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Vascular Endothelial Growth Factor 189 mRNA Isoform Expression Specifically Correlates With Tumor Angiogenesis, Patient Survival, and Postoperative Relapse in Non–Small-Cell Lung Cancer

From the Divisions of Chest Medicine and Cardiovascular Medicine, Department of Internal Medicine; Division of Cardiovascular Surgery, Department of Surgery; and Department of Laboratory Medicine, National Taiwan University Hospital and Institute of Biomedical Science Academia Sinica, Taipei, Taiwan.

Address reprint requests to Pan-Chyr Yang, MD, PhD, National Taiwan University Hospital, No 7, Chung-Shan South Rd, Taipei 100, Taiwan; email pcyang{at}ha.mc.ntu.edu.tw

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: The purpose of this study was to evaluate the correlation between the expression of four different vascular endothelial growth factor (VEGF) mRNA isoforms (VEGF121, VEGF165, VEGF 189, and VEGF206) and the clinicopathologic characteristics, tumor angiogenesis, and outcome of patients with non–small-cell lung cancer.

PATIENTS AND METHODS: We examined the expression of four different VEGF mRNA isoforms in 57 non–small-cell lung cancers using reverse transcriptase polymerase chain reaction and the tumor angiogenesis using immunohistochemical staining.

RESULTS: All 57 lung cancer samples expressed the VEGF121, VEGF165, and VEGF189 mRNA isoforms, and three expressed the VEGF206 mRNA isoform. A high tumoral VEGF189 mRNA isoform expression ratio was associated with a high intratumoral microvessel count (P = .013), short survival (< 24 months; P = .001), and early postoperative relapse (< 12 months; P = .001). Survival and postoperative relapse time were significantly shorter in patients with a high compared with a low tumor VEGF189 mRNA isoform expression ratio (P = .0001 and P = .0086, respectively, log-rank test). In contrast, the VEGF165 and VEGF 206 mRNA isoform expression ratios showed no statistical correlation with tumor angiogenesis, postoperative relapse time, or survival. A high VEGF121 mRNA isoform expression ratio was associated with short survival (< 24 months) and early relapse (< 12 months). Multivariate analysis showed that VEGF 189 mRNA isoform expression, microvessel count, and nodal status were the most important independent prognostic factors for patient survival and postoperation recurrence.

CONCLUSION: The VEGF189 mRNA isoform expression ratio shows a greater correlation with tumor angiogenesis, postoperative relapse time, and survival than do the expression ratios for the VEGF121, VEGF165, and VEGF206 mRNA isoforms and can be used as a prognostic indicator for patients with non–small-cell lung cancers.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
ANGIOGENESIS IS required for tumor growth and metastasis.^1-4 High intratumoral microvessel counts have been shown to correlate with tumor advancement, systemic metastasis, and prognosis in several human solid cancers, including melanoma and breast, colon, and lung cancers.^5-8

Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors and can stimulate the proliferation and migration of endothelial cells under physiologic and pathologic conditions. It has been shown to play an important role in the angiogenesis process in various kinds of human neoplasm. Overexpression of VEGF has been demonstrated in cancer cells from colon,⁹ breast,¹⁰ brain,¹¹ ovary,¹² liver,¹³ and lung cancers,¹⁴ and VEGF expression has been reported to be an independent prognostic factor in patients with breast,¹⁵ gastric,¹⁶ and lung¹⁷ cancer.

Four isoforms of VEGF (VEGF121, VEGF165, VEGF189, and VEGF206) have been reported to be expressed in human tissues.¹⁸ The transcripts for these four isoforms arise from alternative splicing of a single VEGF gene, and the four VEGF isoforms are reported to have different biologic activities.¹⁹ The larger isoforms, VEGF189 and VEGF206, are cell-associated because of their stronger affinity for cell-surface proteoglycan,¹⁹ whereas the smaller isoforms, VEGF121 and VEGF165, are secretory forms. VEGF165, VEGF189, and VEGF206 are heparin-binding proteins, whereas VEGF121 is not.²⁰

Several studies have shown VEGF121 isoform to be the most highly expressed form in human tissues and tumors,²¹ whereas expression of VEGF165, VEGF189, and VEGF206 is variable.^17,21 Only a limited number of studies on the precise expression patterns of the four different VEGF isoform transcripts in different normal and tumor tissues have been performed,^13,22,23 and the role of the different VEGF isoforms in tumor progression and metastasis is still unclear. Tokunaga et al²³ showed that the pattern of the combined expression of the VEGF121, VEGF165, and VEGF189 mRNA isoforms is correlated with liver metastasis, advanced metastatic (M) stage, and poor prognosis in colon cancer, whereas patterns lacking VEGF189 mRNA expression are not. Although the level of expression of VEGF mRNA has been shown to be correlated with prognosis in patients with non–small-cell lung cancers (NSCLC),²¹ the study of the expression patterns of different VEGF isoforms and their clinicopathologic and prognostic significance in lung cancer is still limited.²⁴ The role of VEGF165 and VEGF206 in tumor progression and systemic metastasis in cancer has never been studied. The angiogenic properties of the different VEGF isoforms are still unknown, and the relationship between the expression pattern of the different VEGF isoforms and tumor angiogenesis has received little attention.^25,26

In this study, we used reverse transcriptase polymerase chain reaction (RT-PCR) to quantitate the expression of all four VEGF mRNA isoforms in NSCLC. The expression ratios for VEGF121, VEGF165, VEGF189, and VEGF206 mRNAs were also calculated. The purpose of this study was to evaluate (1) the correlation between the expression patterns of the different VEGF mRNA isoforms and the clinicopathologic characteristics, (2) the relationship between different VEGF mRNA isoform expression patterns and tumor angiogenesis, and (3) the significance of different VEGF mRNA isoform expression patterns as a prognostic indicator in NSCLC.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients and Tissue Specimens
Fifty-nine consecutive patients with clinical stage I, II, or IIIA NSCLC, resected at this institute, were included in this study between May 1994 and May 1996. The tumor samples were frozen and stored at -80°C until required for analysis of VEGF mRNA expression patterns. Paraffin-embedded formalin-fixed surgical specimens were prepared for immunohistochemical (IHC) staining for VEGF protein and intratumoral microvessels. Among them, two were excluded later because the tissue was preserved inappropriately or most of the specimens were necrotic and unsuitable for IHC staining.

Histologic classification was performed according to the World Health Organization criteria.²⁷ Tumor size, local invasion, and lymph node metastasis were determined at pathologic examination. The final disease stage was determined by a combination of surgical and pathologic findings according to the tumor-node-metastasis system for lung cancer staging, as recommended by the American Joint Committee on Cancer.²⁸

The patients consisted of 40 men and 17 women, with a mean age of 61 ± 11 years (mean ± SD). The tumors consisted of 28 squamous cell carcinomas and 29 adenocarcinomas. The surgical-pathologic staging was stage I in 19 patients, stage II in nine, and stage IIIA or IIIB in 29. Tumor status was T1 in 12 patients, T2 in 25, T3 in 19, and T4 in one. Twenty-seven patients had no lymph node metastasis (N0), whereas 30 had regional or mediastinal lymph node metastasis (13 had N1, 15 had N2, and two had N3 disease).

Follow-up continued until May 1999, with the observation period ranging from 36 to 60 months. Survival time was calculated from the date of operation to the date of death. Relapse time was calculated from the date of operation to the date of detection of local recurrence or systemic metastasis. We artificially defined survival times of less than 24 months (median value) as short survival and relapse times of less than 12 months (median value) as early relapse. Twenty-eight patients had an early relapse, and 26 had a short survival.

RT-PCR for Assessing Expression of VEGF mRNA Isoforms
Total cellular RNA was prepared from the frozen tumor tissue using an RNA extraction kit (RNeasy Mini Kit; Qiagen, Valencia, CA). Total RNA (1 µg) was denatured together with random primer (10 mmol) for 10 minutes at 70°C. After chilling on ice for 5 minutes, the mRNA was reverse transcribed for 100 minutes at 37°C in reverse transcriptase solution (50 mmol/L Tris-HCl, 40 µmol/L KCl, 8 mmol/L MgCl_2,), 0.5 mmol/L of each deoxynucleotide triphosphate, 12 units of RNasin, and 100 units of reverse transcriptase (Gibco BRL, Gaithersburg, MD) in a total volume of 25 µL. After incubation for 5 minutes at 95°C to inactivate reverse transcriptase, the cDNA was used as the template for 30 rounds of PCR amplification. Two sets of VEGF primers were used for PCR amplification ( Fig 1):

View larger version (27K): [in this window] [in a new window]   Fig 1. Primers for detection of the isoforms of VEGF mRNA. Primers 1 was designed for PCR of the VEGF121, VEGF165, VEGF189, and VEGF206 isoforms. Primers 2 was designed for PCR of VEGF165, VEGF189, and VEGF206. Probe was designed in exons 4 and 5 of the VEGF mRNA isoforms.

1.    Forward primer (exon 1): 5'-CTG CTG TCT TGG GTG CAT TG-3'
   

      Reverse primer (exon 8): 5'-TGT GAC AAG CCG AGG CGG TGA-3'.
   

      For amplification of VEGF121, VEGF165, VEGF189, and VEGF206.
   

   
   

2.    Forward primer (exon 4): 5'-CGG ATC AAA CCT CAC CAA GGC C-3'.
   

      Reverse primer (exon7): 5'-CTT TCT CCG CTC TGA GCA AGG C-3'.
   

      For amplification of VEGF165, VEGF189, and VEGF206.
   

   
   

The VEGF cDNA fragments (5 µL of 25 µL of reverse transcriptase product) were amplified by 30 rounds of PCR consisting of 1 minute at 94°C, 1 minute at 55°C, and 2 minutes at 72°C using a thermocycler (PTC-100 programmable thermal controller; MJ Research, Waltham, MA) and AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, CA). Serial dilution of VEGF cDNA fragment and PCR amplification for 26, 28, and 32 cycles were also performed to check that the PCR product at 30 cycles was still in the log phase of PCR amplification.

Sequencing of the PCR Products
After electrophoresis on an agarose gel, we collected the four different sized PCR products by the gel-melting method. The DNA sequences of the PCR products were then determined using a DNA autosequencer (ABI Prism DNA sequencer; Perkin-Elmer) and a standard procedure.²⁹

Calculation of the Expression Ratios for the Different VEGF mRNA Isoforms
The level of expression of the four individual VEGF mRNA isoforms was estimated by densitometry. The quality of the RNA was estimated by RT-PCR of beta-actin mRNA and of mRNA for the TATA box binding protein, a less abundant house-keeping gene³⁰ ( Fig 2A and 2B).

View larger version (64K): [in this window] [in a new window]   Fig 2. Integrity of the total RNA isolated from tumor sample examined by RT-PCR of mRNAs of (A) beta-actin and (B) TATA box-binding protein (TBP) gene (lanes 2 through 9). Lane 1, 100–base pair (bp) marker. The size of the PCR products of beta-actin and TBP mRNAs were 300 bp and 89 bp, respectively.

IHC Staining of VEGF Protein and Microvessels
IHC staining of VEGF protein and microvessels was performed using the avidin-biotin peroxidase complex (ABC) method, with modifications.⁷ Five-µm paraffin-embedded sections were mounted on poly-L-lysine–coated slides, dewaxed, and rehydrated. Anti-VEGF (1:40; Upstate Biotechnology, Lake Placid, NY)³¹ or anti-CD34 (1:100; Novocastra, Newcastle, UK) monoclonal antibodies were used to stain, respectively, for VEGF protein or endothelial cells in microvessels. The anti-VEGF antibody is raised against a synthetic peptide corresponding to the consensus sequence of four splicing variants of VEGF. It recognizes all four isoforms of VEGF. All incubations were at room temperature. The primary antibody was applied for 60 minutes, then the sections were washed with phosphate-buffered saline before rabbit antimouse immunoglobulin second antibody (1:10) was applied for 30 minutes, followed by ABC (1:20) for 30 minutes. After washing in phosphate-buffered saline, the color was developed by incubating the slides for 20 minutes with the fast-red substrate (diaminobenzidine tetrahydrochloride; Zymed, San Francisco, CA). The tumor specimen was then counterstained with Mayer’s solution. To confirm that the anti-VEGF antibody specifically recognizes this growth factor, we also performed blocking experiments using VEGF121 and VEGF165 peptides. Anti-VEGF antibody (1:200 dilution, 0.5 µg in 100 µL) in the presence or absence VEGF blocking peptides (5 µg in 100 µL) were used for IHC staining of tumor specimen.

To evaluate the expression of VEGF protein, we established a five-point score corresponding to the staining intensity: grade 0, negative; grade 1, weak; grade 2, moderate; grade 3, high; grade 4, very high.¹⁷ Grade 0 indicates staining intensity equal to that of the negative control (no primary antibody), grade 3 indicates staining intensity equal to that of the positive control (normal bronchial epithelium), and grade 4 indicates staining intensity stronger than that of the positive control. The score was analyzed as both a continuous and dichotomous variable. We selected 3.0 (the median value) as the cutoff value to distinguish weak VEGF protein staining from strong VEGF protein staining.

The number of microvessels in the area of most intense neovascularization^32,33 was counted on three x200 fields (x20 objective with x10 ocular lens, 0.785 mm² per field) and expressed as the average of three readings. Counting was performed by two independent observers without knowledge of the clinical information of the patients; the interobserver agreement for microvessel counts was good (r = 0.88, P < .001). A median value of 120 was used as the cutoff value between low and high microvessel counts.

Statistics
All statistical tests were performed using SPSS software (Version 8.0, SPSS Inc, Chicago, IL). The relationship between the expression of VEGF 165, VEGF189, or VEGF206 mRNAs and that of VEGF121 mRNA was analyzed by linear regression. The correlation between the expression ratios for the different VEGF mRNA isoforms and the clinicopathologic characteristics of the patients was analyzed using the ² and Mann-Whitney tests.³⁴ The ² test was also used to compare microvessel counts in tumors with high and low expression ratios for each of the VEGF121, VEGF165, VEGF189, and VEGF206 mRNA isoforms. The survival curve was obtained using the Kaplan-Meier method,³⁵ and the difference in survival time for tumors with high or low expression ratios for each of the VEGF121, VEGF165, VEGF189, and VEGF206 mRNA isoforms was analyzed using the log-rank test. The relapse-free survival curve and the difference in relapse time were obtained using the same statistical method. Multivariate analysis by stepwise Cox-regression³⁶ was used to identify the most important independent prognostic indicators of survival and relapse for NSCLC patients.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression of Different VEGF mRNA Isoforms
Expression of the VEGF121, VEGF165, and VEGF189 mRNA isoforms was detected in all 57 lung cancer specimens by RT-PCR, whereas VEGF206 mRNA isoform expression was detected in three tumors ( Fig 3A and 3B).

View larger version (69K): [in this window] [in a new window]   Fig 3. VEGF expression in the tumor determined by RT-PCR using (A) primers 1 and (B) using primers 2 (lanes 2 through 9). Lane 1, 100-bp marker. The size of PCR products were 435 bp, 567 bp, and 639 bp for (A) VEGF121, VEGF165, and VEGF189 mRNA isoforms, respectively, and were 132 bp, 204 bp, and 255 bp for (B) VEGF165, VEGF189, and VEGF206 mRNA isoforms, respectively.

View larger version (13K): [in this window] [in a new window]   Fig 4. Correlation between expression levels of (A) VEGF189 and VEGF121 isoform mRNAs and (B) VEGF165 and VEGF121 isoform mRNAs. The values on the x and y axes are the photodensity (expressed as arbitrary units) of the PCR product for the VEGF isoform mRNAs.

View larger version (136K): [in this window] [in a new window]   Fig 5. (A) Immunohistochemical stain showed the VEGF protein mainly located in the cytoplasm of cancer cell, whereas little VEGF was expressed by stroma cell and tissue (ABC method, x400 magnification). (B) IHC stain with anti-CD34 antibody shows the microvessel appearing as brown linear fragments with or without internal lumens in the stroma. Specificity of staining with anti-VEGF antibody (C) in the absence or (D) presence of VEGF 165 blocking peptides.

A high VEGF189 mRNA expression ratio was associated with high intratumoral angiogenesis. Seventeen (64%) of the 26 tumors with a high VEGF189 mRNA expression ratio ( 0.07) had high intratumoral microvessel counts (120 in x200 field), whereas 10 (32%) of 31 tumors with a low VEGF189 mRNA expression ratio had high intratumoral microvessel counts (P = .013, ² test). A high tumor VEGF189 mRNA expression ratio was also associated with short survival (< 24 months) and early recurrence (< 12 months). Twenty (77%) of the 26 patients with a high tumor VEGF189 mRNA expression ratio survived for less than 24 months, whereas only six (19%) of the 31 patients with a low VEGF189 mRNA expression ratio had a short survival (P < .001, ² test). Patients with a high VEGF189 mRNA expression ratio also had a higher incidence of early postoperative recurrence (20 of 26, 77%) than patients with a low VEGF189 mRNA expression ratio (eight of 31, 26%; P < .001, ² test). There was no correlation between the VEGF189 mRNA expression ratio and tumor status, lymph node metastasis, disease stage, histologic type, or other clinicopathologic variables ( Table 1).

Survival and Postoperative Relapse
The median survival of patients with a high VEGF189 mRNA expression ratio was 18.0 months (95% confidence interval [CI], 14.3 to 21.7 months), which is significantly shorter than that for patients with a low VEGF 189 mRNA expression ratio (median, 40 months, 95% CI, 35.1 to 44.9 months) (P = .0001, log-rank test) ( Fig 6A).

View larger version (18K): [in this window] [in a new window]   Fig 6. (A) Survival curve and (B) probability of postoperative relapse curve in patients with NSCLC. Patient survival (P .0001, log-rank test) and time of postoperative relapse (P = .0086, log-rank test) were both significantly shorter in tumors with a high VEGF189 expression ratio ( 0.07) than in those with a low VEGF189 expression ratio(< 0.07).

The difference in survival between tumors with high and low VEGF189 mRNA expression ratios was also statistically significant in the adenocarcinoma (P = .0049, n = 29) and squamous cell carcinoma (P = .0224, n = 28) subgroups, whereas the difference in relapse time was not (P = .075 and P = .100 for adenocarcinoma and squamous cell carcinoma, respectively). There was no statistically significant difference in overall survival and relapse time between patients with high or low tumor mRNA expression ratios for VEGF121 (P = .1458 and P = .0772, respectively, log-rank test), VEGF165 (P = .497 and P = .716, log-rank test), or VEGF206 (P = .8008 and P = .6651, log-rank test).

Patients with strong tumoral VEGF IHC staining had a significantly shorter survival (median, 18 months; 95% CI, 14.17 to 21.83 months) than patients with weak tumoral VEGF IHC staining (median, 36 months; 95% CI, 32.48 to 39.52 months) (P = .0197, log-rank test). The postoperation relapse time was also significantly earlier in patients with strong tumoral VEGF IHC staining than in patients with weak tumor VEGF IHC tumoral staining (median ± SE of 7.0 ± 1.10 months v 26.0 ± 1.89 months; P = .0491, log-rank test).

In terms of the clinicopathologic variables studied, univariate analysis showed that age, nodal status, tumor status, disease stage, microvessel count, and the VEGF189 mRNA expression ratio had prognostic significance in terms of patient survival, whereas age, nodal status, microvessel count, and the VEGF189 mRNA expression ratio were important predicators for patient relapse ( Table 3). When all of these variables were analyzed by multivariate analysis in terms of patient survival and relapse time using the stepwise Cox regression model, the VEGF189 mRNA expression ratio (P = .0001 and P = .0283, respectively), microvessel count (P = .0003 and P = .0081), and nodal status (P = .0004 and P = .0097) remained as independent prognostic factors ( Table 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Four different isoforms of human VEGF have been identified in human tissues. The mRNAs for all four isoforms arise by alternative splicing of the single VEGF gene, and the different proteins have different biologic activities.¹⁹ Different VEGF isoform mRNA expression patterns have been reported in colon cancer,²³ hepatocellular carcinoma,¹³ and CNS neoplasm.²²

The investigations of the significance of the different patterns of VEGF isoform expression in terms of tumor biologic behavior and patient prognosis are limited.^23,24 Tokunaga et al²³ recently showed that one pattern (VEGF121 + VEGF165 + VEGF206) of VEGF mRNA expression is correlated with liver metastasis and a poor prognosis in patients with colon cancer.

In the present study, we used RT-PCR to assess the expression of all four VEGF isoform mRNAs and to quantify their relative expression ratios (VEGF121, VEGF165, VEGF189, or VEGF206 mRNA compared with total VEGF mRNA) in NSCLC. The results show that a high VEGF189 mRNA expression ratio correlated with a high microvessel count in tumors but was not correlated with tumor status, nodal status, or disease stage. A high VEGF189 mRNA expression ratio in the tumor also was correlated with a short survival (< 24 months) and early relapse (< 12 months). In contrast, neither the VEGF165 or VEGF206 mRNA expression ratio showed a statistically significant correlation with tumor advancement, tumor angiogenesis, or patient prognosis. A high VEGF121 mRNA isoform expression ratio was correlated with a short survival and early relapse. Multivariate analysis showed the VEGF189 mRNA expression ratio, microvessel count, and nodal status to be the most important prognostic factors for survival and relapse.

Tokunaga et al²³ showed that the pattern of the combined expression of VEGF121, VEGF165, and VEGF189 mRNAs was associated with a higher incidence of liver metastasis and a poorer prognosis in patients with colon cancer, as compared with the expression pattern of VEGF121 and VEGF165 mRNA, either alone or together. The VEGF121 + VEGF165 +VEGF189 mRNA expression pattern was also associated with higher vein involvement in tumor tissue. In this study, we showed that a high VEGF189 mRNA isoform expression ratio in the tumor was correlated with a high intratumoral microvessel count, which may contribute to the early relapse and short survival of these patients. Oshika et al²⁴ showed that the appearance of the VEGF189 mRNA/protein isoform is closely associated with short survival in squamous cell carcinoma but is not associated with a poor prognosis in adenocarcinoma. However, they could not exclude the possibility that the prognostic significance of VEGF189 mRNA was due to the confounding effect of the concomitant high VEGF121 mRNA isoform expression in the tumors. In this study, to avoid the confounding effect from the most dominant VEGF isoform (VEGF121), we standardized the VEGF165, VEGF189, and VEGF206 mRNA expression levels with respect to the total VEGF mRNA. Our results show that survival was significantly shorter in both squamous cell carcinoma and adenocarcinoma with a high VEGF189 isoform mRNA expression ratio, and the overall relapse time was significantly shorter in tumors with high VEGF189 mRNA expression ratio.

In the study of Tokunaga et al,²³ the VEGF189 or VEGF165 mRNA isoform was found in 52% or 95%, respectively, of colon cancers. In the study of Oshika et al,²⁴ the VEGF189 mRNA isoform was found in 90% of NSCLC samples, whereas all tumors expressed the VEGF121 and VEGF165 mRNA isoforms, and none expressed VEGF206 mRNA. In our study, the VEGF189 mRNA isoform was expressed in all lung cancer specimens (100%), whereas the VEGF206 mRNA isoform was expressed in 5%. These differences may result from different primer design, different PCR efficiency, and the different populations in the three studies.

The angiogenic properties of the four different isoforms of VEGF are still unknown. Park et al¹⁹ showed that VEGF189 protein has the strongest ability to bind to extracellular matrix component. Koura et al³⁸ showed that the VEGF189 isoform was specifically upregulated by an increased cell density during in vitro culture of a colon cancer cell line, and suggested that this upregulation might result in increased angiogenesis, tumor growth, and metastasis. Cheng et al²⁵ showed that rapid blood vessel growth and breakdown occurred, resulting in intracerebral tumor-associated hemorrhage, in tumors caused by a VEGF121- and VEGF165-overexpressing glioblastoma cell line, whereas there was enhanced angiogenesis, but no eruption of surrounding nascent blood vessels, in a tumor caused by VEGF189-overexpressing cells. Cheng et al²⁵ also showed that the blood vessel density was five-fold higher in tumors formed by VEGF189-overexpressing cells than in tumors formed by parental glioblastoma cell lines and that vessel density was higher in tumors caused by VEGF121- or VEGF189-overexpressing cells than in those derived from VEGF165-overexpressing cells. When Tokunaga et al²⁶ inoculated 31 samples of human primary esophageal carcinoma cells into the skin of mice, 19 of the xenografts were accepted; of these, 18 (95%) expressed the VEGF189 isoform. Because only 19 (61%) of the original 31 samples had aberrant VEGF 189 mRNA expression, Tokunaga et al suggested that VEGF189 isoform expression is correlated with the xenotransplantability of human esophageal cancer. Our results support these findings. In this study, the VEGF189 mRNA isoform expression was not high, with photodensity of PCR product representing only approximately 8% (mean) of that of total tumor VEGF mRNA. However, we demonstrated that of the expression ratios for the different VEGF mRNA isoforms, the VEGF189 isoform showed the highest correlation with the intratumoral microvessel count. This result provides indirect in vivo evidence that, in NSCLC, the VEGF189 isoform might be more angiogenic than the other VEGF isoforms.

In the present study, the VEGF189 expression ratios were shown to be more closely correlated with patient prognosis than the VEGF121, VEGF165, and VEGF206 mRNA expression ratios. Cheung et al³⁹ suggested that VEGF189 is important in the maintenance of vascular integrity. Thus VEGF189 would not only increase tumor neovascularization, but also maintain vascular integrity, providing routes for nutrient supply and for systemic metastasis. This might explain the prognostic significance of the VEGF189 isoform in NSCLC.

Although VEGF189 had been considered to be a cell-associated protein, a recent study²⁵ showed that the VEGF189 protein, like VEGF121 and VEGF165, can be released from a cultured cell line into the conditioned medium and that the release of VEGF189 protein is enhanced by heparin. In addition, it is possible that the VEGF189 and VEGF206 isoforms may also be released after cell death due to hypoxia or necrosis and induce angiogenesis toward the hypoxic area to rescue the hypoxic cells. This might be important in squamous cell lung cancer, in which central necrosis in the tumor nest is frequently seen.

Several studies have demonstrated a correlation between upregulation of total VEGF mRNA expression and a poor prognosis in NSCLC patients. The results presented here suggest that not only the level of total VEGF mRNA expression but also the pattern of expression of the different VEGF mRNA isoforms has a significant influence on tumor angiogenesis and prognosis in NSCLC patients.

We conclude that the VEGF189 mRNA isoform expression ratio correlates with tumor angiogenesis, survival, and relapse and is an independent prognostic indicator for NSCLC patients. Examination of VEGF mRNA isoforms patterns will be helpful in predicting the prognosis of such patients.

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Submitted March 29, 2000; accepted September 8, 2000.

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