The expression of CTSG protein was different in non-small cell lung cancer tissues and normal non-cancer tissues

Box plots were drawn based on the levels of CTSG expression in non-small cell lung cancer tissues and normal non-cancerous tissues (Fig. 2A), and the results demonstrated that the CTSG expression level in LUAD tissues (1.02) was significantly lower than that of normal non-cancerous tissues (6.57, log2FC =-1.906, P < 0. 01), and in LUSC tissues CTSG expression level (0.75) was significantly lower than that in normal non-cancerous tissues (6.74, log2FC =-2.149, P < 0. 01). In addition, the expression level of CTSG is significantly different in different stages of NSCLC (F = 5.6, P < 0. 01, Fig. 2B). Whether for LUAD or LUSC, the expression of CTSG in normal tissues was significantly higher than that in tumor tissues of different clinical stages (P < 0.05), and in clinical stages 1, 2, and 3, the more advanced the stage, the lower the expression of CTSG (Fig. 2C and D).

Fig. 2

CTSG expression in non-small cell lung cancer. A: The expression difference of CTSG between tumor (red) and non-tumor (grey) tissues, * P<0.05; B: The expression difference of CTSG in different stages of LUAD and LUSC; C: Expression of CTSG in LUAD based on individual cancer stages; D: Expression of CTSG in LUSC based on individual cancer stages

Effect of CTSG on the prognosis of patients with non-small cell lung cancer

According to the results of survival analysis, for both LUAD and LUSC patients with low CTSG transcripts per kilobase million (TPM), the overall survival (OS) was significantly shorter than that of those with high CTSG TPM (P < 0.05), but the level of CTSG TPM had no significant effect on the disease-free survival (DFS) of both LUAD and LUSC (P > 0.05) (Fig. 3). The results of the analysis predicted that the expression level of CTSG might have some influence on the survival prognosis of patients with non-small cell lung cancer.

Fig. 3

The impact of CTSG expression on overall survival and disease-free survival in patients with non-small cell lung cancer, A: Lung adenocarcinoma; B: Lung squamous carcinoma

Baseline of participants

In our cohort, among the 45 HIV-related patients, 37 patients were male and 8 patients were female, the age of the patients ranged from 25 to 75 years old, with an average age of (60 ± 2) years old. There were 10 cases of LUSC, 31 cases of LUAD, and 4 cases of other pathologic types (including 3 cases of large cell carcinoma and 1case of carcinoid tumor). And 52 cases of simple lung cancer include 35 males and 17 females, and the patients’ age ranged from 40 to 86 years old, with an average age of (68 ± 1) years old. And there were 12 cases of LUSC and 40 cases of LUAD. In order to illustrate the differences in the expression of CTSG protein in different pathological tissues or normal tissues, we tried to collect cases with consistent indicators in baseline data when collecting samples from the two groups, so as to minimize the errors arising from the differences in individual clinical factors. The demographic and clinical characteristics of the 45 HIV-related patients and 52 simple lung cancer are shown in Table 1.

Table 1 Baseline of participantsExpression of CTSG proteins in HIV-related lung cancer tissues

Hematoxylin eosin (HE) staining and immunohistochemistry were used to detect the expression of CTSG protein in 45 cases of HIV-related lung cancer tissues, 52 cases of simple lung cancer tissues and their corresponding adjacent non-tumor tissues. The results showed that CTSG protein was mainly localized on the cell membrane and appeared as tan or brown granules. In non-small cell lung cancer, the expression of CTSG was lower than that in adjacent non-tumor tissues. Meanwhile, the expression of CTSG protein in HIV-related lung cancer tissues was not only significantly lower than that in adjacent non-tumor tissues, but also lower than that in simple non-small cell lung cancer tissues (Fig. 4). 40 of the 45 cases of HIV-related lung cancer tissues were positive for CTSG protein expression, with a positivity rate of 88.9%. The expression score of CTSG protein in 45 tissues was calculated according to the above CTSG immunohistochemical results quantification method, which resulted in the lowest score of CTSG expression in 45 tissues as 1 point, the highest as 18 points, and the average score was 7.8 points. Taking 7.8 points as the cut-off value, those > 7.8 and ≤ 18 points were considered as the high expression group, and those ≥ 1 and ≤ 7.8 points were considered as the low expression group, resulting in 20 cases in the low expression group and 25 cases in the high expression group, with a high expression rate of 55.6%. Additionally, the high CTSG expression rate of HIV-related LUAD was 74.19%, and the high expression rate of HIV-related LUSC was 10%, and the high CTSG expression rate of different pathologic type of HIV-related lung cancer was compared by using χ2 test, which shows that there is a difference in the CTSG expression of different pathologic types (P<0.05) (Table 2).

Fig. 4

The expression of CTSG protein in simple lung cancer tissues, HIV-related lung cancer tissues and their corresponding adjacent non-tumor tissues detected by HE staining and IHC. (Scale bar = 50 μm)

Table 2 Expression of CTSG protein in HIV-related non-small cell lung cancer and paraneoplastic tissuesCorrelation between CTSG protein expression and clinicopathological parameters in HIV-related lung cancer

The statistical results of this experiment showed that the level of CTSG protein expression was not related to the sex, age, smoking history, whether HIV RNA could be detected after the first consultation, and lymph node metastasis of the patients with HIV-related lung cancer (P>0.05); however, it was closely related to the pathological type of HIV-related lung cancer, distant metastasis, and clinical stage, and the difference was statistically significant (P<0.05) (Table 3).

Table 3 Relationship between CTSG protein expression and clinicopathologic parameters in HIV-related lung cancerCorrelation of CTSG protein expression with laboratory test indicators and survival prognosis in HIV-related lung cancer

We collected laboratory test indicators in 45 patients with HIV-related lung cancer, which included CD4 + T cell count, CD8 + T cell count, CD4 + T/CD8 + T cell count, carcinoembryonic antigen (CEA), CA125, pro-gastrin releasing peptide (ProGRP), neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC), and cytokeratin 19 fragment (CYFRA21-1). These indicators are measured and collected at the patient’s first visit to the hospital.

According to the immunohistochemical CTSG expression score of each pathological tissue, 45 samples were divided into high expression group and low expression group, and the t-test was used to compare the laboratory test indicators between the two groups, and the results showed that there were significant differences in the CD4 + T cell count and CD4 + T/CD8 + T cells between the two groups (P < 0.05). The mean value of CD4 + T cell count in the high CTSG expression group was higher than that in the low CTSG expression group (Fig. 5A). Plus, the mean value of CD4 + T/CD8 + T cells in the high CTSG expression group was higher than that in the low CTSG expression group (Fig. 5B). There was no significant difference between the high and low expression groups for the rest of the laboratory test indicators (Table 4).

Fig. 5

Correlation of CTSG protein expression level with laboratory test indicators and survival prognosis in HIV-related lung cancer, A: Statistics of the CD4 + T cell count in the CTSG high expression group and low expression group; B: Statistics of the CD4 + T/CD8 + T cells in the CTSG high expression group and low expression group; C: Survival curves of the CTSG high and low expression groups. *P < 0.05 by t-test

Table 4 Relationship between CTSG protein expression and laboratory indicators in HIV-related lung cancer

After a median follow-up of 15 (1–45) months, of the 45 patients with HIV-related lung cancer included in this study, 16 (35.6%) patients died after discharge from hospital and 4 (8.9%) patients were lost to follow-up. The shortest survival was 1 month and the longest survival was more than 45 months, with a 6-month survival rate of 72.1% (95% CIs: 0.55–0.83) and a 1-year survival rate of 66.4% (95% CIs: 0.4911–0.7898). All causes of death were thought to be related to HIV-related lung cancer. Grouped according to high and low CTSG expression, we performed a log-rank test to do the comparison of survival differences, which showed that the difference between the survival curves of the two groups was not statistically significant (P = 0.123). However, by plotting the survival curves, we could roughly see that the overall survival (OS) of the high CTSG expression group was slightly longer than that of the low CTSG expression group [HR(High) = 0.48], which was consistent with the trend that the overall survival (OS) of patients with low CTSG expression is significantly shorter than that of those with high CTSG expression in NSCLC retrieved by our big data (Fig. 5C).