4.1 PD-L1 expression in NSCLC
The most comprehensive study regarding PD-L1 expression was the EXPRESS study, which included 2,435 patients and evaluated PD-L1 expression in samples obtained from local laboratories located in 45 centers in 18 countries. The percentages of patients with PD-L1 TPS ≥50% and TPS ≥1% were 22% vs 51% in Europe, 22% vs 53% in the Asia-Pacific region, 22% vs 47% in the Americas, and 24% vs 54% in other countries. In our study, the ratio is 14.7% vs 54.3%. The prevalence of PD-L1 was similar across geographic regions and broadly consistent with central testing results from a clinical trial screening population; naturally, our results are no exception.
In 2016, Aggarwal et al. found a larger proportion of patients with TPS < 1% in the subgroup with non-squamous cell carcinoma compared with squamous cell carcinoma (26% vs 19%); that is, in LUADs, patients’ PD-L1 is more likely to be negative compared with LUSCs , which is similar to our findings. In our study, 59.46% of LUADs expressed PD-L1 negatively, while 43.91% of LUSCs expressed PD-L1 negatively. Because PD-L1 is increasingly familiar and recognized by the medical community and testing is affordable, a growing number of patients can access PD-L1 detection. Therefore, with the increase in detection rates, the overall negative rate has also increased, which is understandable. Similarly, in 2017, an association was observed between adenocarcinoma and low PD-L1 expression in NSCLC samples, including Chinese, Japanese, Korean, American, Canadian, British, and French individuals. Additionally, the results indicated that PD-L1 expression was also associated with sex, smoking status, histology, differentiation, tumor size, lymph node metastasis, TNM stage, and EGFR mutation . Moreover, this is one of the few studies that is consistent with our conclusion. However, Chen et al. concluded that a significant difference in PD-L1 expression between LUSCs and LUADs was observed, but in contrast, age, sex, and smoking history were not statistically significant. Gelatti et al. were the first to comprehensively describe PD-L1 expression in the Brazilian population . In China, a study by Song et al. showed that PD-L1 expression was associated with advanced stage, lymph node (LN) metastasis, solid predominant subtype and wild-type epidermal growth factor receptor (EGFR) gene expression .
Taken together, there are some divergences in certain aspects between these studies of patients in various regions. However, this may be due to differences in the regional population, sample size, PD-L1 detection reagent, and PD-L1 expression interpretation threshold . Therefore, we should try to find other experiments with small differences for analogy analysis. Despite the differences, a relatively unified opinion is that the expression of PD-L1 is related to the histological subtype of lung cancer. Generally, the expression of PD-L1 in LUADs is more likely to be lower than that in LUSCs, which is consistent with our research results.
4.2 Statistical differences mainly exist in subtypes and differentiation degree
Until now, no general pathway to control PD‐L1 expression has been discovered. Depending on stimulus and cell type, the expression of PD‐L1 was found to connect with various signaling molecules: p44/42 and/or p38 MAPKs 26, 27, or STAT‐1, STAT‐3, and IRF‐1 28-30 . Also discovered was that STAT3 in LUSC tissues showed increased expression level rather than in LUAD in the Talbot database . Intercellular communication between tumor cells, immune cells, and the stroma characterizes the tumor microenvironment . On the one hand, accumulating data have established that STAT3 directly regulates the expression of PD-L1 [33, 34]; on the other hand, recent findings have shown that STAT3 represses CD8+ T cell chemotaxis and activation . Additionally, PD-L1 expression has bearing on CD8 T cell infiltration within the squamous cell carcinoma microenvironment [36, 37]. Based on these studies, many experiments on the expression of PD-L1, T cells, and STAT3 in squamous cell carcinoma have been carried out. Dun et al. obtained tumor-associated MDSCs derived from patients with head and neck squamous cell carcinoma, and it was found that pSTAT3 was at higher levels of expression to and suppressed T cell proliferation through the actions of arginase 1. Another trial conducted in oral squamous carcinoma cells by Xia revealed that pSTAT3 levels are elevated in CAFs cocultured with oral squamous carcinoma cells . According to these studies, activation of the STAT3 pathway directly upregulates the expression of PD-L1. However, in different lung cancer subtypes, the expression of STAT family members is different. Hence the influence of the STAT family on the expression of PD-L1 in lung cancer and adenocarcinoma is worthy of further discussion.
Consistent with most studies, we concluded that the expression of PD-L1 is related to the type of NSCLC. However, we also believe that a significant difference exists in the differentiation states of lung cancer tissues. We know that cellular differentiation, or simply cell differentiation, is the process through which a cell undergoes changes in gene expression to become a more specific type of cell. These changes are largely due to highly controlled modifications in gene expression and are the study of epigenetics [40-43]. In other words, during the process of cell differentiation, cell morphology tends to exhibit certain histology features, which is the fundamental basis for the microscopic diagnosis of lung squamous cell carcinoma or lung adenocarcinoma. In our study, many statistical results showed that tumor differentiation is related to PD-L1 expression; in particular, a patient with well to medium differentiation, has a 76.0% to 78.4% probability of having a negative PD-L1 expression. Although findings in other studies differ, there are subjective distinctions in the judgment of differentiation in various studies. It is worth noting that our research sample is as large as possible to reduce this error, so we are more confident in our conclusions. Because there is no research on the molecular mechanism as theoretical support for this controversy, more work needs to be conducted.
In our univariate analysis, we also found that PD-L1 expression was significantly different in smoking, sex, and lymph node metastasis. It was found that the efficacy of pembrolizumab, an antibody targeting programmed cell death-1 (PD-1), is correlated with the molecular smoking signature [44-46]. Smoking status may be a predictive marker for better survival as PD-1/PD-L1 inhibitors  and total smoking duration may be a predictor of a PD-L1 TPS ≥ 50% (P= 0.001), which is consistent with our study. In our study, nearly half of the patients were smokers, and there was a significant difference in their PD-L1 expression (P=0.031). We all know that in China, the proportion of male smokers is much larger than that of female smokers, and our data also show the same (the correlation analysis between smoking history and sex: P = 0.015). So, to some extent, it is comprehensible when smoking history and sex are factors related to the expression of PD-L1. However, gender differences themselves have an impact on the diseases [49-51]. Preclinical studies suggest that the expression of PD-L1 is modulated in an estrogen-dependent and sex-dependent manner [49, 50]. Sex-related differences in the anticancer immune response have been described in tumor expression levels of PD-L1 across a large spectrum of tumors, including NSCLC [52-54]. This difference in lung cancer treatment based on gender differences also verifies our experimental results and affirms the significance of our study. In our research, there were 99 female patients who were medium or well differentiated with no lymph node metastasis; their negative expression rate was as high as 78.8% (95% CI: 70.6%-87.0%).
Lymph node metastasis is the most common method of metastasis of lung cancer [55-57]. Recent studies have demonstrated that the expression of PD-L1 may be mediated by the PI3K/AKT/PTEN pathway [58-60]. The activated PI3K-AKT pathway plays a central role in the translation of interferon-γ, which is a key regulator of PD-L1 transcription [60, 61]. Up to 50% of penile cancers positively express PD-L1, and PD-L1 is positively related to LNM [62, 63]. In addition, Hu et al. first showed a positive linear correlation between NLR and PD-L1 in penile cancer. Such a linear correlation was also reported in cholangiocarcinoma . These conclusions combined with our experimental results show that there is a relationship between PD-L1 expression and lymph node metastasis. Although the specific mechanism is not completely clear, it also provides guidance for future research. In our study, the expression of PD-L1 in patients without lymph node metastasis was usually negative or low. In contrast, while a smoker patient was in the N3 stage with no distant metastasis, he showed high expression of PD-L1 (R2=62.5%, 95% CI: 35.9%-89.1%).
4.3 Anti-PD-1/PD-L1 immunotherapy
The biological function of PD-1 is to limit T cell activity in peripheral tissues in the inflammatory response of infection and to limit autoimmunity [65, 66]. However, this function translates into the main immune resistance mechanism in the tumor microenvironment. The PD-1/PD-L1 signaling pathway plays an important role in tumor cells escaping immunosurveillance[67, 68]. To evade host immune surveillance, tumors express PD-L1, which interacts with PD-1 on T cells to decrease immune responses. In the past decade, programmed death-1 receptor (PD-1) and programmed death ligand-1 (PD-L1) monoclonal antibodies have shown great potential in the treatment of lung cancer; they have many advantages, including significant antineoplastic activity, induction of long-lasting responses, and good safety. However, the side effects and factors influencing therapy have also been noticed, and only a subset of lung cancer patients will respond to anti-PD-1/PD-L1 therapy . Although four anti-PD1/PDL1 agents-nivolumab, pembrolizumab, atezolizumab, and durvalumab-have been approved by the FDA as significant therapeutic effects for lung cancer [70-73], they have their challenges. For example, the effective cure ratio is not met. Due to the high expense and long treatment period, there is no doubt that the low effective ratio fails to satisfy doctors and patients. Hence, how to identify patients most likely to benefit from the treatment and how to improve its therapeutic effects are questions that remain to be answered.
In clinical practice, inhibitors targeting programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are gradually being used to treat non-small cell lung cancer, while radiotherapy combined with chemotherapy is the main treatment for small cell lung cancer in China. Early concurrent chemoradiation is the standard of care for limited-stage SCLC (LS-SCLC). A combination of etoposide and cisplatin or carboplatin remains the mainstay of first-line treatment for ES-SCLC, with the addition of atezolizumab, now becoming standard . Although chemotherapy combined with PD-L1 antibody was approved by the FDA in 2019, it has not been widely used, and there is no routine PD-L1 detection in patients with SCLC. Therefore, this study only recruited patients with squamous cell carcinoma and adenocarcinoma in SCLC.