Leukemia & Lymphoma, March 2004 Vol. 45 (3), pp. 499–506
The Interplay Between C-Myc Oncogene Expression and Circulating Vascular Endothelial Growth Factor (sVEGF), Its Antagonist Receptor, Soluble Flt-1 in Diffuse Large B Cell Lymphoma (DLBCL): Relationship to Patient Outcome S. AREFa,*, M. MABEDb, K. ZALATAc, M. SAKRANAb and H. EL ASKALANYb a
Hematology Unit, Clinical Pathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt; bInternal Medicine Department, Mansoura Faculty of Medicine, Mansoura, Egypt; cPathology Department, Mansoura Faculty of Medicine, Mansoura, Egypt (Received 17 July 2003)
The c-Myc is a ubiquitous and multifunctional oncogene. Recently, data obtained from experimental study suggests the involvement of c-Myc oncogene in angiogenesis. In the present study the interrelation of sVEGF, sFlt-1 concentrations and c-Myc oncoprotein expression at diagnosis were assessed in DLBCL and their impact on the patient outcome. Forty-five DLBCL patients beside 10 normal controls were included. C-Myc oncoprotein was assessed by immunohistochemistry. SVEGF and sFlt-1 were determined by enzyme linked immunosorbent assay. C-Myc over-expression was detected in 66.6% of DLBCL. The DLBCL patient group with positive c-Myc over-expression showed significantly higher sVEGF and significantly decreased sFlt-1 as compared to group with negative c-Myc over-expression (P = 0.000 and P = 0.009 respectively). SVEGF was positively correlated to sLDH and s.b2 microglobulin (r = 0.6, P = 0.000, r = 0.69, P = 0.000) respectively. On the other hand sFlt-1 was negatively correlated to sLDH and s.b2 microglobulin (r 7 0.25, P 4 0.05, r 7 0.49, P = 0.001) respectively. The non-living DLBCL group showed significantly higher expression of c-Myc, higher concentration of sVEGF and lower concentration in sFlt-1 level as compared to the living group (P = 0.000 for all). Multivariate analysis revealed that c-Myc over-expression; high sVEGF and normal sFlt-1 levels at diagnosis had independent adverse influence on survival (relative risk: 17.9, 35.7, 29.3, 2.63; P 5 0.0001, P 5 0.0001, and P = 0.03 respectively) In conclusion: C-Myc over-expression significantly associated with high sVEGF and normal sFlt-1 level in DLBCL patients, suggesting a complex interrelationship between c-Myc oncogene expression and angiogenic regulators. C-Myc over-expression, high sVEGF and normal sFLt-1 levels at diagnosis had an independent adverse influence on survival in DLBCL patients and considered bad prognostic markers. Keywords: c-Myc; sVEGF; sFlt-1; DLBCL; Angiogenesis; Outcome
INTRODUCTION Diffuse large B cell lymphomas (DLBCL) are the most frequently occurring types of NHLs. According to WHO classification of neoplastic diseases of the hematopoietic and lymphoid tissues, DLBCL comprises about 40% of adult cases of non-Hodgkin’s lymphoma [1]. This type of NHL is a more heterogeneous group of disorders regarding clinical behavior and histopathologic features than other groups of lymphomas, representing the classical high-grade lymphomas. They typically present with rapidly progressive lymphadenopathy associated with fast cellular proliferation and progressive infiltra-
tion. Some patients have a more aggressive course than others [2]. Despite extensive effort and constant progress in our understanding of NHL pathogenesis, the DLBCL group remains an heterogeneous entity awaiting further clinico-pathological stratification. The working formulation has separated DLBCL into different histologic categories. This histologic distinction is often difficult, not reproducible and does not reflect different clinical outcome. The recently proposed WHO classification of lymphoid neoplasms has grouped all these types in a specific category under the term DLBCL [1]. The clinical heterogeneity of DLBCL is likely to be the clinical reflection of several intrinsic biological characteristics of
*Corresponding author. Tel.: 0 02 050 2343011. Fax: 0 02 050 2267563. E-mail:
[email protected] ISSN 1042-8194 print/ISSN 1029-2403 online # 2004 Taylor & Francis Ltd DOI: 10.1080/10428190310001607151
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tumor [3]. Molecular events in DLBCL are very complicated and the over-expression of c-Myc in DLBCL was stated to be largely independent of c-Myc translocation. The proto-oncogene c-Myc encodes a transcriptional factor that is implicated in various cellular process—cell growth, proliferation, loss of differentiation and apoptosis [4 – 6]. Deregulated expression of c-Myc family genes occurs in a broad range of human cancers and is often associated with poor prognosis, indicating a key role for this oncogene in tumor progression [7 – 10]. The tumor angiogenesis and vasculogenesis are an essential step for tumor growth and metastasis [11 – 13]. Tumor angiogenesis was postulated to be regulated by balance between pro- and anti-angiogenic growth factors [14]. Recent data obtained from in vitro observation [15] indicates that tumor angiogenesis can be induced by cellular c-Myc oncogene, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis [16]. The main angiogenic factor is VEGF which exerts its biological function through its receptor Flt-1(fims-like tyrosine kinase 1). The soluble form was stated to be naturally a produced antagonist [17]. Kendall et al. [18] reported that mRNA for a soluble truncated form of Flt-1(sFlt-1) was generated by alternative splicing in cultured human umbilical vein endothelial cells. To our knowledge the relationship between sVEGF, sFlt-1 concentrations and c-Myc expression in DLBCLs was not previously characterized. The aim of this study was to assess the relation between c-Myc oncogene expression and angiogenic factors, namely VEGF, Flt-1 and their impact on the patient outcome.
SUBJECTS This study includes 45 newly diagnosed DLBCL patients (26 males, 19 females), their ages ranged from 38 to 70 years (median 56) (Table I). The lymph node biopsies were performed at the time of clinical diagnosis. The initial diagnosis was confirmed by two pathologists. All patients included in this study were subjected to examination of past history to verify the presence of B symptoms, a physical examination to determine the presence of lymphadenopathy, or hepatosplenomegaly; standard blood tests which include complete blood counts (CBC), erythrocyte sedimentation rate (ESR), bone marrow aspiration/biopsy, serum LDH, serum b2 microglobulin, assessment of the c-Myc oncogene expression, sVEGF and sFlt-1 concentrations. In addition, radiological investigations were performed which included abdominal ultrasound, computerized tomography (CT), and chest X-ray. The DLBCL patients were followed up to 36 months or until patients’
TABLE I
Patients population (n = 45)
Age 5 60 5 60 Sex < , B-symptoms + 7 Bulky disease + 7 An Arbor stage I/II II/IV IPI score Low/low-intermediate risk High/high intermediate risk Patient state Non-survivors Partial response Remission
No
%
19 26
42.2 57.8
26 19
57.8 42.2
25 20
55.5 44.5
24 21
53.3 46.7
18 27
40.0 60.0
28 17
62.2 37.8
19 8 18
42.2 17.8 40.0
death (Median 18 months). Patients were classified according to the international prognostic index (IPI) (age, clinical stage, performance status, high sLDH, and number of extra nodal site). IPI was scored as lowintermediate low (scores 0, 1, 2) and high-intermediate high score (scores, 3, 4, 5). The DLBCL patients were classified according to the patients’ clinical state at 36 months follow-up into non-survivors (n = 19) and survivors (n = 26). The state of the living group was as follows: 18 patients in complete remission (CR), which was defined as normalization of physical and radiological findings 4 weeks after the last cycle of chemotherapy. Eight patients were in partial response (PR), which was defined as 50% reduction of the initial tumor mass. Nineteen patients who did not fulfil the above-mentioned criteria were considered non-responders (NR). All patients were treated with antracyclincontaining regimen. CHOP chemotherapy as cyclophosphamide 750 mg/m2 i.v. day 1, Doxorubicin 50 mg/m2 i.v. day 1, Vincristine 1.4 mg/m2 day 1, and Predinisone 100 mg p.o. days 1 – 5, was employed as front-line treatment. Ten normal subjects of matched age and sex were taken as a control group.
METHODS Venous blood samples (8 ml) were collected at diagnosis by venipuncture from the anticubital vein with minimal hemostasis and were divided as follow: 1 ml blood was added to EDTA tube for complete blood count assay (Coulter counter, Onyx); 3 ml were added to heparinized
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tube to separate platelets free plasma which was stored at 7 808C until sVEGF and sFlt-1 assay; 2 ml to sodium citrate tube for erythrocyte sedimentation rate assay (Westergreen method); the remaining blood sample was left to clot. The serum was obtained from clotted sample for determination of sLDH (Autoanalyzer, American Corp, IN, USA) and s.b2 microglobulin (ELISA). Enzyme Linked Immunosorbent Assay for SVEGF and SFlt-1 Plasma VEGF and Flt-1 were measured by ELISA using commercially available reagents and recombinant (R&D systems, Abingdon, UK) as described in the manufacturers sheet. The VEGF assay has a minimum sensitivity of 10 pg/ml, with an intra-assay coefficient of variation (CV) of 4.9% and inter assay CV of 6.4% at 1.6 pg/ml. The sFlt-1 assay has minimal sensitivity of 50 pg/ml, an intra-assay CV of 3.7%, and inter-assay CV of 8.8% at 10 ng/ml. Enzyme Linked Immunosorbent Assay of b2 Microglobulin Serum b2 microglobulin was determined by a kit obtained from Eurogenetics (Germany) according to manufacturer’s instructions. Briefly, predilution of the samples into 10 (10 ml serum 100 ml diluent) were done, then 25 ml samples and standards and 100 ml of enzyme conjugate were added into precoated wells and incubated for 30 min at room temperature. After 5 washes, 100 ml of buffer solution containing hydrogen peroxide and 100 of tetramethylbenzedine were added, and color was allowed to develop within 10 min at room temperature. The reaction was stopped by adding 50 ml of 2 N HCl, and the absorbence of each well was determined at 450 nm. Determination of C-Myc Expression by Immunohistochemistry C-Myc was searched for by specific monoclonal antibody IgG2 (clone 9 E11 from Novocastra Laboratories Ltd, Newcastle, UK). The detection kit (chromogen) was obtained from that laboratory of Vision, Fremont (CA, USA).
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20 min, then block by normal horse serum for 5 min at 378C (to block non specific back ground staining). The monoclonal antibody with 1 : 100 dilution was applied for 1 h in humid medium at room temperature, then washed 4 times in PBS, followed by a biotinylated goat antipolyvalent secondary antibody for 15 min at 378C and washed 4 times in PBS, then Streptavidin peroxidase was applied for 100 min at room temperature, then washed 4 times in a buffer. One to two drops of Diaminobenezidine (DAB) chromogen was added for 20 min at room temperature. Slides were counterstained with hematoxylin, dehydrated and cover slipped. Burkitt’s lymphoma specimen was used as positive control. Interpretation of the Results C-Myc positive staining was seen as fine brownish granules. The expression was scored semiquantitatively by analysis of 5 – 10 higher power fields by 2 independent observers. The results were scored as negative (0): if no staining was observed in any cell, or the positivity was detected in non-neoplastic cells or only in the cytoplasm. In contrast, results were scored as positive if neoplastic cells exhibited nuclear or paranuclear staining. The intensity of c-Myc expression was graded according to Pagnano et al. [19] into ( + ) if 1 – 9% of the cells were stained; ( + + ) if 10 – 50% of the cells were stained or ( + + + ) if more than 50% of the cells were stained. Fig. 1 shows different degrees of c-Myc expressions (grades 0, + , + + , + + + ). Statistical Analysis Data were analyzed using SPSS (statistical for social sciences) version 9. Qualitative data were presented as numbers and percent. Fisher’s exact test was used to test significant difference between groups. Quantitative data were presented as means + SEM. Student’s t-test was used for comparison between means. Correlation between data was done by Spearman’s rank correlation. Multivariate survival analysis was performed with Cox proportional – hazards model entering the cMyc expression, sVEGF, sFlt-1, IPI, sb2 microglobulin, and sLDH level at diagnosis. All P values were two tailed. P 5 0.05 was considered to be statistically significant.
Procedure For each lymph node biopsy block, 2 mm-thick sections were cut on neoprene-coated slides. The slides were deparaffinized and blocked for endogenous peroxidase (Which may produce non-specific background staining) with hydrogen peroxides for 20 min; then washed twice in phosphate buffer solution (PBS). Antigen retrieval was performed using Biogenex antigen retrieval citra solution in a 908C water bath for 30 min. The slides were then allowed to cool for
RESULTS Soluble VEGF, SFlt-1, Sb2 Microglobulin, and SLDH Concentration Levels SVEGF, sFlt-1, b2 microglobulin, and sLDH concentrations were significantly elevated in DLBCL patients as compared to normal controls (P = 0.000, P = 0.007, P = 0.001, and P = 0.000 respectively) (Table II).
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FIGURE 1 CT scan at the time of diagnosis of the B-ALL showing the different degree of c-Myc oncoprotein expression by immunostaining in DLBCL lymph nodes: (A) Negative (o); (B) Positive (+); (C) Positive (++); (D) Positive (+++).
TABLE II Group Control (n = 10) DLBCL (n = 45) P
s.VEGE, s.Flt-1, s.b2 microglobulin and s.LDH in DLBCL vs. normal controls
s.VEGF (pg/ml)
s.Flt-1 (ng/ml)
s.b2 microglobulin (ng/ml)
s.LDH (m/l)
133.0 + 18.5 671.6 + 53.4 0.000
36.4 + 1.8 90.6 + 9.1 0.007
2.0 + 0.18 4.7 + 2.3 0.001
233.8 + 14.2 524.1 + 23.1 0.000
Interrelation between C-Myc Over-expression, SVEGF, and SFlt-1 Levels SVEGF was significantly higher among DLBCL patients with positive c-Myc over-expression as compared to those with negative c-Myc over-expression (P = 0.000). In contrast sFlt-1 was significantly higher in c-Myc negative DLBCL group as compared to DLBCL patients with positive c-Myc over expression (P = 0.009) (Table III). When sVEGF and sFlt-1 were classified into normal and high levels (more than mean normal control + 2 SEM), all the patients with c-Myc over-expression have high VEGF. While, in negative c-Myc over-expression group; 5 patients have high sVEGF and 10 have normal sVEGF. Moreover all c-Myc positive patients have normal sFlt-1. On the other hand, among patients
group with c-Myc negative over-expression, 14 patients have normal sFlt-1 and 16 have high sFlt-1. This means that c-Myc over-expression always associated with increase in sVEGF, and absence of c-Myc overexpression always associated with increased sFlt-1 (Table IV; Fig. 1). Relation of C-Myc Over-expression, SVEGF, and SFlt-1 to Severity Markers in DLBCL SVEGF was correlated positively with severity markers in DLBCL being an older age group ( 5 60 years) as compared to a younger age group (P = 0.000); in DLBCl with bulky disease as compared to non-bulky disease (P = 0.000), in stage (3 + 4) vs. stage (1 + 2) (P = 0.000); in high- high-intermediate IPI score, vs. low- lowintermediate score (P = 0.000), but sVEGF did not differ
C-MYC ONCOGENE EXPRESSION TABLE III s.VEGF and sFlt-1 concentration levels among DLBCL patients positive for c-Myc vs. those negative for c-Myc over expression Group C-Myc over expression + 7
sVEGF
sFlt-1
875.0 + 45.7 264.7 + 22.3 P = 0.000
74.1 + 10.9 123.6 + 12.9 P = 0.019
TABLE IV Interrelation between c-Myc over expression and s.VEGF and s.Flt-1 concentration levels in DLBCL patients C-Myc over expression Parameters sVEGF Normal High sFlt-1 Normal High
Negative (n = 15)
Positive (n = 30)
10 5
– 30
– 15
14 16
between DLBCL with positive B symptoms as compared to those with negative B symptoms (P 4 0.05). In contrast, Flt-1 was correlated negatively with severity markers in DLBCL being lower in the higher age group ( 5 60 years) as compared to lower age group (P = 0.000); in DLBCl with bulky disease as compared to non bulky disease (P = 0.000), in stage (3 + 4) vs. stage (1 – 2) (P = 0.007); in high- high-intermediate IPI score, vs. low-intermediate low score (P = 0.013), but sFlt-1 did not differ between DLBCL with positive B symptoms as compared to those with negative B symptoms (P 4 0.05) (Table V). C-Myc over-expression was significantly higher in DLBCL patients aged 5 60 as compared to those 5 60 years (P = 0.00), in bulky disease vs. non-bulky disease (P = 0.002), in stage (3 + 4) vs. (1 + 2) (P = 0.00), in high-high intermediate IPI score as compared to low-low intermediate IPI score (P = 0.01). However, C-Myc overexpression was not significantly different in DLBCL patients with positive B symptoms as compared to those with negative B symptoms (P 4 0.05 (Table VI). VEGF was positively correlated with sLDH and b2 microglobulin (r = 0.6, P = 0.000, r = 0.69, P = 0.000 respectively); but negatively correlated to sFlt-1 (r 7 0.58, P = 0.000). On the other hand, sFlt-1 was negatively correlated to sLDH, and sB2 microglobulin (r 7 0.25, P 4 0.05, r 7 0.48, P = 0.001) (Table VII). Relation of C-Myc, SVEGF, SFlt-1 to Patients Outcome SVEGF and c-Myc over expression were significantly higher in non-living DLBCL patients as compared to
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TABLE V Relation between s.VEGF and s.Flt-1 concentrations and severity markers in DLBCL patients
Age 5 60 5 60 B-symptoms + 7 Bulky disease + 7 IPI score Low/low-intermediate risk High/high intermediate risk DLBCL stage I/II III/VI
s.VEGF
s.Flt-1
967.9 + 64.9 455.0 + 45.0 P = 0.000
52.1 + 8.5 118.8 + 11.8 P = 0.000
720.2 + 73.2 610.8 + 77.7 P 4 0.05
87.4 + 12.5 94.7 + 13.5 P 4 0.05
845.2 + 68.7 473.1 + 59.5 P = 0.000
66.1 + 9.8 118.7 + 13.8 P = 0.000
541.6 + 58.7 885.6 + 81.0 P = 0.001
107.9 + 11.5 62.2 + 12.2 P = 0.013
397.6 + 49.0 854.3 + 61.5 P = 0.000
119.9 + 12.0 71.2 + 11.6 P = 0.007
TABLE VI Relation of c-Myc over-expression to severity markers in DLBCL patients c-Myc over expression
Age 5 60 years 5 60 years B-symptoms 7 + Bulky disease 7 + IPI score Low High DLBCL stage I/II III/VI Patient outcome Non-survivors Survivors
Negative
Positive
P value
14 1
12 18
8 7
12 18
12 3
9 21
0.002
13 2
15 15
0.01
11 4
7 23
0.001
– 15
19 11
0.000
0.001
4 0.05
living patients (P = 0.000 for both). In contrast, Flt1 was significantly higher in living DLBCL patients as compared to non-living group. C-Myc over expression and elevated sVEGF were present in all deceased patients (n = 19). Moreover, in the nonliving group 12 out of 19 have normal sFlt-1 (Table VIII).
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TABLE VII Correlation between concentration levels of s.VEGF, s.Flt-1, s.LDH and b2 microglobulin
s.VEGF (pg/ml) s.Flt-1 (ng/ml)
TABLE IX analysis
s.Flt-1
s.LDH
s.b2 microglobulin
r 7 0.58 P 0.000 ----
r 0.6 P 0.000 r 7 0.25 P 4 0.05
r 0.7 P 0.000 r 0.62 P 0.00
TABLE VIII Relation between s.VEGF and s.Flt-1 concentration levels to DLBCL patients status Non survivors (n = 19) s.VEGF s. Flt-1
Partial response (n = 8)
990.0 + 53.9 551.9 + 74.5 P = 0.000 45.9 + 4.5 113.1 + 20.7 P = 0.000
Remission (n = 18) 388.7 + 52.8 127.9 + 14.6
Influence of sVEGF, sFlt-1, c-Myc expression, IPI, sb2 microglobulin and sLDH on survival of DLBCL patients in a multivariate
Variables sVEGF ( 5 vs. 5 725 pg/ml) cMyc (positive vs. negative) IPI (low/low intermediate vs. high/high intermediate) sb2 microglobulin ( 5 5.3 vs. 5 5.3) sFlt-1 ( 5 vs. 5 99.7 ng/ml) sLDH ( 5 vs. 5 577.9)
b coefficient
P value
Relative risk (eb)
95% CI for relative risk
3.36 3.11 2.47 2.17 73.37 0.31
5 0.0001 5 0.0001 0.002 0.006 0.031 0.09
35.7 17.9 11.91 10.51 2.63 1.77
16.14 – 207.14 5.8 – 25.6 2.82 – 207.31 2.79 – 180.1 1.58 – 4.39 0.75 – 5.17
b, estimated coefficient of hazard function; 95% CI, 95% confidence interval; sVEGF, serum vascular endothelial growth factor; sFlt-1, vascular endothelial growth factor; IPI, international prognostic index; sLDH, serum lactate dehyrogenase.
Multivariate Survival Analysis To determine whether c-Myc expression, sVEGF, and sFlt-1 have independent influence on survival, they were entered into a multivariate survival analysis together with IPI, sb2 microglobiulin, and sLDH level at diagnosis as shown in Table IX. The DLBCL patients were classified into two groups, those with high sVEGF, sFlt-1, sb2 microglobulin and sLDH, vs. those with lower level. The cut-off value for patient’s classification was the normal control group mean + 2 SEM. In this model, a high pretreatment concentration of sVEGF, sFLt-1, and cMyc over-expression had an independent influence on survival.
DISCUSSION C-Myc is a multifunctional proto-oncogene, and its constitutive expression results in oncogenic activation, and contributes to progression of a wide range of human and animal tumors. However, the known c-Myc functions which are cell cycle control and regulation of apoptosis was claimed to be insufficient to promote tumorigenesis, and that c-Myc oncoprotein must provide other functions that intiate and or/sustain malignancy. Recently, Baudino et al. [15], in an experimental study, reported a novel angiogenic function for c-Myc. The objective of this study was to assess this function in clinical samples. The c-Myc oncogene over-expression was observed in 66.6% in the studied DLBCL patients group. Whilst Pagnano et al. [19] reported similar findings, Kawaski et
al. [20] reported lower c-Myc oncogene expression. This variation could be attributed to differences in the methods of detection. The role of c-Myc oncoprotein in lymphomagenesis is complex. In addition to established function of c-Myc over-expression that leads to cell proliferation by promoting the expression of a set of cell cycle activators (cyclins D1, D2, E, and cdK4); transcriptional repression of cell cycle/growth arrest genes (gas1, p15, p21, p27 and gadd34, 7 45, and 7 153) [5]; and abolishment of differentiation and apoptosis, recently angiogenic function was stated [15]. b2 microglobulin and sLDH were significantly higher in the DLBCL group as compared to controls. This finding is in agreement with that of Alici et al. [21]. SLDH and b2 microglobulin were reported to be indirect indicators of tumor mass as well as markers of disease activity [22,23]. Angiogenesis (the formation of new blood vessels), is controlled by balance between positive and negative endothelial regulatory factors. Soluble VEGF and sFlt-1 were significantly elevated in DLBCL patients as compared to controls. This finding is in accordance partially with the results of Belgore et al. [24] who measured by ELISA sVEGF and sFlt-1 in hematological cancer and breast cancer, and concluded that sVEGF, not sFlt-1 levels were significantly higher in both diseases as compared to controls. Belgore et al. [24] also stated that the levels of VEGF were higher in hematological cancer as compared to breast cancer. Moreover, Barleon et al. [25] reported the presence of sFlt-1 in normal human serum and suggests that sFlt-1 plays an important role as a naturally occurring VEGF antagonist in the regulation and availability of VEGF-mediated biological activities in vivo.
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In the present study VEGF was significantly higher in c-Myc positive DLBCL patients as compared to cMyc negative patients. Moreover, all patients with cMyc over-expression are associated with elevated sVEGF. On the contrary, Flt-1 was significantly lowered in c-Myc positive patients as compared to those patients with negative c-Myc over-expression. In addition, all patients with normal or low sFlt-1 were positive for c-Myc over-expression. This means that cMyc over-expression associated with elevation of angiogenic factors and suppresses the angiogenic inhibitors. These findings support the in vitro reported findings by Baudino et al. [15]. Furthermore, Toi et al. [26] assessed the expression of VEGF and VEGFR1 (Flt-1) in breast cancer, and found a high expression of VEGF and VEGFR, and concluded that the intratumoral balance between sVEGFR1 and VEGF levels might be crucial for the progression of breast cancer. The precise mechanism by which c-Myc controls the expression of angiogenic factors has not been resolved, but VEGF regulation appears to be indirect. It was reported that c-Myc indirectly regulates VEGF RNA levels by affecting the turnover of VEGF RNA. Regulation of VEGF RNA stability during hypoxia and in other scenarios is well documented [27,28]. However, Baudino et al. [15] suggested that VEGF gene might be a trnascriptional target for c-Myc oncoprotein, and reported that c-Myc over-expression augment VEGF expression. Moreover, c-Myc augments angiogenesis through disabling the expression of Flk-1, and Flt-1. The increased soluble sFlt-1 in c-Myc DLBCL negative patients may have biological function so as to regulate plasma VEGF levels [18,29,30]. The inhibitor function of sFlt-1 to VEGF has been emerged in several studies. Goldman et al. [30] reported that native soluble VEGFR inhibits tumor growth, metastasis, and mortality rate. In addition Hasumi et al. [31] stated that soluble Flt-1 expression suppresses carcinomatous ascities in nude mice bearing ovarian cancer, and suggested that inhibition of VEGF activity by sFlt-1 expression may provide a mean to control carcinomatous ascites and angiogenesis of malignant ascites tumors. All patients with increased sVEGF were present among the c-Myc positive group. On the other hand, all c-Myc negative patients had high sFlt-1. This supports the in vitro-based suggestion that c-Myc oncogene might regulate angiogenesis through inducing expression of VEGF and suppression of sFLt-1. However among the c-Myc positive patients there were 16 of these patients who had high sFlt-1 levels; this may point to another mechanism controlling sFlt-1 expression or the degree of expression of c-Myc was not sufficient to suppress sFlt-1. C-Myc over-expression, and sVEGF were significantly higher among the non-living group as compared to the living group. In contrast, the living group had significantly higher sFlt-1 as compared to the non-living group.
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These findings are consistent with previous studies [32 – 35]. Multivariate analysis revealed, that pretreatment cMyc over-expression, high sVEGF at diagnosis had high adverse influence on DLBCL patients’ survival compared to IPI, sb2 microglobulin and sLDH. This finding suggests that coupling between angiogenesis-promoting and angiogenesis-inhibiting factors with c-Myc overexpression occurs in DLBCL patients and has a strong influence on the patient’s outcome. In conclusion, C-Myc over-expression significantly associated with high sVEGF and normal sFlt-1 level in DLBCL patients, suggested a complex interrelationship between c-Myc oncogene expression and angiogenic regulators. High sVEGF, c-Myc over-expression, and normal sFLt-1 levels at diagnosis had an independent, adverse influence on survival in DLBCL patients and were considered poor prognostic markers.
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