Edited by: Sacha I. Rothschild, Universität Basel, Switzerland
Reviewed by: Janaki Deepak, University of Maryland, Baltimore, United States; Matteo Giaj Levra, Centre Hospitalier Universitaire de Grenoble, France
This article was submitted to Thoracic Oncology, a section of the journal Frontiers in Oncology
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Lung cancer is the most common and deadliest cancer worldwide (
In the last decade, inhibitors of PD-1 and its ligand PD-L1 were introduced into clinic. As a cell surface protein, PD-L1 is wildly expressed in variety of cells (such as immune cells, endothelial cells and tumor cells) and suppress immune-response lead by lymphocytes (
Like PD-L1, vascular endothelial growth factor (VEGF) is known as an immunosuppressive factor and plays an important role in tumor immune through promoting a tumor immunosuppressive microenvironment (
Few studies reported that the connection between PD-L1 and VEGFA expression in patients with resected LADC. In the present study, we retrospectively reviewed on the data of patients with resected tissue of LADC and investigated the relationship between PD-L1 and VEGFA expression to further explore their potential efficacy-predictive value in anti-angiogenic therapy with immunotherapy and their prognostic significance.
We retrospectively reviewed 129 patients with LADC (56 with wild type, 56 with EGFR mutations, 13 with KRAS mutations, 4 with ALK mutations) surgically treated in Tianjin Medical University Cancer Institute and Hospital between December 2011 and September 2016. The postoperative follow-up period ranged from 21 to 79 months (median: 47 months). The patients' inclusion criteria included: (1) complete following-up date; (2) without neoadjuvant therapy before surgery; Regarding to preoperative staging assessment, all patients underwent positron emission tomography-computed tomography (PET-CT) or enhanced chest CT, contrast-enhanced computed tomography (ECT) and brain magnetic resonance imaging (MRI) if they were necessary. Twelve patients with stage IVA refer to incidental pleural metastases detected at surgery and 20 patients with stage III were found incidental N2 nodes during surgery. The clinical data included age at diagnosis, sex, smoking history, gene mutation status, histological subtypes, clinical stage, and postoperative chemotherapy; all parameters were retrieved based on electronic medical records. Progression-free survival (PFS) was defined as the interval between the date of resection and the date of disease progression or death. The follow-up for patients' survival with documented disease progression were censored on the last follow-up date. Overall survival (OS) was defined as the interval between the date of resection and either death due to any cause or the last follow-up. All clinical information was obtained from medical records. The study was approved by the Tianjin Medical University Cancer Institute and Hospital's Ethics Committee and informed consent was obtained from all patients in verbal form.
Four micrometer thickness of tumor sections were deparaffinized and rehydrated. Antigen retrieval was performed at 130°C for 2 min, using EDTA solution (PH 11.0) for PD-L1, or citrate buffer (PH 6.0) for VEGFA. After blocking with 3% hydrogen peroxide and normal goat serum, the sections were incubated with primary antibody against PD-L1/CD274 antibody (66248-1-lg, Proteintech, USA) at dilution 1:1,200 or anti-VEGFA antibody (ab1316, Abcam, USA) at dilution 1:300 at 4°C overnight, followed by EIVISON plus (kit-9903, MXB, China), DAB kit (ZL1-9019, ZSGB-BIO, China) per the manufacturers protocol were used for coloration. Finally, the tumor sections were stained with hematoxylin and evaluated under a light-field microscope.
To measure the PD-L1 and VEGFA expression by IHC, stained sections were digitally analyzed at x400 resolution using an Olympus BX-UCB. PD-L1 and VEGFA expression were scored by H-score system. An H-score (range, 0–300) was calculated as the sum of the product of the highest intensity of staining (0, negative; 1, weak positive; 2, moderate positive; 3, strong positive) and percentage of tumor cells positive (0–100%; with any intensity of positive staining) (
Images of immunohistochemical staining for PD-L1 and VEGFA expression in LADC. The staining strength of PD-L1 expression:
Statistical analyses were performed using SPSS v.21 (IBM Corp, Armonk, NY, USA) and survival curve was performed with GraphPad Prism 6 (USA, GraphPad Software). The relationships between PD-L1/VEGFA expression and clinical features were compared using Fisher's exact test; the linear relationship between PD-L1 and VEGFA expression was determined using Pearson's test. Patients' survival was estimated by the Kaplan-Meier method and compared using the log-rank test. Multivariate and univariate analysis were performed using the cox proportional hazards regression model to calculate the hazard ratio (HR) and 95% CI. Two-tailed
Baseline clinic-pathological characteristics of 129 LADC cases were summarized in
Clinical characteristics of 129 patients with LADC.
Male | 61 (52.7) |
Female | 68 (47.3) |
<65 | 104 (80.6) |
≥65 | 25 (19.4) |
Yes | 57 (44.2) |
No | 72 (55.8) |
I | 73 (56.6) |
II | 19 (14.7) |
III | 25 (19.4) |
IV | 12 (9.3) |
Lepidic predomiant | 47 (36.4) |
Acinar predomiant | 41 (31.8) |
Papillary/micropapillary predomiant | 23 (17.8) |
Solid predomiant | 7 (5.5) |
Other | 11 (8.5) |
T1 | 72 (55.8) |
T2 | 40 (31.0) |
>T2 | 17 (13.2) |
N0 | 86 (66.7) |
N1 | 8 (6.2) |
N2 | 35 (27.1) |
M0 | 117 (90.7) |
M1 | 12 (9.3) |
WT | 56 (43.4) |
EGFR | 56 (43.4) |
Other mutations | 17 (13.2) |
H-socre ≤ 50 | 64 (49.6) |
H-socre >50 | 65 (50.4) |
H-socre ≤ 100 | 76 (58.9) |
H-socre >100 | 53 (41.1) |
Chemotherapy/chemoradiotherapy | 46 (35.7) |
Chemotherapy+TKI/TKI | 7 (5.4) |
Non-treatment | 76 (58.9) |
Relationship between VEGFA/PD-L1 expression and the clinical characteristics in 129 patients with LADC.
0.703 | 0.250 | |||||
Male | 37 (48.7) | 24 (45.3) | 27 (42.2) | 34 (52.3) | ||
Female | 39 (51.3) | 29 (54.7) | 37 (57.8) | 31 (47.7) | ||
0.902 | 0.532 | |||||
<65 | 61 (80.3) | 43 (81.1) | 53 (82.8) | 51 (78.5) | ||
≥65 | 15 (19.7) | 10 (18.9) | 11 (17.2) | 14 (21.5) | ||
0.383 | 0.690 | |||||
Yes | 36 (47.4) | 21 (60.4) | 27 (41.3) | 30 (46.2) | ||
No | 40 (52.6) | 32 (39.6) | 37 (58.7) | 35 (53.8) | ||
0.867 | 0.733 | |||||
I | 45 (59.2) | 28 (52.8) | 35 (54.7) | 38 (58.5) | ||
II | 11 (14.5) | 8 (15.2) | 11 (17.2) | 8 (12.3) | ||
III | 13 (17.1) | 12 (22.6) | 11 (17.2) | 14 (21.5) | ||
IV | 7 (9.2) | 5 (9.4) | 7 (10.9) | 5 (7.7) | ||
0.529 | 0.350 | |||||
Lepidic predominate | 29 (38.1) | 18 (34.0) | 25 (39.1) | 22 (33.8) | ||
Acinar predominate | 26 (34.2) | 15 (28.3) | 15 (23.4) | 26 (40.0) | ||
Papillary/micropapillary predominate | 10 (13.2) | 13 (24.5) | 13 (20.3) | 10 (15.4) | ||
Solid predominate | 5 (6.6) | 2 (3.8) | 4 (6.3) | 3 (4.6) | ||
Other | 6 (7.9) | 5 (9.4) | 7 (10.9) | 4 (6.2) | ||
0.051 | 0.281 | |||||
T1 | 42 (55.3) | 30 (56.6) | 40 (62.5) | 32 (49.2) | ||
T2 | 28 (36.8) | 12 (22.7) | 16 (25.0) | 24 (37.0) | ||
>T2 | 6 (7.9) | 11 (20.7) | 8 (12.5) | 9 (13.8) | ||
0.721 | 0.700 | |||||
N0 | 50 (65.8) | 36 (67.9) | 43 (67.2) | 43 (66.2) | ||
N1 | 6 (7.9) | 2 (3.8) | 5 (7.8) | 3 (4.6) | ||
N2 | 20 (26.3) | 15 (28.3) | 16 (25.0) | 19 (29.2) | ||
0.760 | 1.000 | |||||
M0 | 68 (89.5) | 49 (92.5) | 58 (90.6) | 59 (90.8) | ||
M1 | 8 (10.5) | 4 (7.5) | 6 (9.4) | 6 (9.2) | ||
0.081 | 0.417 | |||||
WT | 33 (43.4) | 23 (43.4) | 28 (43.8) | 28 (43.1) | ||
EGFR | 29 (38.2) | 27 (50.9) | 30 (46.9) | 26 (40.0) | ||
Other mutations | 14 (18.4) | 3 (5.7) | 6 (9.3) | 11 (16.9) | ||
0.693 | 0.472 | |||||
Chemotherapy/chemoradiotherapy | 28 (36.8) | 18 (34.0) | 22 (34.4) | 24 (36.9) | ||
Chemotherapy+TKI/TKI | 3 (3.9) | 4 (7.5) | 2 (3.1) | 5 (7.7) | ||
Non-treatment | 45 (59.3) | 31 (58.5) | 40 (62.5) | 36 (55.4) | ||
H-score ≤ 50 | 29 (38.2) | 35 (66.0) | ||||
H-score >50 | 47 (61.8) | 18 (34.0) |
However, we found that expression of VEGFA was negatively correlated with expression of PD-L1 (
PD-L1 and VEGFA expression were measured in 129 patients wherein 53 patients (41.1%) had high expression PD-L1 (H-score >100) and 65 patients (50.4%) had high expression VEGFA (H-score >50). In all patients, PD-L1high group showed a significant negative impact on the OS (
Association with PD-L1 expression and OS or PFS. There was a significant poor OS
Therefore, we further investigated the association between the expression of PD-L1 and survival in different subgroups. Firstly, we found high PD-L1 expression was not significantly correlated with adverse survival in patients of clinical stage I/II, in contrast, significantly correlated with poor OS (
Kaplan–Meier curves showing OS
Finally, we found that high PD-L1 expression was as a poor prognostic role for PFS (
Kaplan–Meier curves showing OS
In a univariate analysis on the all patients, six clinical characteristics were determined as unfavorable prognostic factors for PFS: clinical stage III/IV [HR = 6.552 (95% CI 3.852–11.145),
Univariate and multivariate cox analysis of factors for progression-free survival and overall survival in patients with LADC.
Gender (female vs. male) | 1.451 | 0.819–2.571 | 0.202 | 1.199 | 0.716–2.007 | 0.490 |
Age (<65 vs. ≥65) | 1.461 | 0.744–2.870 | 0.271 | 1.065 | 0.634–1.790 | 0.812 |
Smoking history (no vs. yes) | 1.286 | 0.729–2.267 | 0.385 | 1.046 | 0.543–2.018 | 0.892 |
T factor (T ≤ 2 vs. T >2) | 3.735 | 1.997–6.985 | 3.778 | 2.073–6.855 | ||
N factor (N0 vs. >N0) | 2.798 | 1.580–4.955 | 3.674 | 2.178–6.194 | ||
M factor (M0 vs. M1) | 3.254 | 1.568–6.750 | 3.710 | 1.863–7.391 | ||
Gene mutations (WT vs. EGFR) | 1.099 | 0.662–1.942 | 0.745 | 1.277 | 0.761–2.141 | 0.354 |
Histological subtypes (acinar vs. non-acinar) | 0.599 | 0.337–1.063 | 0.113 | 0.706 | 0.544–0.916 | 0.089 |
Stage (I/II vs. III/IV) | 4.801 | 2.698–8.542 | 6.552 | 3.852–11.145 | ||
Postoperative therapy (else vs. adjuvant chemotherapy) | 0.917 | 0.492–1.709 | 0.785 | 1.036 | 0.587–1.828 | 0.903 |
VEGFA expression (≤50 vs. >50) | 1.470 | 0.823–2.624 | 0.193 | 1.297 | 0.711–2.183 | 0.327 |
PD-L1 expression (≤100 vs. >100) | 1.924 | 1.080–3.427 | 3.143 | 1.106–1.864 | ||
Co-expression (else vs. PD-L1+/VEGFA+) | 3.114 | 1.564–6.201 | 2.907 | 1.082–4.067 | ||
T factor | 2.313 | 1.130–4.733 | 1.389 | 0.694–2.778 | 0.353 | |
N factor | 1.546 | 0.720–3.316 | 0.263 | 1.806 | 0.919–3.550 | 0.086 |
M factor | 1.730 | 0.756–3.956 | 0.194 | 1.642 | 0.738–3.655 | 0.224 |
Stage | 2.657 | 1.085–6.504 | 4.064 | 1.815–9.097 | ||
PD-L1 expression | 1.494 | 0.735–3.036 | 0.267 | 2.163 | 1.124–4.163 | |
Co-expression | 3.230 | 1.388–7.518 | 1.360 | 0.621–2.980 | 0.443 |
Recently, some clinical trials (
In the present study, we analyzed the correlation of PD-L1 and VEGFA expression and patients' prognosis. There was no significant correlation between VEGFA expression and survival found, as similar as the results in other studies (
The interesting finding from our study is that PD-L1 expression inversely related to the expression of VEGFA. About this issue, the traditional view is that VEGFA high expression will lead to vascular abnormalities, further aggravation of hypoxia and the activation of HIF-1a pathway in tumor tissues, which could result in increased expression of PD-L1 on tumor cells. According to this viewpoint there seems to be a close positive association between PD-L1 and VEGF expression in tumor tissues. Several studies also found that VEGF expression is positive associated with PD-L1 expression in clear cell renal cell carcinoma (
Finally, we found that only 14.0% (18/129) patients revealed VEGFA+&PD-L1+, far fewer than the other types, but these patients had the worst prognosis compared to other groups. As far as we know, no study has reported this phenomenon in LADC, so we proposed another hypothesis: some LADC tumor cells in patients with co-expression of VEGFA and PD-L1 may have strong ability to evade from the attacks by immune cells, so even if few immune cells infiltrate into tumor tissue, through abnormal vasculature caused by high-expressed VEGFA, they can also cause “high-response” tumor cells to express high PD-L1. This explanation indicated that co-expression of PD-L1 and VEGFA may be as a predictor for high recurrent risk and poor prognosis. For these patients, the combination of anti-VEGF and anti-PDL1 could be an interesting treatment strategy. It also provides a theoretical possibility for screening optimal population to combination of anti-VEGFA and anti-PD-1/PD-L1 therapy. We also analyzed the relationship between co-expression of PD-L1 and VEGFA and clinical features, but failed to found a significant correlation between them and clinical features maybe due to only 18 patients with co-expression of PD-L1 and VEGFA.
Our study still has several limitations: 1. A similar limitation with other studies is the lack of standardized cut-off value of PD-L1 and VEGFA expression; 2. The amount of patient samples collected retrospectively were relatively small; 3. It should be emphasized that it is the initial and immature study to explore the correlation of VEGFA and PD-L1 expression in LADC; we demonstrated the correlation between them, but the underlying mechanisms is still unclear.
High expression PD-L1 is a poor factor on PFS and OS in patients with WT, clinical stage III/IV or acinar adenocarcinoma, but has no significant impact on patients with early stage and EGFR mutations. Expression of VEGFA is negatively correlated with the expression of PD-L1, but patients with co-expression of PD-L1 and VEGF will lead to significantly poor prognosis than negative ones. Our study also provides the theoretical possibility to screen optimal population of combination with anti-PD-L1/PD-1 and anti-VEGF therapy in LADC.
SL: design of study and manuscript preparation; YJ: data statistics; TQ and KL: critical revision of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
Scatter diagram showing the correlation of VEGFA expression and PD-L1 expression based on the results of H-score.
Kaplan–Meier curves showing OS
Correlation of expression of VEGFA and/or PD-L1 and the clinical characteristics in 129 patients with LADC.
Correlation of co-expression of VEGFA and PD-L1 and the clinical characteristics in 129 patients with LADC.