Abstract

Vascular endothelial growth factor (VEGF ) is a secreted endothelial cell-specific mitogen, which is induced by hypoxia and is angiogenic in vivo. Recently, elevated serum concentrations of VEGF (S-VEGF ) have been reported in patients with cancers of various histologies. However, the prognostic significance of S-VEGF in human cancer is unknown and the origin of S-VEGF remains unsettled. We measured S-VEGF by enzyme-linked immunosorbent assay from sera taken from 82 patients with non-Hodgkin's lymphoma before treatment and stored for 9 to 15 years at −20°C. All but one of the patients had been followed-up for at least 5 years or until death. S-VEGF ranged from 15 to 964 pg/mL; median, 228 pg/mL; mean, 291 pg/mL. A higher than the median S-VEGF level was associated with a poor World Health Organization performance status, a high International Prognostic Index, a high serum lactate dehydrogenase level, and a large cell histology. Patients with lower than the median S-VEGF at diagnosis had a 71% 5-year survival rate in comparison with only 49% among those with a higher than the median S-VEGF. We conclude that a high pretreatment S-VEGF level is associated with poor outcome in non-Hodgkin's lymphoma.

ANGIOGENESIS, the sprouting of new capillaries from preexisting ones, is an important component in many physiological and pathological processes. In healthy adults extensive angiogenesis occurs only in the female reproductive system. Angiogenesis may also take place in some pathological conditions, such as wound healing, rheumatoid arthritis, diabetic retinopathy, and tumor growth (reviewed by Folkman1 ).

During tumorigenesis, the vasculature can become activated to grow new capillaries in response to an appropriate stimulus (reviewed by Hanahan and Folkman2 ). One such stimulus is vascular endothelial growth factor (VEGF ), also called vascular permeability factor (VPF ), which is a soluble, dimeric 46-kD protein that is active as an endothelial cell-specific mitogen and as a vascular permeability factor (reviewed by Ferrara3 and Dvorak et al4 ). As a result of alternative splicing of VEGF mRNA, there exist at least three isoforms of VEGF encompassing 121, 165, and 189 amino acid residues.5 The two shorter forms are efficiently secreted from cells, whereas the longer one remains mostly cell associated. VEGF is induced by hypoxia in vitro and in vivo.6-10 Tumor cells have been shown to express both VEGF mRNA and protein in a variety of human cancers,11-14 and it has been concluded that VEGF has an important role in tumor biology and in the process of tumor angiogenesis (reviewed by Dvorak et al4 ). Expression of VEGF has been detected also in lymphoma.15 Furthermore, VEGF protein purified from a human histiocytic lymphoma cell line promoted dermal blood vessel leakage in guinea pigs at a dose of 20 ng and promoted in vitro endothelial cell growth at concentrations as low as 50 pmol/L.16 Expression of VEGF has been shown to increase tumor growth and angiogenesis in vivo in a nude mouse model.17-19 Similarly, anti-VEGF antibodies have the ability to inhibit the growth of several tumor cell lines in nude mice.20-22 

Recently, elevated serum VEGF concentrations (S-VEGF ) have been observed in patients with various histological types of cancer.23,24 High S-VEGF has also been associated with a large size of the primary breast tumor and a short tumor volume doubling time in advanced colorectal cancer.23,25 However, little is known about the clinical significance of S-VEGF, and there are no studies available on the prognostic value of S-VEGF in any type of human cancer. In the present study we measured S-VEGF from lymphoma patients' sera stored for years at −20°C and found that S-VEGF values were associated with survival and several important clinical variables.

MATERIALS AND METHODS

Patients

S-VEGF was measured in 82 randomly selected adult patients with non-Hodgkin's lymphoma diagnosed and treated in the Department of Oncology, Helsinki University Central Hospital in 1981 to 1987, of whom frozen serum taken at the time of diagnosis but before possible lymphoma treatment was available. Histological classification was not reviewed in conjunction with the present study, but 18 lymphomas (22%) had been classified as low-grade, 40 (49%) intermediate-grade, and 22 (27%) as high-grade lymphoma according to the Working Formulation scheme26 by a pathologist with a special interest in lymphoma, and 2 (2%) cases were considered unclassifiable.

Thirty-eight (46%) of the patients were men, and the median age at diagnosis was 60 years. Clinical staging was done according to the Ann Arbor classification system. The clinical status, a chest radiograph, computed tomography (CT) scans of the mediastinum and abdomen, and a bone marrow biopsy were taken as staging examinations. Twenty-two (27%) of the patients had stage I, 24 (29%) stage II, 14 (17%) stage III, and 22 (27%) stage IV disease at diagnosis. Fifteen patients (18%) had B symptoms (weight loss, unexplained fever, or night sweats). The International Prognostic Index (IPI)27 could be determined in 79 (96%) cases.

A total of 68 patients were treated with combination chemotherapy. The patients with intermediate- or high-grade lymphoma and disseminated disease were treated usually with bleo-CHOP (bleomycin, cyclophosphamide, doxorubicin, vincristine, and prednisone), M-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone), or another anthracycline containing a combination chemotherapy regimen. Low-grade lymphomas were usually treated with a single agent chlorambucil if symptomatic. Twenty-eight patients received megavoltage radiotherapy.

All patients were regularly followed-up with a few months intervals in an outpatient department. The median follow-up time was 84 months (range, 33 to 136 months), and all but one patient were followed up over 60 months or until death. During the follow-up time 38 patients died.

Methods

Venous blood samples. Peripheral venous blood samples were collected in sterile test tubes a few hours or a few days before starting lymphoma therapy, centrifuged at 2,000g for 10 minutes, and then stored at −20°C.

Table 1.

The Effect of Freezing on the VEGF Concentrations in 12 Serum Samples

Subject S-VEGF (pg/mL) 
 One Freeze-Thaw Cycle Two Freeze-Thaw Cycles 
 
23 22 
36 42 
931 866 
86 94 
435 432 
1150 1303 
37 33 
1389 1464 
1143 1155 
10 38 40 
11 2703 3114 
12 67 68 
Subject S-VEGF (pg/mL) 
 One Freeze-Thaw Cycle Two Freeze-Thaw Cycles 
 
23 22 
36 42 
931 866 
86 94 
435 432 
1150 1303 
37 33 
1389 1464 
1143 1155 
10 38 40 
11 2703 3114 
12 67 68 

Abbreviations: VEGF, vascular endothelial growth factor; S-VEGF, serum vascular endothelial growth factor.

Table 2.

Association Between the Pretreatment S-VEGF Level and Nine Clinicopathological Factors in Non-Hodgkin's Lymphoma

Factor No. of Cases S-VEGF* P 
  228 pg/mL N (%) >228 pg/mL N (%)  
 
WHO performance status 
0-1 67 39 (58%) 28 (42%)  
 
2-4 15 2 (13%) 13 (87%) .002 
 
International Prognostic Index 
0-2 57 34 (60%) 23 (40%) .003 
 
3-4 22 5 (23%) 17 (77%)  
 
Serum LDH level 
Normal 53 32 (60%) 21 (40%) .005 
> Normal 26 7 (27%) 19 (73%)  
 
Gender 
Male 38 23 (61%) 15 (39%) .08 
 
Female 44 18 (41%) 26 (59%)  
 
Age at diagnosis 
≤60 42 22 (52%) 20 (48%) .66 
 
>60 40 19 (48%) 21 (52%)  
 
No. of extranodal sites 
≤0-1 74 38 (51%) 36 (49%) .71 
 
>1 3 (38%) 5 (63%)  
 
Presence of B-symptoms 
No 67 33 (49%) 34 (51%) .78 
 
Yes 15 8 (53%) 7 (47%)  
 
Histological grade 
Low 18 10 (56%) 8 (44%) .82 
 
Intermediate 40 20 (50%) 20 (50%)  
 
High 22 10 (45%) 12 (55%)  
 
Ann Arbor stage 
22 10 (45%) 12 (55%) .85 
 
II 24 11 (46%) 13 (54%)  
 
III 14 8 (57%) 6 (43%)  
 
IV 22 12 (55%) 10 (45%)  
Factor No. of Cases S-VEGF* P 
  228 pg/mL N (%) >228 pg/mL N (%)  
 
WHO performance status 
0-1 67 39 (58%) 28 (42%)  
 
2-4 15 2 (13%) 13 (87%) .002 
 
International Prognostic Index 
0-2 57 34 (60%) 23 (40%) .003 
 
3-4 22 5 (23%) 17 (77%)  
 
Serum LDH level 
Normal 53 32 (60%) 21 (40%) .005 
> Normal 26 7 (27%) 19 (73%)  
 
Gender 
Male 38 23 (61%) 15 (39%) .08 
 
Female 44 18 (41%) 26 (59%)  
 
Age at diagnosis 
≤60 42 22 (52%) 20 (48%) .66 
 
>60 40 19 (48%) 21 (52%)  
 
No. of extranodal sites 
≤0-1 74 38 (51%) 36 (49%) .71 
 
>1 3 (38%) 5 (63%)  
 
Presence of B-symptoms 
No 67 33 (49%) 34 (51%) .78 
 
Yes 15 8 (53%) 7 (47%)  
 
Histological grade 
Low 18 10 (56%) 8 (44%) .82 
 
Intermediate 40 20 (50%) 20 (50%)  
 
High 22 10 (45%) 12 (55%)  
 
Ann Arbor stage 
22 10 (45%) 12 (55%) .85 
 
II 24 11 (46%) 13 (54%)  
 
III 14 8 (57%) 6 (43%)  
 
IV 22 12 (55%) 10 (45%)  

Abbreviations: S-VEGF, serum vascular endothelial growth factor; WHO, World Health Organization; LDH, lactate dehydrogenase.

*

The median was used as the cut-off value

S-VEGF immunoassay. S-VEGF concentrations were determined as S-VEGF immunoreactivity by using a quantitative sandwich enzyme immunoassay technique (Quantikine R; R & D Systems, Minneapolis, MN). The system uses a solid phase monoclonal antibody and an enzyme-linked polyclonal antibody raised against recombinant human VEGF. For each analysis 100 μL of serum was used. All analyses and calibrations were performed in duplicate. The calibrations on each microtiter plate included recombinant human VEGF standards. Optical densities were determined by using a microtiter palate reader (Multiscan RC Type 351; Labsystems, Helsinki, Finland) at 450 nm. The blank was subtracted from the duplicate readings for each standard and sample. A standard curve was created using StatView 4.02 (Abacus Concepts Inc, Berkeley, CA) by plotting the logarithm of the mean absorbance of each standard versus the logarithm of the VEGF concentration. Concentrations are reported as pg/mL. The coefficient of variation of interassay determinations reported by the manufacturer vary from 6.2% to 8.8% when the S-VEGF concentrations range between 50 and 1,000 pg/mL. When we measured 12 serum samples twice in two separate assays, the interassay variation ranged from 2% to 9% within the same concentration range. The possible effect of freezing on S-VEGF was investigated by measuring S-VEGF before and after a freeze-thawing cycle in serum samples taken from six cancer patients and from six healthy volunteers. S-VEGF levels were determined without any knowledge of the survival or other clinical data except age and sex of the patients, which could be found on the test tube labels.

Statistical analysis. Statistical analysis was done by using the BMDP software (BMDP Statistical Software Inc, Los Angeles, CA). Cumulative survival was estimated with the product-limit method from the date of diagnosis. The Mantel-Cox log-rank test and the generalized Wilcoxon test were used to compare survival between different groups. The relative importance of different variables was analyzed using Cox's proportional hazard model (BMDP 2L). Frequency tables were analyzed with the χ2 test or Fisher's exact test. The effect of freezing on S-VEGF was analyzed with the Wilcoxon signed rank test. All P values are two tailed.

RESULTS

The Effect of Freezing on VEGF Concentrations of Serum Samples

The effect of freeze thawing was studied in 12 serum samples. Before freezing, S-VEGF of the 12 samples tested ranged from 23 to 2,703 pg/mL; median, 261 pg/mL; mean, 670 pg/mL, and after freezing and thawing, the range was from 22 to 3,114 pg/mL; median, 263 pg/mL; mean, 719 pg/mL (P > .1; Table 1). Hence, freezing and thawing of the serum samples did not have any marked effect on S-VEGF values.

Table 3.

Association Between Pretreatment S-VEGF and the Histological Type of Non-Hodgkin's Lymphoma by the Working Formulation Scheme

Histological Type No. S-VEGF3-150 
 of Cases ≤228 >228 
  pg/mL pg/mL 
Small cell lymphocytic 
Small cell plasmacytoid 
Follicular small cleaved cell 10 
Follicular mixed cell 
Diffuse small cleaved cell 
Diffuse mixed cell 
Diffuse large cell 27 10 17 
Immunoblastic 14 
Lymphoblastic 
Small round cell 
Burkitt type 
Histiocytic 
Other 
Unclassifiable 
Histological Type No. S-VEGF3-150 
 of Cases ≤228 >228 
  pg/mL pg/mL 
Small cell lymphocytic 
Small cell plasmacytoid 
Follicular small cleaved cell 10 
Follicular mixed cell 
Diffuse small cleaved cell 
Diffuse mixed cell 
Diffuse large cell 27 10 17 
Immunoblastic 14 
Lymphoblastic 
Small round cell 
Burkitt type 
Histiocytic 
Other 
Unclassifiable 
F3-150

The median was used as the cut-off value

Fig. 1.

Survival of 82 patients with non-Hodgkin's lymphoma by the pretreatment S-VEGF level. The median (228 pg/mL) was used as the cut-off value. Survival rates at 24, 60, and 96 months are given.

Fig. 1.

Survival of 82 patients with non-Hodgkin's lymphoma by the pretreatment S-VEGF level. The median (228 pg/mL) was used as the cut-off value. Survival rates at 24, 60, and 96 months are given.

Serum VEGF in Non-Hodgkin's Lymphoma

Serum VEGF concentrations ranged from 15 to 964 pg/mL (median, 228 pg/mL; mean 291 pg/mL) among the 82 patients with non-Hodgkin's lymphoma. One third of the patients had S-VEGF lower than 122 pg/mL and one third higher than 314 pg/mL.

If the median S-VEGF concentration was used as the cut-off value, a high S-VEGF was strongly associated with a poor World Health Organization (WHO) performance status (>1, P = .002), a higher than the normal serum lactate dehydrogenase (S-LDH) level measured at the time of the diagnosis (P = .005), a high IPI (>2, P = .003), and a possible association was found with the female gender (P = .08; Table 2). No association between a high pretreatment S-VEGF and age at diagnosis, Ann Arbor stage, or the presence of B-symptoms was found (P > .10). Similarly, no association between S-VEGF and the Working Formulation grading was found (P = .82), but patients with large cell lymphoma (either large cell diffuse, G, n = 27; immunoblastic, H, n = 14; lymphoblastic, I, n = 2; or histiocytic lymphoma, n = 1) had more often a higher than the median pretreatment S-VEGF level in comparison with the rest of the patients (P = .03; Table 3).

S-VEGF and Survival

Patients with higher than the median S-VEGF had inferior survival in comparison with those with lower than the median pretreatment value (P = .04 by the log-rank test and .01 by the generalized Wilcoxon test; Fig 1). The 2- and 5-year survival rates of the patients with a lower than the median S-VEGF level were 85% and 71%, respectively, and those of the patients with a higher than the median level, 59% and 49%, respectively. A high S-VEGF level was associated with inferior survival also when tertiles and quartiles were used as cut-off values (Table 4). A higher than the median S-VEGF was associated with poor overall survival among patients with low-grade lymphoma (5-year survival 56% v 100%; P = .04) and those with high-grade lymphoma (5-year survival 25% v 50%; P = .03), but in intermediate-grade lymphomas the difference was not statistically significant (62% v 67%; P > .1). When survival in the subgoup of large cell diffuse and immunoblastic lymphomas (n = 41) was analyzed separately, patients with a higher than the median S-VEGF (269 pg/mL) had inferior survival in comparison with patients with lower than the median S-VEGF, but the difference was not statistically significant (2-year survival 55% v 71%; 5-year survival 44% v 52%; P > .1).

Table 4.

Prognostic Factors in Univariate Analyses

Factor 5-Year Survival (%) P 
   Wilcoxon Test Log-Rank Test 
International Prognostic Index 
   
0-2 57 74 <.0001 <.0001 
3-4 21 23   
WHO performance status 
0-1 67 69 <.0001 <.0001 
2-4 15 18   
Serum LDH level 
Normal 53 72 .001 .0007 
>Normal 26 34   
Ann Arbor stage 
I-II 46 72 .006 .01 
III-IV 36 44   
No. of extranodal sites 
0-1 74 63 .008 .008 
>1 25   
Serum VEGF at diagnosis 
   
≤228 pg/mL (median) 41 71 .01 .04 
>228 pg/mL 41 49   
≤122 pg/mL (tertiles) 27 70 .07 .15 
123-314 pg/mL 27 63   
>314 pg/mL 28 46   
≤95 pg/mL (quartiles) 20 75 .03 .04 
96-228 pg/mL 21 67   
229-410 pg/mL 20 60   
>410 pg/mL 21 38   
Presence of B-symptoms 
No 67 64 .01 .08 
Yes 15 40   
Histological grade 
Low 18 78 .02 .03 
Intermediate 40 65   
High 22 36   
Age at diagnosis 
≤60 42 69 .02 .03 
>60 40 50   
Gender 
Male 38 61 .74 .86 
Female 44 59   
Factor 5-Year Survival (%) P 
   Wilcoxon Test Log-Rank Test 
International Prognostic Index 
   
0-2 57 74 <.0001 <.0001 
3-4 21 23   
WHO performance status 
0-1 67 69 <.0001 <.0001 
2-4 15 18   
Serum LDH level 
Normal 53 72 .001 .0007 
>Normal 26 34   
Ann Arbor stage 
I-II 46 72 .006 .01 
III-IV 36 44   
No. of extranodal sites 
0-1 74 63 .008 .008 
>1 25   
Serum VEGF at diagnosis 
   
≤228 pg/mL (median) 41 71 .01 .04 
>228 pg/mL 41 49   
≤122 pg/mL (tertiles) 27 70 .07 .15 
123-314 pg/mL 27 63   
>314 pg/mL 28 46   
≤95 pg/mL (quartiles) 20 75 .03 .04 
96-228 pg/mL 21 67   
229-410 pg/mL 20 60   
>410 pg/mL 21 38   
Presence of B-symptoms 
No 67 64 .01 .08 
Yes 15 40   
Histological grade 
Low 18 78 .02 .03 
Intermediate 40 65   
High 22 36   
Age at diagnosis 
≤60 42 69 .02 .03 
>60 40 50   
Gender 
Male 38 61 .74 .86 
Female 44 59   

Abbreviations: WHO, World Health Organization; LDH, lactate dehydrogenase; VEGF, vascular endothelial growth factor.

Several other factors correlated strongly with overall survival in univariate survival analyses in the present series (Table 4). Therefore, S-VEGF using the median as the cut-off value was tested in a multivariate analysis together with the IPI score, gender, and the presence of B symptoms using Cox's proportionate hazard model. In this analysis, only the IPI score had independent influence on survival (P < .001, the relative risk of death 2.3, 95% confidence interval from 1.7 to 3.2).

DISCUSSION

In the present study we found many patients with non-Hodgkin's lymphoma to have an elevated pretreatment S-VEGF concentration. The pretreatment S-VEGF of non-Hodgkin's lymphoma patients ranged from 15 to 964 pg/mL (median, 228 pg/mL; mean, 291 pg/mL). In comparison, the S-VEGF in 184 healthy individuals reported by Yamamoto et al23 varied from values that were under the detection limit to 228 pg/mL. The results of Yamato et al are in line with our experience, because we have only rarely measured VEGF concentrations higher than 150 pg/mL in sera of presumably healthy individuals (range from 1 to 177 pg/mL; n = 113).24 

We found a high pretreatment S-VEGF to be associated with poor overall survival and a few established adverse prognostic factors in non-Hodgkin's lymphoma. S-VEGF did not have independent prognostic value when the IPI was included in a multivariate analysis, but it should be noted that in a univariate analysis S-VEGF was about as strong a prognostic factor as age at diagnosis, which is currently included as a component together with stage, S-LDH level, the number of extranodal sites, and the WHO performance status in the IPI.27 Although first designed for aggressive non-Hodgkin's lymphomas, the IPI has been shown to be useful in all grades of lymphoma.28 However, the present findings should be interpreted with some caution because the series is heterogeneous and includes several types of non-Hodgkin's lymphoma.

In lymphoma and several other types of human cancer the cancer cells produce VEGF.4,15 Hypoxia has been considered to be an important stimulus for VEGF production in tumor cells, but also various cytokines such as interleukin-6, epidermal growth factor, transforming growth factor-β1, keratinocyte growth factor, and tumor necrosis factor-α have been shown to induce the expression of VEGF in cultured cells.29,30 Lymphocytes infiltrating human cancers, macrophages, and peripheral blood T lymphocytes and platelets also contain VEGF.31-34 Therefore, circulating VEGF found in patients with cancer may originate from cancer cells but also from tumor infiltrating benign cells and circulating peripheral blood cells. However, it should be noted that not all of the proteins stored in blood cells are endogenously synthesized, but instead may originate primarily from plasma.35,36 Consequently, it is possible that VEGF secreted by cancer cells or by tumor infiltrating inflammatory cells escapes to the circulation and accumulates in the blood cells, such as platelets and lymphocytes.

In the present study we measured higher than the median S-VEGF values more frequently from large cell lymphomas than from small cell or mixed cell lymphomas, and high S-VEGF levels were also associated with a poor WHO performance status, a high serum LDH level, and the IPI. These findings are not in contradiction with the hypothesis that high serum VEGF levels are associated with growth of lymphoma and active angiogenesis. The biological role of circulating VEGF in cancer progression and dissemination may be of importance. VEGF in the tumor microenvironment not only stimulates proliferation of tumor blood vessels, but also increases vascular permeability (reviewed by Dvorak et al4 ), thus, possibly contributing to tumor cell extravasation and metastasis formation. Moreover, VEGF has recently been found to inhibit maturation of dendritic cells, which are important antigen-presenting cells.37 Hence, exposure of the immune system to high levels of local and possibly also circulating VEGF may play a role in allowing tumors to avoid induction of an immune response.

In conclusion, these data indicate that a high S-VEGF level is associated with poor outcome in non-Hodgkin's lymphoma. A high pretreatment S-VEGF may be associated with active tumor angiogenesis and tumor growth, and it is possible that similar associations with unfavorable survival can be found in other types of human cancer as well. Further studies are now needed in non-Hodgkin's lymphoma to find out whether S-VEGF is a prognostic factor in single histological types of the disease and whether S-VEGF values are associated with the proliferation rate of lymphoma. It will be of particular interest to find out whether S-VEGF levels predict response to antiangiogenic drugs in lymphoma and in other types of human malignancy. In addition, further studies are required to investigate the origin of circulating VEGF as well as its levels in conditions such as infectious and inflammatory states.

ACKNOWLEDGMENT

We thank Elina Roimaa for technical assistance.

Supported by grants from the Finnish Academy, the Finnish Cancer Foundation, and the Helsinki University Central Hospital Research Funds.

Address reprint requests to Heikki Joensuu, MD, PhD, Department of Oncology, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.

REFERENCES

REFERENCES
1
Folkman
J
Angiogenesis in cancer, vascular, rheumatoid and other disease.
Nature Med
1
1995
27
2
Hanahan
D
Folkman
J
Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.
Cell
86
1996
353
3
Ferrara
N
The role of vascular endothelial growth factor in pathological angiogenesis.
Breast Cancer Res Treat
36
1995
127
4
Dvorak
HF
Brown
LF
Detmar
M
Dvorak
AM
Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis.
Am J Pathol
146
1995
1029
5
Tischer
E
Mitchell
R
Hartman
T
Silva
M
Gospodarowicz
D
Fiddes
JC
Abraham
JA
The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.
J Biol Chem
266
1991
11947
6
Shweiki
D
Itin
A
Soffer
D
Keshet
E
Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.
Nature
359
1992
843
7
Shweiki
D
Neeman
M
Itin
A
Keshet
E
Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: Implications for tumor angiogenesis.
Proc Natl Acad Sci USA
92
1995
768
8
Namiki
A
Brogi
E
Kearney
M
Kim
EA
Wu
T
Couffinhal
T
Varticovski
L
Isner
JM
Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells.
J Biol Chem
270
1995
31189
9
Banai
S
Shweiki
D
Pinson
A
Chandra
M
Lazarovici
G
Keshet
E
Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: Implications for coronary angiogenesis.
Cardiovasc Res
28
1994
1176
10
Pe'er
J
Shweiki
D
Itin
A
Hemo
I
Gnessin
H
Keshet
E
Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases.
Lab Invest
72
1995
638
11
Brown
LF
Berse
B
Jackman
RW
Tognazzi
K
Manseau
EJ
Dvorak
HF
Senger
DR
Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas.
Am J Pathol
143
1993
1255
12
Brown
LF
Berse
B
Jackman
RW
Tognazzi
K
Manseau
EJ
Senger
DR
Dvorak
HF
Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract.
Cancer Res
53
1993
4727
13
Plate
KH
Breier
G
Weich
HA
Mennel
HD
Risau
W
Vascular endothelial growth factor and glioma angiogenesis: Coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms.
Int J Cancer
59
1994
520
14
Weninger
W
Uthman
A
Pammer
J
Pichler
A
Ballaun
C
Lang
I
Plettenberg
A
Bankl
HC
Stürzl
M
Tschachler
E
Vascular endothelial growth factor production in normal epidermis and in benign and malignant epithelial skin tumors.
Lab Invest
75
1996
647
15
Dvorak
HF
Sioussat
TM
Brown
LF
Berse
B
Nagy
JA
Sotrel
A
Manseau
EJ
Van de Water
L
Senger
DR
Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: Concentration in tumor blood vessels.
J Exp Med
174
1991
1275
16
Connolly
DT
Heuvelman
DM
Nelson
R
Olander
JV
Eppley
BL
Delfino
JJ
Siegel
NR
Leimgruber
RM
Feder
J
Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.
J Clin Invest
84
1989
1470
17
Ferrara
N
Winer
J
Burton
T
Rowland
A
Siegel
M
Phillips
HS
Terrell
T
Keller
GA
Levinson
AD
Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells.
J Clin Invest
91
1993
160
18
Zhang
HT
Craft
P
Scott
PA
Ziche
M
Weich
HA
Harris
AL
Bicknell
R
Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells.
J Natl Cancer Inst
87
1995
213
19
Claffey
KP
Brown
LF
Aguila
L
Tognazzi
K
Yeo
KT
Manseau
EJ
Dvorak
HF
Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis.
Cancer Res
56
1996
172
20
Kim
KJ
Li
B
Winer
J
Armanini
M
Gillett
N
Phillips
HS
Ferrara
N
Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo.
Nature
362
1993
841
21
Asano
M
Yukita
A
Matsumoto
T
Kondo
S
Suzuki
H
Inhibition of tumor growth and metastasis by an immunoneutralizing monoclonal antibody to human vascular endothelial growth factor/vascular permeability factor121.
Cancer Res
55
1995
5296
22
Warren
RS
Yuan
H
Matli
MR
Gillett
NA
Ferrara
N
Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis.
J Clin Invest
95
1995
1789
23
Yamamoto
Y
Toi
M
Kondo
S
Matsumoto
T
Suzuki
H
Kitamura
M
Tsuruta
K
Taniguchi
T
Okamoto
A
Mori
T
Yoshida
M
Ikeda
T
Tominaga
T
Concentrations of vascular endothelial growth factor in the sera of normal controls and cancer patients.
Clin Cancer Res
2
1996
821
24
Salven
P
Mäenpää
H
Orpana
A
Alitalo
K
Joensuu
H
Serum VEGF is often elevated in disseminated cancer.
Clin Cancer Res
6
1997
647
25
Dirix
LY
Vermeulen
PB
Hubens
G
Benoy
I
Martin
M
De Pooter
C
Van Oosterom
AT
Serum basic fibroblast growth factor and vascular endothelial growth factor and tumour growth kinetics in advanced colorectal cancer.
Ann Oncol
7
1996
843
26
National
Cancer Institute sponsored study on classification of non-Hodgkin's lymphomas
Summary and description of a working formulation for clinical usage.
Cancer
49
1982
2112
27
The International Non-Hodgkin's Lymphoma Prognostic Factors Project. A predictive model for aggressive non-Hodgkin's lymphoma. N Engl J Med 14:987, 1993
28
Hermans
J
Krol
AD
van Groningen
K
Kluin
PM
Kluin-Nelemans
JC
Kramer
MH
Noordijk
EM
Ong
F
Wijermans-PW
International Prognostic Index for aggressive non-Hodgkin's lymphoma is valid for all malignancy grades.
Blood
86
1995
1460
29
Cohen
T
Nahari
D
Cerem
LW
Neufeld
G
Levi
BZ
Interleukin 6 induces the expression of vascular endothelial growth factor.
J Biol Chem
271
1996
736
30
Frank
S
Hubner
G
Breier
G
Longaker
MT
Greenhalgh
DG
Werner
S
Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing.
J Biol Chem
270
1995
12607
31
Salven P, Heikkilä P, Joensuu H: Enhanced expression of VEGF in metastatic melanoma. Br J Cancer (in press)
32
Freeman
MR
Schneck
FX
Gagnon
ML
Corless
C
Soker
S
Niknejad
K
Peoples
GE
Klagsbrun
M
Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: A potential role for T cells in angiogenesis.
Cancer Res
55
1995
4140
33
Berse
B
Brown
LF
van de Water
L
Dvorak
HF
Senger
DR
Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors.
Mol Biol Cell
3
1992
211
34
Möhle
R
Green
D
Moore
MAS
Nachman
RL
Rafii
S
Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.
Proc Natl Acad Sci USA
94
1997
663
35
Harrison
P
Wilbourn
B
Debili
N
Vainchenker
W
Breton
Gorius J
Lawrie
AS
Masse
JM
Savidge
GF
Cramer
EM
Uptake of plasma fibrinogen into the alpha granules of human megakaryocytes and platelets.
J Clin Invest
84
1989
1320
36
Harrison
P
Cramer
EM
Platelet alpha-granules.
Blood Rev
7
1993
52
37
Gabrilovich
DI
Chen
HL
Girgis
KR
Cunnigham
HT
Meny
GM
Nadaf
S
Kavanaugh
D
Carbone
DP
Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dentritic cells.
Nature Med
2
1996
1096