Grb14 protein expression is upregulated in lung adenocarcinoma clinical specimens.
The clinical characteristics of all patients are shown in Table 1. Grb14 protein expression in the TMA was detected by IHC. In this TMA, the expression profile and localization of Grb14 in 75 lung adenocarcinoma and 75 adjacent lung adenocarcinoma tissues were examined by immunohistochemical analysis (Figure 1A). The expression level of Grb14 in the lung adenocarcinoma tissues was significantly higher than that in the noncancerous lung adenocarcinoma tissues (lung adenocarcinoma 6.07±1.01 vs. benign, 4.80±1.22; P<0.01) (Figure 1B). Immunohistochemical staining revealed that Grb14 immunostaining occurred mainly in the cytoplasm of the cells from the lung adenocarcinoma tissue (Figure 1C and D). The intensity of Grb14 staining was high in the cytoplasm of cancer cells (Figure 1E), while it was low in the cytoplasm of normal lung tissue samples Figure 1F).
Table 1. Clinical characteristics of all patients
Clinical characteristics TMA TCGA
|
Lung adenocarcinoma (cases)
|
75 230
|
Age (mean)
< 65
≥ 65
Pathological stage
T1
T2
T3
T4
Metastasis
NO
YES
Tumor stage
Stage I
Stage II
Stage III
Stage IV
|
48 84
27 122
20 60
41 130
11 17
3 14
71 158
4 8
37 115
18 47
16 46
4 9
|
|
|
TMA, tissue microarray (tissues analyzed by immunohistochemistry). The TCGA dataset was contributed by EA et al13. All 230 patients in the TCGA dataset were given a follow-up examination ranging from 0 to 224 months (median, 13.35 months). The primary analysis endpoint for the cohort of patients was overall survival. All patients who succumbed to diseases other than lung adenocarcinoma or unexpected events were excluded from the cohort.
High Grb14 mRNA levels in the TCGA dataset are associated with disease progression and poor prognosis in lung adenocarcinoma. To further assess the prognostic value of high Grb14 expression in lung adenocarcinoma patients, Kaplan-Meier analysis was performed to compare prognosis between patients with high and low Grb14 expression levels (Figure 2). The mean Grb14 expression level in the TCGA dataset was used as a cut-off to divide cases into high and low Grb14 expression groups. Interestingly, overall survival in the high and low Grb14 expression groups was 41.59 ± 5.2 and 88.67 ± 16.69 months, respectively, indicating that lung adenocarcinoma patients with high Grb14 expression experienced shorter survival times than the low expression group (log rank = 7.714, P = 0.005). Association of Grb14 protein and mRNA expression with the clinicopathological characteristics of patients with lung adenocarcinoma. As shown in Table 2, the results from immunostaining with the limited clinical information from the TMA failed to reveal any significant association between Grb14 expression and the clinicopathological characteristics of the patients with lung adenocarcinoma tissues. Since the increased expression of Grb14 protein in lung adenocarcinoma tissues did not correlate with the clinicopathological characteristics of our TMA cohort, we proceeded to analyze Grb14 expression at the mRNA level. However, we failed to find any significant association between Grb14 expression and the clinicopathological characteristics of the patients with lung adenocarcinoma in the TCGA dataset. The reason may be related to the limited amount of clinical information.
Table 2. Correlation of GRB14 expression with the clinicopathological characteristics of lung adenocarcinoma
Clinical characteristics
|
TMA
|
|
Grb14 expression in TCGA dataset
|
Cases
|
Mean ± SD
|
P-value
|
Cases
|
Mean ± SD
|
P-value
|
|
Grb14 expression
Benign
Cancer
|
75
75
|
4.80±1.22
6.07±1.01
|
<0.01
|
|
|
-
-
|
|
Age (years)
<65
≥65
Metastasis
NO
YES
Pathological stage
≤T3A
|
48
27
71
4
61
|
6.13±1.02
5.96±1.16
6.06±1.08
6.25±0.96
6.00±1.11
|
0.533
0.727
0.263
|
|
84
122
158
8
207
|
137.71±205.06
185.88±218.41
177.49±317.71
129.55±88.93
175.21±304.08
|
0.112
0.672
0.633
|
>T3A
Overall survival
Alive
Deceased
|
14
-
-
|
6.36±0.84
|
|
|
14
155
67
|
215.18±275.39
175.98±335.78
179.27±202.98
|
0.941
|
-, indicates lack of relative information of patients. TCGA dataset contributed by Collisson EA et al13.
Prognostic implications of Grb14 expression in lung adenocarcinoma.
As shown in Table 3, univariate analysis and multivariate analysis revealed that there was a significant difference in the survival rates between the patients with high Grb14 expression and those with low Grb14 expression [hazard ratio (HR), 1.960; 95% confidence interval (CI), 1.212-3.299; P<0.001] and (HR 3.418; 95% CI 1.610-7.528). In addition, univariate analysis revealed that higher pathological stage [hazard ratio (HR), 1.925, 95% CI, 0.912-6.301; P<0.05], higher tumor stage (HR, 2.436, 95% CI, 1.659-5.551; P<0.001), higher surgical margin resection status (HR, 3.035, 95% CI, 1.305-37.51; P<0.01) and prior diagnosis status (HR, 0.4893, 95% CI, 0.1818-0.8197; P<0.01) were independent predictors for poor survival. Multivariate analysis also revealed that higher pathological stage (HR, 3.730, 95% CI, 1.784-7.796; P<0.01), higher surgical margin resection status (HR, 6.914, 95% CI, 2.063-23.171; P<0.01) and prior diagnosis status were related to poor survival.
Table 3. Prognostic value of Grb14 expression for overall survival by Cox proportional hazards model.
|
Overall survival
|
|
|
Overall survival
|
|
|
Hazard ratio (95% CI)
|
P-value
|
|
Hazard ratio (95% CI)
|
P-value
|
Univariate analysis
|
|
|
Multivariate analysis
|
|
|
Grb14
|
1.960 (1.212-3.299)
|
<0.001
|
Grb14
|
3.418 (1.610-7.258)
|
<0.001
|
Age
Metastasis
Pathological stage
|
1.271 (0.474-3.626)
1.925 (0.912-6.301)
|
0.079
0.625
<0.05
|
Age
Metastasis
Pathological stage
|
0.292 (0.093-0.913)
3.730 (1.784-7.796)
|
0.088
0.532
<0.01
|
Tumor stage
Surgical margin resection status
Prior diagnosis
|
2.436 (1.659-5.551)
3.035 (1.305-37.51)
0.4893 (0.1818-0.8197)
|
<0.001
<0.05
<0.01
|
Tumor stage
Surgical margin resection status
Prior diagnosis
|
-
6.914 (2.063-23.171)
0.449 (0.200-1.008)
|
<0.01
<0.01
<0.05
|