher than the cutoff value of 5 cells per 7.5 mL of blood was associated with a poor prognosis (both in terms of PFS and OS) (2,9,22,23). Patients with HR + HER2 − MBC have their own characteristics. A previous study also reported that the CTC count is closely associated with the molecular classification of BC (24). Specifically, the study reported that patients with the luminal-like HER2 − subtype were less likely to be CTC positive than those with HER2 + or triple-negative breast cancer (TNBC) (24). CTC positivity was also shown to be an independent prognostic factor for shorter PFS and OS in HR + HER2 − patients (23). Thus, we specifically analyzed the HR + HER2 − group. The positivity of CTC was 69.8% (37/53), which showed that the Pep@MNP system had a comparable capture efficiency to that of the Cell Search system (25). In our study, we also found that the CTC count had significant prognostic value for PFS but not OS in the all patients. This study sought to explore the role of the CTC count in guiding treatment selection for metastatic patients.
The STIC CTC trial was the 1st study which reported that CTC could guide treatment choice for HR + HER2 − MBC patients (21). This phase 3 non-inferiority study was designed to treat 778 enrolled patients who were randomly allocated to groups based on either the physician's choice or the CTC count-driven choice for their 1st-line treatment. The patients' CTC counts were assessed using the Cell Search system. In the CTC count-driven arm, patients with ≥5 CTCs were treated with CT, and patients with <5 CTCs were treated with ET. Patients in the 2 arms had a similar median PFS; that is, 13.9 (95% CI: 12.2-16.3) months in the physician's-choice arm and 15.5 (95% CI: 12.7-

17.3) months in the CTC count-driven arm.
For the patients with discordant treatment recommendations (i.e., those that were clinically low-risk but CTC-high and clinically high-risk but CTC-low), PFS and OS were longer in the patients receiving CT than ET, which indicates that the CTC count had better predictive value than other clinical characteristics. In our study, the patients with a CTC count ≥2 who received 1st-line CT had a significantly longer PFS than those who received 1st-line ET. In the CTC count <2 group, the median PFS of the patients who received CT or ET did not differ significantly. The clinical benefit rate (CBR) [complete response (CR) + partial response + SD] in the CT group was 84.2% (32/38) and the patients achieving CR, partial response, or SD were switched to maintenance hormone therapy, which might account for the longer PFS in the CT group (P=0.031).
Previous studies have shown that CTCs are highly enriched with mesenchymal markers. Additionally, mesenchymal CTCs are associated with the therapeutic  results of MBC studies (26). The total CTC counts did not differ significantly between the luminal-like breast cancer and TNBC patients. The CTCs of luminal-like breast cancer mainly comprise the epithelial cell type, while those of TNBC predominantly comprise the mesenchymal cell type (27). In our study, we did not detect the mesenchymal phenotype of CTCs, which may have more prognostic value for MBC patients. However, the STIC CTC trial assessed CTCs using the Cell Search system, which also detected the epithelial type CTCs.
This study had several limitations. First, the sample size was small. Second, most of the patients received CT rather than ET. Third, the 1st-line ET for this group of patients was administered in combination with the CDK4/6 inhibitors; however, CDK4/6 inhibitors were not used in this study. Finally, the Pep@MNP CTC detection platform failed to identify mesenchymal-like CTCs. Thus, the molecular characterization of CTCs was not performed in our study; however, this may have predictive value in the selection of treatments.

Conclusions
The present study showed that CTC count determined by the Pep@MNP system confirmed the prognostic value in the HR + HER2 − MBC patients. And it might be helpful in