Percutaneous microwave ablation (MWA) increased the serum levels of VEGF and MMP-9 in Stage I non-small cell lung cancer (NSCLC)

Abstract

Background: Lung cancer is the leading cause of cancer death around the world. Percutaneous microwave ablation (MWA) is an emerging treatment strategy for medically inoperable early-stage non-small cell lung cancer (NSCLC). In this study, we investigated the association of MWA and serum angiogensis promoters VEGF and MMP-9 in these patients subgroup.

Methods: We enrolled 52 patients with Stage I NSCLC patients in this study. For each patient, blood samples were drawn by venous puncture, one immediately prior to MWA and the others on Post-Procedure Days (PPD) 1, 3, 5, 7, 10 and 14. Serum samples were analysed for VEGF and MMP-9 levels with use of commercially available enzyme-linked immunosorbent assay. Also, blood samples of 28 healthy volunteers were set as the healthy controls.

Results: We did not observe a significant difference of serum VEGF and MMP-9 between NSCLC patients and healthy controls. The VEGF levels increased on the first day (256.0 ± 6.16 pg/ml, p < 0.05) after MWA and peaked on the PPD3 (418.0 ± 14.54 pg/ml, p < 0.05). Although it gradually reduced afterwards, its levels on PPD14 (141.2 ± 4.41 pg/ml, p < 0.05) was still higher than pre-procedure level. The serum MMP-9 level was significantly elevated from PPD1 (231.3 ± 7.93 ng/ml, p < 0.05) until PPD10 (155.3 ± 5.62 ng/ml, p < 0.05), while it normalised to pre-procedure level on PPD14 (90.78 ± 3.36 ng/ml, p > 0.05). The highest MMP-9 level was observed on PPD5 (399.7 ± 17.70 ng/ml, p < 0.05).

Conclusion: Our preliminary results indicated that percutaneous MWA resulted in increased serum levels of VEGF and MMP-9 in Stage I NSCLC patients. Antiangiogenesis approaches may be helpful for patients defending against metastases during the immediate post-ablation time window.

Keywords:

• Microwave ablation (MWA)
• VEGF
• MMP-9
• non-small cell lung cancer (NSCLC)

Introduction

Lung cancer remains the leading cause of cancer death around the world [1]. Surgical resection is considered as the standard of curative therapy for early-stage resectable non-small cell lung cancer (NSCLC). However, patients with coexistent medical morbid conditions are not suitable for surgery, limiting the management options of their lung malignancy [2,3]. Over the past decade, thermal ablation as a minimally invasive therapy has emerged as a viable alternative strategy to surgery in medical inoperable NSCLC patients. In pulmonary tumours, microwave ablation (MWA) therapy is considered superior to other thermal ablation options with respect to its physical properties [4–7]. We recently showed that MWA is safe and effective for the treatment of Stage I peripheral NSCLC [8,9].

Nevertheless, even after apparent complete ablation, some patients still develop local or distant recurrence. There are multiple variables that, together, determine whether or not a metastasis develops. The formation of new blood vessels from existing vasculature, termed as angiogenesis, plays a critical role in the growth and development of tumours [10], as tumour enlargement requires an increased blood supply to feed the growing mass of cells. Many solid tumours cannot grow beyond 2–3 mm without neovascularisation [11,12]. Angiogenesis, growth of new blood vessels, is regulated by at least in part by the balance of numerous pro- and anti-angiogenesis factors. Of note, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) are two of the most potent factors involved in this process.

VEGF is identified as one of the important potent and specific growth factor of angiogenesis, with direct effects on endothelial cell proliferation, migration and vascular permeability [13]. Elevated expression of VEGF was observed in many types of malignancies including colon, breast and NSCLCs [14–16]. Furthermore, NSCLC patients with higher serum VEGF level have a shorter overall survival than those with a lower serum level [17]. The MMPs belong to a family of zinc-dependent neutral endopeptidases. Under physiological conditions, they are capable of degrading extracellular matrix and basement membrane components. The malignant tumour cell metastasis requires several steps, in which the first phase is the destruction and penetration of the basement membrane. There are more than 20 members of the MMP family [18]. The gelatinases MMP-9, formerly known as gelatinase B or 92 kDa Type IV collagenase, have been shown to be strongly correlated with tumour aggressiveness and metastatic potential [16,19]. Pre-treatment serum MMP-9 level was also a novel prognostic marker in patients with NSCLC [16]. Interesting, surgical trauma is shown to be associated with altered circulating angiogenesis factors levels in NSCLC patients, including VEGF and MMP-9 [20–23]. However, the effects of MWA, as an alternative treatment strategy, on these serum angiogenesis factors have not been studied.

To our knowledge, this is the first study to examine the serum VEGF and MMP-9 concentrations before and after MWA in patients with early-stage NSCLC. We found that the serum VEGF and MMP-9 levels were significantly increased after local MWA.

Materials and methods

Patients

From July 2010 to Jun 2013, 52 patients with primary Stage IA or IB peripheral NSCLC underwent computer tomography-guided percutaneous MWA were enrolled in this study. Standardised pre-treatment investigations included contrast computer tomography of the thorax or positron emission tomography (PET) scan when necessary. All patients were cytologically or histologically confirmed with NSCLC. Patients in this study met the following criteria: (i) Stage IA or IB (T1: 2N0M0), patients with poor lung function (FEV1 < 1 L, FEV1%<50%, MVV <50%); (ii) patients who were medically inoperable with renal or heart dysfunction and other comorbid medical conditions (such as severe diabetes); (iii) patients who refused surgery; and (iv) patients who have had previous therapy to the treated lesion were excluded. All patients were treated only with MWA, and no adjuvant chemotherapy or radiotherapy was applied. In addition, 28 healthy volunteers were enrolled in this study as controls. Ethical approval was given by the ethics committee of the Shandong Provincial Hospital Affiliated to Shandong University. Informed consent for the study was obtained from all patients.

Methods

Blood samples were drawn by venous puncture from each patient, one immediately prior to MWA and the others on Post-Procedure Days (PPD) 1, 3, 5, 7, 10 and 14. Ten millilitres venous blood was taken and subsequently centrifuged at 2000 rpm for 10 min, and aliquots were frozen at −80 °C for later analysis.

Serum samples were analysed for VEGF and MMP-9 levels with use of commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D System Inc., Minneapolis, MN) according to the manufacturer’s instructions.

MWA treatment procedures

Instrumentations used in our medical centre are listed as below: (i) MTC-3C microwave ablation system [Nanjing Qi Ya Research Institute of Microwave Electric, China; registration standard: YZB/country 1408–2003; No. SFDA (III) 20073251059] or (ii) ECO-2450B microwave ablation system [Nanjing, ECO Microwave Institute, China; registration standard: YZB/country1475–2013; No: SFDA (III) 20112251456]. The detailed procedures were similar as previously described [24]. Generally, the microwave emission frequency was set at 2450 ± 50 MHz and ablation power between 60 and 80 W. The effective length of the antenna is 100–180 mm and 14–20G outside diameter. In addition, we applied a water circulation cooling system to reduce the antenna surface temperature. For tumours ≤3.5 cm and 3.6–5.0 cm, we used single or double antennae, respectively. The whole procedures of MWA were performed under CT guidance and the detailed procedures were described in our previous publications [8,9,25]. All patients received a CT scan immediately after MWA to monitor the shape and size of the lesions, as well as to determine any potential complications including pneumothorax, bleeding or others. The proposed ablative margin was 0.5 cm.

Statistical analysis

The results are reported as mean ± standard error unless otherwise stated. The VEGF and MMP-9 levels at the varying time points within a group were compared using Wilcoxon’s matched pairs test. Intergroup analysis was performed using the Mann–Whitney U-test. All statistical analyses were performed using Graph Pad Prism Version 4.1 software for Windows (GraphPad software Inc., San Diego, CA). A p values <0.05 was considered statistically significant.

Results

Concentrations of circulating VEGF and MMP-9 and clinicopathological characteristics in NSCLC patients and healthy controls

The serum of 52 patients (33 men and 19 women; median age 72 years, range 47–82 years) with Stage IA or IB peripheral NSCLC were sampled and analysed. As shown in Table 1, there was no statistically significant difference of circulating VEGF and MMP-9 in any of the clinical markers in terms of patients’ sex, age, lesion location, histopathological types and tumour size. In addition, 28 healthy volunteers consisting of 18 men and 10 women were enrolled in the study as control group. The median age of the control group was 69 (range 46–79 years). There was no significant difference in terms of age and sex between the two groups. As shown in Table 2, the circulating concentrations of VEGF and MMP-9 were similar in Stage I NSCLC patients and healthy control groups (VEGF: 114.6 ± 4.979 versus 117.4 ± 6.523 pg/ml; MMP-9: 88.82 ± 3.584 versus 86.71 ± 4.697 ng/ml, p > 0.05).

Table 1. Relationship between clinicopathological characteristics and circulating VEGF and MMP-9 in 52 Stage I NSCLC patients.

		Circulating cytokine concentration
		VEGF (pg/ml)			MMP-9 (ng/ml)
Clinicopathological characteristics	Numbers (%)	Mean	25–75% Quartile	p Value	Mean	25–75% Quartile	p Value
Total	52	114.6	104.6–124.6		88.82	81.6–96.0
Gender
Male	33 (63.5)	112.7	99.6–125.9		86.9	78.6–95.1
Female	19 (36.5)	117.9	101.4–134.5	0.59	92.2	77.6–106.8	0.60
Age
≥65	40 (76.9)	112.9	101.9–123.9		87.5	78.8–96.3
<65	12 (23.1)	120.4	93.7–147.0	0.71	93.0	80.6–105.4	0.43
Lesion location
Left lung	29 (55.8)	119.8	105.9–133.8		93.7	83.4–104.0
Right lung	23 (44.2)	108.1	93.2–123.0	0.23	82.7	72.6–92.8	0.13
Tumour histology
Adenocarcinoma	31 (59.6)	115.1	102.6–127.6		89.7	78.6–100.9
Squamous cell carcinoma	14 (26.9)	125.0	100.3–149.6		89.6	78.8–100.4
Others	7 (13.5)	92.0	73.6–110.3	0.15	83.1	67.4–98.8	0.65
Tumour size
>3 cm	33 (63.5)	111.5	98.8–124.1		89.0	81.9–96.1
≤3 cm	19 (36.5)	120.1	102.5–137.8	0.44	88.5	72.0–105.1	0.41

Table 2. Expression levels of serum VEGF and MMP-9 for healthy controls and patients after MWA.

	Circulating cytokine concentration
	VEGF (pg/ml)			MMP-9 (ng/ml)
Time point	Mean	25–75% Quartile	SE	Mean	25–75% Quartile	SE
Healthy controls	117.4	104.0–130.8	6.52	86.71	77.1–96.3	4.70
Pre-OP	114.6	104.6–124.6	4.98	88.82	81.6–96.0	3.58
POD1	256.0*	243.7–268.4	6.16	231.3*	215.4–247.3	7.93
POD3	418.0*	388.8–447.2	14.54	389.2*	350.3–428.1	19.37
POD5	399.8*	369.9–429.8	14.92	399.7*	364.2–435.2	17.69
POD7	240.1*	223.8–256.4	8.12	239.5*	222.1–256.9	8.67
POD10	164.0*	154.3–173.7	4.84	155.3*	144.0–166.6	5.62
POD14	141.2*	132.4–150.1	4.41	90.78	84.03–97.52	3.36

*p < 0.05.

Changes in circulating VEGF and MMP-9 levels after MWA

Serum VEGF levels at each time point after MWA were significantly increased compared to the pre-procedure levels. The VEGF levels increased on the first day (256.0 ± 6.16 pg/ml, p < 0.05) after MWA and peaked on the PPD3 (418.0 ± 14.54 pg/ml, p< 0.05). Although it gradually reduced afterwards, its levels on PPD14 (141.2 ± 4.41 pg/ml, p < 0.05) was higher than pre-procedure level.

The serum MMP-9 level was significantly elevated from PPD1 (231.3 ± 7.93 ng/ml, p < 0.05) until PPD10 (155.3 ±5.62 ng/ml, p < 0.05), while it normalised to pre-procedure level on PPD14 (90.78 ± 3.36 ng/ml, p > 0.05). The highest MMP-9 level was observed on PPD5 (399.7 ± 17.70 ng/ml, p < 0.05). The detailed VEGF and MMP-9 levels on each time point were shown in Table 2. The magnitude of the VEGF and MMP-9 increases varied widely. No significantly correlation or association was observed between the magnitude of the increases and any patients characteristics.

Discussion

Although surgery resection is identified as the preferred standard treatment for Stage I NSCLC [26], patients with insufficient cardiopulmonary function, advanced age, other medical comorbidities, and metastatic pulmonary tumours are not suitable candidates for surgery. Image-guided percutaneous MWA was considered as a safe and promising option of treating the particular patients subgroup [8,9,27,28].

We did not find a significant increase of VEGF and MMP-9 in Stage I NSCLC patients compared with healthy controls. Moreover, no relationship was observed between serum levels of MMP-9 as well as VEGF and different clinicopathological characteristics, which is consistent with precious published literatures [26,29]. In addition, we found that the mean serum VEGF and MMP-9 levels were elevated immediately from PPD1 until post-procedure Week 2 for the first time. The mean serum VEGF level increased steadily from PPD1 and peaked on PPD3 after MWA. The serum VEGF levels on PPD14 were still significantly higher than pre-procedure levels. The mean MMP-9 levels were also up-regulated consistently on PPD1 and peaked on PPD5. Notably, although increased over the pre-ablative baseline, the PPD14 level was not statistically significant.

Percutaneous tumours local MWA results in multiple physiologic alterations, some of which, in theory, may be deleterious to cancer patients [27,28]. VEGF and MMP-9 are two of the most important promoters involved in angiogenesis. The serum levels of both VEGF and MMP-9 in early-stage NSCLC (Stage I and II) were shown to be significantly lower than those in advanced stage. In addition, patients with Stage I/II disease and a lower pre-treatment circulating VEGF or MMP-9 levels had a significantly longer survival than those with a higher serum levels [16,26,29]. It may be problematic for cancer patients because the angiogenesis factors are necessary for both wound healing and tumour growth. We found the increased VEGF for at least 14 days and MMP-9 for at least 10 days post-procedurely. Both of these changes are proangiogenic. Similarly, post-procedure circulating VEGF and MMP-9 levels were also significantly higher than pre-procedure levels in early-stage NSCLC patients after receiving open thoracotomy or video-assisted thoracic surgery (VATS) [20–23]. Ng et al. [20] reported that serum MMP-9 levels rose consistently on PPD1 and fell to pre-procedure levels on PPD3 in NSCLC patients receiving thoracotomy or VATS. In addition, their data indicated that post-procedure plasma VEGF elevation and duration in patients undergoing VATS was less significant than major lung resection group [21]. Zhang et al. [23] found that the circulating VEGF levels after thoracotomy (from PPD1 to PPD5) were markedly higher than the day before surgery, while no difference was observed in VATS patients. The oncologic significance of the short-term elevations of these tumour promoters has yet to be elucidated. Nonetheless, in vitro and in vivo experiments suggested that overexpression of VEGF caused by insufficient radiofrequency ablation may promote cancer cell proliferation. Combination of RFA with sorafenib therapy resulted in a synergistic reduction in tumour growth [30,31]. Although currently unproven, it is possible to assume that the increased VEGF and MMP-9 expressions altered by local MWA might trigger the growth of the residual tumour micrometastases which persist after primary lesions ablation, leading to cancer local recurrence or distant metastasis.

Hypoxia, metabolic changes and inflammation are proposed to be the major causes of the production of angiogenic promoters in cancer cells as well as in normal cells (e.g. immune cells and fibroblasts) [32]. With regard to thermal ablation, radiofrequency ablation was shown to stimulate the outgrowth of tumour cells at the ablation lesion periphery, and hypoxia/hypoxia-inducible factor (HIF)-activated pathways were likely to be critical for this process [33]. Furthermore, in contrast to surgery trauma, MWA is more likely a regional burn injury, resulting in local inflammation and effusion. Hence, ablation-related inflammation is another crucial contributor to angiogenic response. The infiltrating macrophages and leukocytes, immature dendritic cells and carcinoma-activated fibroblasts are recruited to the ablation zone and release numerous angiogenic factors. The anti-inflammation treatment with aspirin or celecoxib can block the inflammation-mediated distant tumour growth [34,35].

In conclusion, percutaneous MWA of tumours resulted in increased serum levels of VEGF and MMP-9 in Stage I NSCLC patients. It is possible that the elevated angiogenesis factors after MWA may be associated with an unfavourable prognosis in early-stage NSCLC. Adjuvant and/or neoadjuvant antiangiogenesis approaches might be helpful for defending the patients against metastases during the immediate post-ablation time window. Further studies are warranted to explore the clinical significance and underling mechanism of these changes.

Acknowledgments

The work was supported by National Natural Science Foundation of China (81502610) and Shandong Provincial Natural Science Foundation, China (ZR2014HQ028).

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

The work was supported by National Natural Science Foundation of China (81502610) and Shandong Provincial Natural Science Foundation, China (ZR2014HQ028).