Prognostic value of circulating tumor cells associated with white blood cells in solid cancer: a systematic review and meta-analysis of 1471 patients with solid tumors

Metastasis remains the leading cause of cancer-related mortality. Circulating tumor cells (CTCs), which are shed by either a primary tumor or metastasis into the blood and lymphatic systems, are precursors of metastasis in various solid cancers [1, 2]. While coping with a new and challenging microenvironment, newly disseminated cancer cells may be particularly vulnerable to immune surveillance [3]. Indeed, CTCs face a hostile environment within the bloodstream. To colonize distant organs, CTCs must overcome many obstacles, including evading immune defenses, adapting to supportive niches, infiltrating foreign tissue, surviving as latent tumor-initiating seeds, and eventually emerging to replace host tissue [3, 4]. Under particular conditions, CTCs may be found within the bloodstream in clusters with nonmalignant cells such as white blood cells (WBCs), mesenchymal stem cells (MSCs), cancer-associated fibroblasts (CAFs), and platelets [5, 6]. CTC-WBC clusters act as "hitchhikers" and are transported throughout the body by neutrophils via various mechanisms [7]. For example, neutrophils aid in tumor metastasis by deploying neutrophil extracellular traps (NETs) containing nuclear DNA, which effectively entrap CTC-WBC clusters and facilitate the progression of cancer [8]. More importantly, neutrophils play a crucial role in modulating cell cycle progression and promoting the metastatic capacity of CTCs through their systemic interaction with CTCs [9].

The prognostic value of the CTC-WBC cluster, a combination of “soil” and “seed”, has been demonstrated in multiple malignant solid tumors [10, 11]. According to the 8th edition of the American Joint Committee on Cancer (AJCC) cancer staging manual, the presence of CTC-WBCs is deemed a negative predictor for the prognosis of patients with primary or metastatic breast cancer [12]. However, some studies have indicated that preoperative CTC-WBCs do not correlate with progression-free survival (PFS) [13]. Therefore, to determine the prognostic value of CTC-WBCs and to interpret the results of available studies statistically, we performed this meta-analysis to evaluate the diagnostic accuracy and prognostic value of testing for CTC-WBC clusters in several solid cancers, including hepatocellular carcinoma (HCC) [11, 14], renal cell carcinoma (RCC) [10, 13], metastatic breast cancer (MBC) [15, 16], non-small cell lung cancer (NSCLC) [17], gastric cancer (GC) [18], colorectal cancer (CRC) [19], and small cell lung cancer (SCLC) [20].

To our knowledge, this study constitutes the first pooled analysis evaluating the prognostic significance of CTC-WBCs. Our systematic review and meta-analysis included three prospective [15, 16, 20] and seven retrospective studies [10, 11, 13, 14, 17‐19], involving a total of 1471 patients with seven different types of solid cancer. The main findings of the current study indicate that the presence of CTC-WBCs is significantly associated with worse OS and PFS/DFS/MFS/RFS in patients with solid tumors. The HRs were 2.44 (95% CI: 1.74–3.40, P < 0.001) for OS and 1.83 (95% CI: 1.49–2.24, P < 0.001) for disease progression endpoints. Similar results were obtained in subgroup analyses based on sampling time, treatment method, detection method, detection system, cancer type and study type. Moreover, sensitivity analyses confirmed the reliability of the findings, and no significant indication of publication bias was found. These results suggest that the presence of CTC-WBCs could indicate tumor spread and predict a more advanced tumor stage. Monitoring CTC-WBC levels before and after treatment may provide valuable information for predicting disease progression and determining appropriate treatment strategies.

Considering the potential impact of sampling time on study outcomes, we conducted a stratified analysis based on sampling time. The results of the included studies showed considerable heterogeneity when stratifying by sampling time. Our sensitivity analysis showed that Jansson's research had the most significant impact on the overall results. It was found that this study utilized the CellSearch detection system and employed immunocytochemistry (ICC) for CTC-WBC detection. Interestingly, this was the sole study that utilized ICC, potentially contributing to the observed heterogeneity in the results. In addition, it is important to consider that CellSearch may underestimate CTCs that have undergone epithelial-to-mesenchymal transition (EMT). Therefore, we excluded Jansson's study and found that the results for OS and PFS/DFS/MFS/RFS were statistically significant both before and after treatment. Our analysis showed that the presence of CTC-WBCs before treatment indicates tumor spread and can predict a later tumor stage. On the other hand, the CTC-WBCs remaining after clinical intervention can act as a "seed" of a tumor, leading to earlier recurrence and worse prognosis. These findings suggest that monitoring levels of CTC-WBCs before and after treatment can provide valuable information for predicting disease progression and prognosis.

Regarding treatment options, our meta-analysis demonstrated that CTC-WBC positivity can serve as an excellent prognostic indicator for both local and systemic therapies. Chemotherapy or targeted therapy can impact the phenotype of tumor cells by targeting those cells with proliferative activity, causing their death. However, cancer cells with weak proliferative activity may survive and develop resistance to systematic therapy, which can lead to tumor recurrence through immunoediting [24, 25]. According to Davies et al. [26], liquid biopsy of CTCs can provide real-time information about the heterogeneous nature of a tumor, making it a potentially ideal biomarker for predicting response to docetaxel. In addition, some studies have suggested that surgery can temporarily disseminate CTC-WBCs, resulting in subsequent spread of CTCs and worse survival outcomes [27‐29]. Thus, our findings indicate that CTC-WBCs can provide valuable information not only for predicting prognosis but also for determining appropriate treatment strategies.

Accurate and reliable detection methods and systems are essential for identifying CTC-WBCs, minimizing misdiagnosis and missed detection. Each method offers unique technical advantages, providing valuable molecular or morphological information. By selecting comparable methods and systems, researchers ensure consistent and comparable results, enabling effective comparison and integration of findings across studies. In subgroup analysis, we found consistent prognostic value of CTC-WBCs across analyses stratified by detection method and system. Based on available literature, RNA-ISH and SE-iFISH offer the advantage of high sensitivity and specificity, allowing for both molecular characterization and enumeration of CTC-WBCs [30‐32]. In the realm of detection systems, both CanPatrol and Cytelligen offer cutting-edge tools for CTC-WBC analysis. CanPatrol excels in molecular characterization through RNA-ISH, while Cytelligen specializes in immunomagnetic enrichment and high-quality ICC for detailed morphological information. Researchers can choose between these high-end systems based on their specific goals, whether it's a focus on molecular insights or intricate morphological analysis of CTC-WBCs [33, 34].

To investigate the prognostic value of CTC-WBCs across different types of cancer, subgroup analyses were performed for various cancer types. According to the results, CTC-WBC clusters may not have significant prognostic value for patients with MBC, possibly due to the limited sample size and study design. However, the heterogeneity observed in subgroup analyses between two studies might also be attributed to differences in patient sampling time and treatment plans.

We also conducted stratified analyses based on study design, categorizing studies as either prospective or retrospective. Both prospective and retrospective studies consistently demonstrated a significant association between CTC-WBC clusters and OS. This consistency reaffirms the robustness of CTC-WBC clusters as a reliable prognostic marker, unaffected by inherent research biases. However, when examining PFS, DFS, MFS, and RFS within prospective studies, a notable impact of CTC-WBC clusters on these outcomes was not observed. This divergence may be attributed to differences in prospective study design, such as the variability in disease progression rates, the timing and sensitivity of outcome measurements, or the influence of intervening clinical factors that are more dynamically controlled in a prospective setting.

In terms of clinicopathological features, no significant association was observed with TNM stage, depth of tumor invasion or lymph node metastases. In general, patients at late stages and with lymph node metastases have poorer prognoses [35]. A possible reason for the lack of association is the limited number of cases, and more studies are required to explain the inconsistent result. For HCC, CTC-WBCs in blood were significantly associated with larger tumor size and higher AFP levels. The larger the size of a tumor is, the faster it progresses or the longer it grows. The level of AFP also to a certain extent reflects tumor size, and its dynamic change has a particular relationship with cancer. AFP is reportedly a sensitive indicator for treatment effect and prognosis [36].

Understanding the characteristics of the interaction between cancer cells and immune cells is essential for developing new cancer treatment methods. The number of CTC-WBCs is typically low, usually in single cells or clusters [18]. As mentioned above, some studies have found that CTC-WBCs can exist in association with leukocytes (a large class of immune cells, including neutrophils, eosinophils, basophils, monocytes and lymphocytes) [37]. As the predominant white blood cell population in the bloodstream of humans, neutrophils are a critical component of the innate immune system. Tumor-associated neutrophils (TANs) play a significant role in the growth and metastasis of cancer cells within the tumor microenvironment. They exert their influence through both direct and indirect mechanisms. Directly, TANs interact with cancer cells, while indirectly, they modify the tumor microenvironment to support cancer cell proliferation and dissemination [38]. These TANs exhibit a gene expression pattern similar to that of pretumor cells, expressing genes that facilitate angiogenesis, remodel the surrounding tissue, promote cancer cell metastasis, and suppress the immune system's response, ultimately promoting tumor growth [37, 39]. In 2019, Szczerba et al. [7]conducted a groundbreaking study focused on isolating CTC-WBCs and corresponding cancer cells from breast cancer patients and mouse models. They performed meticulous transcriptome analyses, comparing the profiles of CTC-WBCs associated with neutrophils to those of CTC-WBCs alone. This investigation revealed differentially expressed genes that play crucial roles in cell cycle progression and efficient metastasis facilitation. Importantly, the presence of CTC-WBC clusters was found to be significantly associated with poor patient outcomes, highlighting the urgent need for effective strategies in treating tumor metastasis. However, despite these significant findings, many aspects regarding the features, functions, and molecular characteristics of WBCs related to CTCs remain unclear. The precise nature of CTC-WBC clusters and the underlying principles governing the interplay between CTCs and WBCs during hematogenous spread remain largely unexplored. Therefore, further extensive research efforts are necessary to unravel the intricate mechanisms involved and to enhance diagnostic accuracy in this field.

Our analysis reveals a significant link between CTC-WBCs and unfavorable survival outcomes. However, it's important to acknowledge the limitations of our meta-analysis. One limitation is the variation in CTC-WBC sampling standard and detection systems across the included studies, which may impact positive rates and survival analyses. The absence of a standardized definition for a positive sample also poses challenges in accurately interpreting findings. Furthermore, the consolidation of survival outcomes like PFS, DFS, MFS and RFS in our meta-analysis was necessitated by limited data for each endpoint. Despite their clinical differences, aggregating these metrics was crucial for maintaining statistical robustness and providing a holistic view of disease progression. Consequently, a comprehensive large-scale prospective clinical trial with an extended follow-up period is crucial. More research is needed to understand the mechanisms, establish standardized cutoff points for detecting CTC-WBCs, and explore tumor-immune cell interactions. This includes studying the molecular characteristics, functional roles, and therapeutic potential of CTC-WBC clusters, as well as how monitoring them can guide treatment decisions.

In conclusion, this comprehensive meta-analysis underscores the prognostic significance of circulating tumor cell associated white blood cell CTC-WBC clusters in various solid cancers. The presence of CTC-WBC clusters is consistently associated with unfavorable prognosis, highlighting their potential as valuable biomarkers for predicting cancer prognosis and guiding treatment decisions. The findings emphasize the intricate interplay between tumor cells and the immune microenvironment during metastasis. Despite the heterogeneity in detection methods and cancer types, the consistent trends observed in subgroup analyses suggest the clinical relevance of CTC-WBC clusters across different contexts. For CTC-WBC clusters to be widely accepted as prognostic tools, it is critical to standardize how we detect them and define meaningful cutoff points. Exploring how these clusters form and their role in cancer will further this goal. Conducting large, long-term studies will confirm their relevance in clinical settings, ultimately improving how we predict and treat advanced cancer for better patient outcomes.