Circulating and tumor-associated caspase-4: a novel diagnostic and prognostic biomarker for non-small cell lung cancer

Abstract

Late diagnosis limits therapeutic options and survival rate of non-small cell lung cancer (NSCLC) patients. Therefore the identification of biomarkers represents an emerging medical need. A highly sensitive and specific test was developed to identify/quantify a novel/selective diagnostic biomarker for NSCLC patients, caspase-4. This test was validated by using i) plasma from 125 NSCLC patients and 79 healthy (non-pathological) subjects, ii) plasma from 139 smokers and iii) from 70 chronic-obstructive pulmonary disease (COPD) patients. Caspase-4 quantification was also assessed in the lung tumor mass of 98 paired NSCLC patients compared to 10 non-tumor lung tissues (i.e. tuberculosis). Circulating caspase-4 was detected in both healthy and NSCLC patients; however at different range values: 2.603-3.372 ng/ml for NSCLC patients (95% CI) compared to 0.3994-0.6219 ng/ml for healthy subjects (95% CI). The sensitivity of the test ranged from 97.07% to 100%; the specificity was 88.1% with a positive predictive value of 92.54%, accuracy of 95.19% and AUC of 0.971. Smokers (95% CI, 0.3947-0.6197 ng/ml) and COPD patients (95% CI, 1.703-2.995 ng/ml) showed intermediate values of circulating caspase-4. Tissue levels of caspase-4 in the tumor mass showed that 72 (72.7%) out of 99 patients were positive. More importantly, higher levels (cut-off value = 0.307 ng/ml) of caspase-4 in the tumor mass were associated to reduced overall survival (median 0.92 years) compared to NSCLC patients with lower levels (median 3.02 years). We report for the first time caspase-4 as a novel diagnostic and prognostic biomarker, opening new therapeutic perspectives for NSCLC patients.

Keywords: NSCLC; biomarker; diagnosis; non-small cell lung cancer; prognosis.

Figure 1. Circulating caspase-4 was in the plasma of NSCLC patients

(A) Levels of caspase-4 were detected in the plasma of healthy (H, n = 79) and NSCLC (LC, n = 125) patients. H subjects were non-pathological. LC-derived blood was obtained before surgical resection of the tumor mass. Similarly, blood from patients who were not diagnosed of NSCLC or COPD (lung pathologies, n = 10) showed low levels of circulating caspase-4 (B). Levels of caspase-4 were analyzed according to the histotype (C) and stage (D) of NSCLC. (E) ROC analysis (H subjects vs LC) was performed to define the diagnostic features of caspase-4. Data are expressed as median ± interquartile range, showing outliers as dots. One Way ANOVA followed by Bonferroni’s post test was applied to (B, C and D). Mann Whitney test was performed for Figure A.

Figure 2. Circulating caspase-4 was solely present in the plasma of NSCLC patients

Levels of circulating caspase-4 in NSCLC were compared to other solid (A) (KE, endometrial cancer, n = 12; KO, ovarian carcinoma, n = 12; K colon, colon carcinoma, n = 16; K liver, liver carcinoma, n = 5, K bladder, bladder carcinoma, n = 6; melanoma, n = 9) and liquid (B) (chronic lymphocytic leukaemia, CLL, n = 12; non-Hodgkin lymphoma, NHL, n = 12; multiple myeloma, MM, n = 12). Data are expressed as median ± interquartile range, showing outliers as dots. One Way ANOVA followed by Bonferroni’s post test.

Figure 3. Circulating caspase-4 was detectable in the plasma of smokers and COPD patients

Levels of circulating caspase-4 was analyzed in smokers, n = 139, (A) according to the age (B) (smokers, SM < 60 and SM > 60 years old). Similarly, levels of caspase-4 were analyzed in the plasma of COPD patients, n = 70 (C). Data are expressed as median ± interquartile range, showing outliers as dots. One Way ANOVA followed by Bonferroni’s post test.

Figure 4. Tissue caspase-4 was detectable in the tumor mass of NSCLC patients

(A) Levels of caspase-4 were detected in the tumor mass of NSCLC (LC, n = 99) patients compared to lung tissues obtained from non-cancer and non-COPD patients (i.e. tubercolosis) (lung pathologies, n = 10). Levels of tumor-associated caspase-4 were analyzed according to the stage (B) and histotype (C) (D) ROC analysis (lung pathologies vs LC) was performed to define the diagnostic features of caspase-4 starting from tissue samples. Data are expressed as median ± interquartile range, showing outliers as dots. One Way ANOVA followed by Bonferroni’s post test was applied.

Figure 5. NSCLC patients with higher levels of caspase-4 in the tumor mass showed lower survival rate

Tissue levels of caspase-4 were correlated to the available survival rate of NSCLC patients (n = 73). In particular, 59 NSCLC patients (80.8%) showed levels of tumor-associated caspase-4 higher than the cut-off (0.377 ng/ml); 14 NSCLC patients (19.2%) showed lower levels of caspase-4 than the chosen cut-off. Median survival of patients with high expression (red line) was 0.92 years vs median survival of patients with lower expression (blue line), 3.02 years. Log-rank Mantel-Cox and Wilcoxon test were performed to statistically analyze the survival rate between the two groups.