In clinical trials, PD-L1 inhibitors have benefited selected patients. However, targeted molecular therapy requires verified biomarkers to determine indication. Previous research on these issues has led to inconsistent results. For example, Baptista et al. [15] reported that PD-L1 expression emerged as a positive prognostic biomarker in breast cancer, while others disagreed [16]. Furthermore, the best cell type for determining the prognostic and predictive values of PD-L1 in breast cancer has not been established definitively. Herein, PD-L1 on TCs, TIICs, and CTCs were considered for this purpose. The present study found that patients with high levels of PD-L1 on CTCs had a lower overall survival rate. This can serve as a reference for selection of a PD-L1 inhibitor and monitoring during the entire treatment course.
We did not find PD-L1 expression on TCs, which is in accord with Ali et al.’s report (17), but this differed from the percentages found by the IMpassion130 trial (18). This may be due to the small sample size, and including patients with other than triple-negative breast cancer. Some previous studies reported that PD-L1 on TCs in breast cancer indicates a bad prognosis, as it does for NSCLC and melanoma (16,19), although Baptista et al. determined a good prognosis (15). We found no significant association between PD-L1 tissue expression and progression free survival or overall survival.
What is more, a recent study by Guiu et al. reported a 32.1% positive for TC PD-L1 rate and 36.1% positive for CTC PD-L1 rate and showed that the PD-L1 rate on CTCs has been considered a poor independent prognostic indicator in metastatic breast cancer, meanwhile professor Maria reported the same result (20–21), relative to that of TCs. Our results are consistent with these studies.
CTC evaluation appears of interest in this setting, as it probably faithfully reflects the PD-L1 status of the whole metastatic burden, and could represent an easy biomarker throughout the disease evolution. We were able to detect CTCs (whatever their PD-L1 status) in 73.68% of our patients, 31.58% of the patients presenting with 5 or more CTCs, a percentage in line with published MBC series and the published pooled analysis (22). We found an association between baseline CTCs and survival, as classically described in MBC (22–24).
Interestingly, in accord with the present study, Ali et al. (17) showed that PD-L1 protein in breast cancer is rare, but enriched in basal-like tumors and associated with infiltrating lymphocytes. Schott et al. (25) reported a very high number (94.5%) of breast cancer patients who were positive for PD-L1 on CTCs. This suggests that breast cancer cells may have little PD-L1 in the primary tumor but high PD-L1 levels on CTCs. The discrepancy could be due to the mechanism of tumor development, or differences in detection methods, antibodies used, or scoring systems. To date, there is no guideline for antibody selection, not a standard scoring system in breast cancer. Therefore, the heterogeneity of data must be considered cautiously, when obtained via different means such as the CellSearchVR or Parsortix cell separation systems, or others.
Most of the detection systems have been applied in lung, head, and neck cancers; very few studies have focused on PD-L1 on CTCs of breast cancer. The quantification of PD-L1 on CTCs involves both CTC and PD-L1 detection, respectively. To detect CTCs, available systems include the CellSearch CTC Test (based on positive selection), Parsortix cell separation (physical properties), and isolation by size of epithelial TCs (ISET) (26–27). Combined systems are also widely used (28). It has been shown that Parsortix can improve the total detection rate of CTCs in NSCLC patients, compared to other systems (described above) (29). Some scholars think that this is because of the heterogeneity of some CTCs and the low expression of epithelial cell adhesion molecules (EpCAMs). A cell search based on EpCAMs may not be able to detect some subsets, such as EpCAM-negative CTCs (30, 31).
Considering the advantages of the above detection methods, we chose the Canpatrol CTC typing detection system (Yishan Biotechnology) which combines a variety of technologies to detect the presence of different CTC subtypes, and used nanotechnology to filter after lysis of red blood cells. The markers of epithelial cells (epithelial adhesion molecules and cytokeratins) and interstitial cells (vimentin and twist) were identified by multiple nucleic acid in situ hybridization. This not only guaranteed accuracy of the quantity of CTCs detected by the system, similar to that of Parsortix cell separation, but also the specificity by using the characteristics of CTC (32). To detect PD-L1, techniques such as western blot, flow cytometry, immunohistochemistry, and miRNA gene hybridization have been reported. In the present study, in situ hybridization of multiple nucleic acids was employed to detect PD-L1 mRNA expression on CTCs, which were counted under a fluorescence microscope (33). Currently there is no standard for CTC detection, but many methods are in the exploration stage.
The present study is the first to evaluate the intensity of PD-L1 on CTCs both qualitatively and quantitatively, as there is no standard method. The only other study concerning the detection of PD-L1 on the surface of breast cancer CTCs employs a PD-L1 immune score. Researchers have scored PD-L1 in SKBR3 TCs and SKBR3 TC clusters quantitatively as nil, 1, or 2, indicating nil, low, and high, respectively (34). In several other studies, more than three CTCs with PD-L1 were recorded as CTC PD-L1-positive. In another study, more than one PD-L1+ CTC was considered as CTC PD-L1 positivity (28).
The present study clinically evaluated the PD-L1 intensity on CTCs both qualitatively and quantitatively. PD-L1 levels on CTCs was categorized as nil, low, medium, or high based on the number of indications, scored respectively as nil, 1, 2, or 3. For each patient, the total score was the sum of the scores on all CTCs. Thus, a total score of ≤ 5 points was considered low PD-L1 levels, while > 5 points was high. So far, only a few studies have analyzed the levels of PD-L1 on CTCs for cancers of the breast (34), head and neck (35–37), prostate (38), and lung (39–40), and other solid tumors. The study that to analyze the levels of PD-L1 on CTCs in patients with breast cancer, and the association between PD-L1 on CTCs in such patients and prognosis is rare. Our research will deeply affect PD-L1+ CTC research in the future and will be an indicator for monitoring during immune therapy. In most studies of PD-L1 on CTCs, different CTC enrichment and detection techniques, PD-L1 detection methods, and patient cohorts were used. Therefore, the comparison of clinical significance between these studies must be carefully explained, while our findings are consistent with the data of most studies.
Previous relevant studies have varied with regard to the timepoints of detection of PD-L1 on CTCs—for example, before and after surgery, before and after chemotherapy, during treatment, and disease progression. Most of the findings are in accord with ours. For example, after 6 months of treatment, all patients with NSCLC with progressed disease, and those who had died, had PD-L1+ CTCs, while none of the responding patients showed this positivity (41). This is the evidence that PD-L1+ CTCs are associated with a bad prognosis and poor response to treatment. In addition, in a study of 35 patients with different gastrointestinal tumors, 95% of those with advanced stage had high PD-L1+ CTCs (i.e., 18/19 with progression) (42). In the past, NSCLC and head and neck cancers were the most relevant data concerning the significance of PD-L1+ CTCs. Several studies indicated that PD-1+ and PD-L1+ CTCs could be detected in metastatic NSCLC patients before and after first-line chemotherapy. All of the studies show a high number of CTCs positive for PD-1 before treatment and an association with patients’ poor prognosis (20, 21).
The current study is limited, in that all the patients received only conventional treatment, and no one received immunotherapy. This is because immune suppressants such as PD-L1 inhibitors had not been approved for breast cancer treatment, and most of them were in clinical trial at the time. In addition, PD-L1 on CTCs should be detected at various treatment timepoints and locations (tumor, lymph node, and site of metastasis), to clarify whether the treatment itself affects the status of PD-L1 on CTCs at different site, and thus prognosis.
Most importantly, this study determined that high levels of PD-L1 on CTCs may be a prognostic factor that can predict a poor prognosis, especially compared with that of TCs and TIICs. Our data warrants a larger cohort in a randomized clinical trial to investigate the value of PD-L1+ CTCs for predicting response to immunotherapy and its association with prognosis. To continue the analysis of PD-L1 on CTCs as a potential biomarker for breast cancer, large-scale validation research must address the optimal method of collecting CTCs; the detection and evaluation criteria of PD-L1 on CTCs; and the optimal sites and timepoints for monitoring.