Demographic characteristics of study participants
We enrolled at total of 121 study participants, 44 treatment-naïve early-stage NSCLC patients, 20 non-malignant pulmonary diseases and 57 healthy individuals. The median age of early-stage NSCLC patients was 63.5 years [range 34–83]. Seventy-two percent of patients were female. All patient had ECOG performance of 0–1. A majority (97.7%) of histology was adenocarcinoma. Pathological stage I disease accounted for 68.2%. All patients received curative attempt operation such as lobectomy and bilobectomy. The median age for the non-malignant pulmonary diseases and healthy controls were 48.5 years [range 30–76] and 58 years [range 30–86], respectively. Ninety-five percent of non-malignant pulmonary disease patients presented with pulmonary nodule mimic lung cancer and had operative procedure. A majority (75%) of resected lung tissue histology was infection and inflammation. Details of patient demographics and disease characteristics at study enrollment are provided in Table 1.
Table 1
Baseline clinical characteristics of early-stage NSCLC patients, non-malignant pulmonary diseases and healthy control participants.
Characteristics
|
Early NSCLC patients
(n = 44) (%)
|
Non-malignant pulmonary disease (n = 20) (%)
|
Healthy Controls
(n = 57) (%)
|
Age
≥ 60 year
< 60 year
|
30 (68)
14 (32)
|
7 (35)
13 (65)
|
27 (47.3)
30 (52.7)
|
Sex, n (%)
Male
Female
|
12 (27.3)
32 (72.7)
|
8 (40)
12 (60)
|
20 (36)
37 (64)
|
Smoking, n (%)
Current
Former
Never
|
5 (11.3)
7 (15.9)
32 (72.8)
|
5 (25)
7 (35)
8 (40)
|
2 (3.5)
3 (5.2)
52 (91.3)
|
Histology; malignancy, n (%)
Adenocarcinoma
Adenosquamous cell carcinoma
|
43 (97.7)
1 (2.3)
|
N/A
|
N/A
|
Histology; non-malignancy, n (%)
Granulomatous inflammation
Nodular amyloidosis
Bronchiectasis with chronic inflammation
Chronic and acute inflammation
Hyalinized fibrous nodule
Necrotizing lymphoid hyperplasia
|
N/A
|
12 (60)
2 (10)
1 (5)
3 (15)
1 (5)
1 (5)
|
N/A
|
Staging by AJCC version 7th edition
IA
IB
IIA
IIB
IIIA
IIIB
|
21 (47.7)
9 (20.5)
6 (13.6)
1 (2.3)
6 (13.6)
1 (2.3)
|
N/A
|
N/A
|
N/A = non-applicable |
Quality control assessment and storage time effect
Specific CD3 positive lymphocyte was our population of interest. Automated CD3 positive expression flow cytometry could detect over 10,000 PBMC of 3 protein expression assays: C5AR1, NLRP3 and CLEC4A in 86.7%, 83.4% and 85.1%, respectively. Median collected freezing time of PBMC to automated flow cytometry testing was 19.6 months [range 0-100.4 month; 95% CI 15.9–40]. There was no difference in freezing time of PBMC for both over and under 10,000 PBMC detection of C5AR1, NLRP3 and CLEC4A protein expression (Mann-Whitney U test; p-value 0.5, 0.8 and 0.8, respectively). Long-term storage in -196 °C did not affect the quality of protein detection.
Protein expression on PBMC in early-stage lung cancer patients and healthy controls
The potential of protein expression as a biomarker for early lung cancer detection was first explored comparing to healthy control. The ratio of specific antibody staining to CD3 positive was calculated from the number of specific antibody staining to CD3 positive. Median ratio of C5AR1, NLRP3 and CLEC4A expression in early-stage NSCLC patients were 0.65 [range 0.27–0.96; 95% CI: 0.55–0.70], 0.83 [range 0.27–0.99; 95% CI: 0.73–0.86] and 0.75 [range 0.21–0.98; 95% CI: 0.65–0.81], respectively. While, median ratio of C5AR1, NLRP3 and CLEC4A expression in healthy controls were 0.21 [range 0.05–0.81; 95% CI: 0.23–0.42, p-value < 0.001], 0.32 [range 0.04–0.94; 95% CI: 0.28–0.46, p-value < 0.001] and 0.22 [range 0.04–0.97; 95% CI: 0.27–0.48, p-value < 0.001], respectively. The number of those CD3 positive cells with specific antibody expressions were higher in early-stage NSCLC patients than healthy controls.
In addition to the number of positive staining, flow cytometry also enables the determining of the fluorescence intensity on the cell surface of each specific antibody-stained cell. The average intensity of C5AR1, NLRP3 and CLEC4A expressions in early-stage NSCLC patients were 528.6 [range 342.9-1065.6; 95% CI: 472-554.4], 622.1 [range 440.7–1227; 95% CI: 605-730.7] and 614.2 [range 350.8-1128.3; 95% CI: 575.9-695.3], respectively. While, average intensity of C5AR1, NLRP3 and CLEC4A expression in healthy controls were 619.6 [range 305.8-2465.8; 95% CI: 575.1–725, p-value 0.002], 535.1[range 311.2-1852.7; 95% CI: 520.6-616.5, p-value 0.008] and 532.1 [range 356.3-1364.8; 95% CI: 528.7-631.9, p-value 0.14], respectively. The average adjusted specific protein expression was calculated from average intensity expression × ratio of specific antibody staining to CD3 positive. Average adjusted expression of C5AR1, NLRP3 and CLEC4A in early-stage NSCLC patients were 317.8 [range 104.2-1023.8; 95% CI: 273.5-386.4], 536.1 [range 171.8–1217; 95% CI: 464.1-604.1] and 466.7 [range 135.9-928.4; 95% CI; 410.5-530.2]. The average adjusted expression of C5AR1, NLRP3 and CLEC4A in healthy controls were 140.8 [range 33.2-2002.7; 95% CI: 141.3-303.1, p-value 0.004], 190 [range 12.7–1754; 95% CI: 147.5-299.1, p-value < 0.001] and 133.8 [range 15.3-1325.7; 95% CI: 138-312.4, p-value < 0.001] (Table 2). Both ratio and average adjusted intensity of C5AR1, NLRP3 and CLEC4A were significant higher in early-stage NSCLC patients compared to healthy controls. This suggests that the number of specific protein expression of C5AR1, NLRP3 and CLEC4A on immune cell surface changes in the presence of cancer. The comparison of ratio and average adjusted fluorescence intensities of specific protein expression on CD3 positive lymphocytes between early NSCLC patients and healthy controls are shown in Fig. 1.
Table 2
Shows results of C5aR1, NLRP3 and CLEC4A protein expression of circulating T- lymphocyte of 121 study participants including 44 early-stage NSCLC patients, 20 non-malignant pulmonary diseases and 57 healthy controls.
|
Total (n = 121)
|
Early-stage NSCLC (n = 44)
|
Non-malignant pulmonary disease (n = 20)
|
Healthy control (n = 57)
|
C5aR1 expression
- Pass QC*
- Ratio# [range]
- Average expression [range]
- Adjusted expression [range]
|
86.7%
0.50 [0.05–0.99]
565.9 [305.8-2465.8]
271 [33.2-2002.7]
|
86.3%
0.65 [0.27–0.96]
528.6 [342.9-1065.6]
317.8 [104.2-1023.8]
|
89.4%
0.75 [0.34–0.99]
538.6 [370.9-1177.4]
348.3 [190.3-1172.4]
|
85.9%
0.21 [0.05–0.81]
619.6 [305.8-2465.8]
140.8 [33.2-2002.7]
|
NLRP3 expression
- Pass QC
- Ratio [range]
- Average expression [range]
- Adjusted expression [range]
|
83.4%
0.68 [0.04–0.99]
587.8 [311.2-1852.7]
400.6 [12.7–1754]
|
90.9%
0.83 [0.27–0.99]
622.1 [440.7–1227]
536.1 [171.8–1217]
|
70%
0.87 [0.32–0.99]
627.1 [464.4-1091.9]
532.7 [201.2-797.1]
|
82.4%
0.32 [0.04–0.94]
535.1 [311.2-1852.7]
190 [12.7–1754]
|
CLEC4A expression
- Pass QC
- Ratio [range]
- Average expression [range]
- Adjusted expression [range]
|
85.1%
0.67 [0.04–0.99]
578.9 [350.8-1364.8]
379.6 [[15.3-1325.7]
|
88.6%
0.75 [0.21–0.98]
614.2 [350.8-1128.3]
466.7 [135.9-928.4]
|
75%
0.80 [0.19–0.99]
631.8 [396.3-919.1]
499 [77-918.5]
|
85.9%
0.22 [0.04–0.97]
532.1 [356.3-1364.8]
133.8 [15.3-1325.7]
|
*Pass QC accounting percentage of specimen that had more than 10,000 CD3 + in each specific antibody |
#Ratio calculated by percentage positive cells of specific antibody to CD3 |
Protein expression on PBMC as potential candidate to discriminate early-stage non-small cell lung cancer patients from healthy controls
To assess the potential to discriminate early-stage NSCLC patients from healthy controls, optimal cut-off ratio of each specific antibody staining positive with CD3 positive was conducted. The C5AR1 expression ratio cut-off value at 0.28 could distinguish early-stage NSCLC patients from healthy controls at 100% sensitivity and 50.9% specificity (0.77 area under the ROC curve, accuracy rate 71.4%). The NLRP3 expression ratio cut-off value at 0.58 could distinguish early-stage NSCLC patients from healthy controls at 87.5% sensitivity and 64.7% specificity (0.83 area under the ROC curve, accuracy rate 74%). The CLEC4A expression ratio cut-off value at 0.34 could distinguish early-stage NSCLC patients from healthy controls at 97.4% sensitivity and 57.1% specificity (0.80 area under the ROC curve, accuracy rate 75%) (Fig. 1). Specific protein expression ratio to CD3 positive staining showed better discrimination power than average adjusted protein expression.
Protein expression on PBMC in early-stage lung cancer and non-malignant pulmonary disease
An important clinical issue in the discovery of a biomarker is the ability to discriminate non-malignant pulmonary nodules. We explored specific antibody expression staining to CD3 positive in 20 non-malignant pulmonary diseases compared to early-stage NSCLC patients. C5AR1, NLRP3 and CLEC4A expression ratios in non-malignant pulmonary disease were 0.75 [range 0.34–0.99; 95% CI: 0.60–0.71, p-value 0.2], 0.87 [range 0.32–0.99; 95% CI: 0.66–0.92, p-value 0.62] and 0.80 [range 0.19–0.99; 95% CI: 0.63–0.87, p-value 0.57], respectively. Average intensity of C5AR1, NLRP3 and CLEC4A expression in non-malignant pulmonary disease were 538.6 [range 370.9-1177.4; 95% CI: 471.4-604.9, p-value 0.48], 627.1 [range 464.4-1091.9; 95% CI: 556.8-778.1, p-value 0.92] and 631.8 [range 396.3-919.1; 95% CI: 528.5-696.3, p-value 0.69], respectively. Neither ratio nor intensity expression specific antibody staining in CD3 positive revealed a difference to early-stage NSCLC patients. Immune cell reaction of C5AR1, NLRP3 and CLEC4A could not discriminate non-malignant process to malignant process.