Patient characteristics and survival analyses
Seventy-one young LUAD patients were selected for inclusion in our study. The basic characteristics of the patients are summarized in Table 1. The mean age was 40.4 years, ranging from 27 to 45 years. In the cohort of young LUAD patients from our center, we found obvious heterogeneity between our cohort and the population of lung cancer worldwide. In our cohort, we detected that 73.24% of young patients harbored EGFR or ALK mutations. The most frequent three genomic alterations were deletion mutations of EGFR 19 exon (31%), EGFR 21 exon L858R point mutation (23%) and echinoderm microtubule associated protein like 4 (EML4)-ALK fusion (14%), which indicates that young patients may be more likely to benefit from EGFR-tyrosine kinase inhibitors (EGFR-TKIs) or ALK-TKIs. The basic information of the genomic alterations is shown in Figure S1E. In addition, 472 LUAD patients and 809 lung cancer patients from the TCGA database were also included in our study.
As shown in Fig. 1, the IHC sections of all variables were collected for MOD calculation. The positive and negative staining comparisons of PD-L1 and CD28 are shown in Fig. 1A, 1B, 1D and 1E. CD3 was involved in the representation of total lymphocytes, and the positive and negative staining IHC images are shown in Fig. 1G and Fig. 1H, respectively. In our cohort, there was no significant difference in DFS between the CT and IM groups. The IHC sections of PD1 and CD8 are shown in Figure S1A-D. In this study, we compared the MOD between the progression group (disease recurring before the end of follow-up) and the stable group and found a significant difference between them. Figure 1C demonstrates that a higher mean MOD of PD-L1 was detected in the progression group than in the stable group (P = 0.0032), which is similar to the results of CD28 (P < 0.0001), as shown in Fig. 1F. This finding revealed that PD-L1 and CD28 may be associated with rapid progression in young LUAD patients. However, the MOD of CD3 showed a different distribution, and a higher mean MOD was detected in the stable group (P < 0.0001), as shown in Fig. 1I, which indicated that TILs may be a biomarker of long DFS in young LUAD patients. The mean MODs of PD1 and CD8 were calculated, and no significant differences were found between the two groups.
Survival analyses based on five variables related to TIME, including CD28, PD-L1, PD1, CD3 and CD8, were performed in this study. Thereafter, PD-L1 + CD28 (MODPD−L1+CD28) and the CD3/CD8 ratio (MODCD3/CD8) were also included in the survival analyses. The results revealed that TIME-related molecules are closely associated with the prognosis of LUAD and lung cancer, as shown in Figure S2, Fig. 3A-D and Fig. 4A-D, especially in young LUAD patients, as shown in Fig. 2A to Fig. 2F. Patients with a higher expression of CD28 had a shorter DFS (young LUAD: P < 0.0001; LUAD: P = 0.0011; lung cancer: P = 0.0001) but a longer OS (LUAD: P = 0.016; lung cancer: P = 0.0282). Higher expression of PD-L1 was associated with a worse prognosis for lung cancer patients both in DFS (young LUAD: P = 0.0382; LUAD: P = 0.0002; lung cancer: P = 0.0001) and OS (lung cancer: P = 0.0032). Moreover, we found that PD-L1 + CD28 may be a better biomarker for DFS in young LUAD patients and that a higher MODPD−L1+CD28 was associated with a shorter DFS (P = 0.0004), which is more persuasive than a single variable in predicting DFS according to the AUC of these three variables. TIL-related variables such as CD3 and CD8 were also found to be associated with DFS among young LUAD patients. CD3, which represents the total lymphocytes, was a convincing variable for predicting a long DFS in young LUAD patients (P < 0.0001) but a short DFS in LUAD patients (P = 0.0003) and lung cancer patients (P < 0.0001), which indicated that young LUAD patients showed a different response to TILs, which resulted in a different outcome. A higher CD3/CD8 ratio was associated with a longer DFS (P = 0.0005) in young LUAD patients, which suggests that a higher CTL proportion in total lymphocytes may be related to a worse prognosis in young LUAD patients. Although PD1 and CD3 showed no statistical significance in survival analysis for LUAD and lung cancer patients, we found that these two variables were associated with a good long-term prognosis. Higher expression of the two variables was associated with a longer OS in LUAD and lung cancer patients. The ROC curves of these variables are shown in Figure S1.
Nomograms based on TIME
Eight variables were involved in the Cox regression, and five variables (P < 0.2), including TNM stage, EGFR or ALK mutation status, CD28, PD-L1 and CD3, were selected for the construction of the DFS nomogram for young LUAD patients, as shown in Fig. 2G. Here, we constructed a novel type of nomogram, and the basic characteristics of the patients involved are clearly shown in the nomogram represented by the yellow peak called “density”. We provide an example here to explain our DFS nomogram. One of our patients represented by red dots in the DFS nomogram was involved in the verification of the prognostic model. After the quantification of the basic characteristics, including stage, mutation and the expression of TIME-related variables, we obtained a total score of 359 on the score axis, and the corresponding probability (DFS shorter than 41 months) was 50.9%. The C-index of the DFS nomogram was 0.913, as shown in Table 2, which revealed that our model for DFS has satisfactory and robust discrimination ability compared to any single variable involved.
Table 2
The discrimination ability of ROC curves and nomograms.
| Young LUAD patients | LUAD patients | Lung cancer patients |
| AUC or C-Index | P value | AUC or C-Index | P value | AUC or C-Index | P value |
CD28 | 0.777 | < 0.0001 | 0.594 | 0.001 | 0.556 | 0.007 |
PD-L1 | 0.713 | 0.007 | 0.531 | 0.256 | 0.547 | 0.025 |
PD-1 | 0.537 | 0.639 | 0.541 | 0.141 | 0.545 | 0.031 |
PD-L1 + CD28 | 0.81 | < 0.0001 | 0.545 | 0.107 | 0.52 | 0.33 |
CD3 | 0.832 | < 0.0001 | 0.574 | 0.008 | 0.554 | 0.01 |
CD8 | 0.506 | 0.942 | 0.626 | 0.345 | 0.535 | 0.095 |
CD3/CD8 | 0.814 | < 0.0001 | 0.531 | 0.259 | 0.549 | 0.018 |
Nomograms | 0.913 | | 0.678 | | 0.625 | |
Three variables, including age, TNM stage and CD28, and another three variables, including TNM stage, CD28 and PD-L1, were involved in the construction of the OS nomograms for LUAD patients and lung cancer patients, as shown in Fig. 3E and Fig. 4E, respectively. The probabilities of OS less than 1 year, 3 years and 5 years were all evaluated by the OS nomograms. The C-indexes of the OS nomograms were 0.678 and 0.625, as shown in Table 2. Every single variable involved in these nomograms provides a score, and the total score can easily be calculated to estimate the probability of OS less than 1 year, 3 years and 5 years.
Validation of nomogram performance
Harrell’s C-indexes revealed that the nomograms for young LUAD DFS (C-index = 0.913), LUAD OS (C-index = 0.678) and lung cancer OS (C-index = 0.625) showed satisfactory discrimination. Calibration of the nomograms was evaluated by calibration plots, as shown in Fig. 2H, Fig. 3F-H and Fig. 4F-H. The calibration plots showed that the probabilities of our prognostic models agreed with the accuracy probabilities on acceptable scales (the dashed lines in the calibration plots correspond to a 10% margin of error), except for the 5-year OS in the LUAD OS nomogram (Fig. 3H). Generally, it proved that our prognostic models based on the nomograms showed satisfactory and robust ability in the discrimination and calibration of DFS for young LUAD patients and OS for LUAD and lung cancer patients.
The dual effect of CD28 on lung cancer prognosis
Although PD-L1 plays an important role in tumor immune suppression, we conducted PCA analysis and detected the important role of CD28 associated with TIME. Figure S3A-6D describes the correlation between CD28 and other TIME-related variables derived from GEPIA based on the TCGA database [15]. Linear correlations were confirmed between CD28 and other variables, including CD3 (R = 0.66), CD8 (R = 0.43), PD-L1 (R = 0.22) and PD1 (R = 0.42). Low CD28 expression is associated with a higher proportion of type II TIME, which is proven to be sensitive to immune checkpoint inhibitor (ICI) treatment. The TIME type distribution based on CD28 expression is shown in Figure S3E. Based on the TCGA database, we found a group of genes positively correlated with CD28, and the heatmap of these genes is shown in Figure S3F. Then, enrichment analysis was performed to detect the possible cell signaling pathways related to these genes through “METASCAPE” [16], as shown in Figure S3G, and CD28 is closely associated with the immune-related cell signaling pathways in LUAD, especially the T cell activation pathway. According to the TCGA database, the expression level of CD28 is higher in primary tumor tissue than in normal control tissue, and a higher CD28 expression level is associated with an earlier TNM stage and less nodal metastasis, as shown in Figure S3H-J. The dual role of CD28 in the prognosis of lung cancer is shown in Table 3.
Table 3
The dual role of CD28 in the prognosis of lung cancer.
CD28 | LUAD | LUNG CANCER |
DFS | OS | DFS | OS |
Advantage group | low expression | high expression | low expression | high expression |
Log-rank P | 0.0011*** | 0.016* | 0.0001**** | 0.028* |