INTRODUCTION
Sarcoma is a rare malignant aggressive tumor originating from mesenchymal elements [1]. It can occur in children and adults [2]. The incidence of sarcoma is approximately < 1% of all malignancies in adults and 15% of all malignancies in children [3]. The American Cancer Society estimates the number of newly diagnosed sarcomas in the United States in 2020 to be 13,130. Sarcomas occur in nearly 10,000 people in the United States each year, and about 40% of patients die from local metastases or distant metastases [4]. Based on Hospital Information System (SIRS) data at Hasan Sadikin Hospital in Bandung, the number of
sarcomas from 2014 to 2018 was 1,818 patients. High- grade sarcoma has a poor prognosis due to frequent recurrences and distant metastases [5]. High-grade sarcoma has a recurrence rate of 30% and distant metastases of 50% after surgery. The mortality rate of high-grade sarcoma is 50–75% [6].
In patients with high-grade sarcoma, radiotherapy is needed instead of surgery. Combining radiotherapy with surgery has decreased the recurrence rate by <
10% and reduced metastases considerably by 21–29%
when compared to surgery alone [7]. The recurrence incident is a clinical manifestation of cancer cell resistance to radiotherapy. Therefore, it is necessary to find molecular markers to predict radiotherapy sensitivity
Association of Vascular Endothelial Growth Factor (VEGF) and Mammalian Target of Rapamycin (mTOR) with
Radiotherapy Response in High-Grade Sarcomas
Eny Soesilowati
1*, Afiati
1, Herry Yulianti
1, Bethy S Hernowo
1, Adji Kusumadjati
21 Department of Anatomical Pathology, Faculty of Medicine, Universitas Padjadjaran/Hasan Sadikin General Hospital, Bandung, Indonesia
2 Department of Radiotherapy, Faculty of Medicine, Universitas Padjadjaran/Hasan Sadikin General Hospital, Bandung, Indonesia
A R T I C L E I N F O Received : 18 August 2021 Reviewed : 27 September 2021 Accepted : 10 November 2021 Keywords:
high-grade sarcoma, VEGF, mTOR, radiotherapy
A B S T R A C T
Background: Sarcoma is a rare malignant aggressive tumor originating from mesenchymal elements. High-grade sarcoma has a poor prognosis. The mortality rate of high-grade sarcomas is 50–75%. Radiotherapy is needed in high-grade sarcoma. Radioresistance in the high-grade sarcoma is still common. Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) are molecular markers that play a role in radioresistance. This study aimed to analyze the association of VEGF and mTOR with radiotherapy response in high-grade sarcoma.
Methods: This study was an analytic observational study with a cross-sectional design. The minimum samples based on Lameshow’s formula included 40 selected paraffin-embedded tissue blocks from biopsy high-grade sarcoma, consisting of 20 samples of radioresistance and 20 samples of radiosensitive. Immunohistochemistry staining for VEGF and mTOR were performed on all samples. Statistical analysis used the Chi-Square test. The significance of the data was obtained when p-value <0.05.
Results: High immunoexpression of VEGF (OR = 17, p < 0.05) and mTOR in high-grade sarcoma showed a significant association with radiotherapy response (OR = 16, p < 0.05). The stepwise logistic regression analysis revealed that both VEGF and mTOR immunoexpression influenced radiotherapy response simultaneously.
Conclusions: In this study, which used a minimum sample, it can be concluded that the higher VEGF and mTOR immunoexpression showed the higher radioresistance in high-grade sarcoma.
*Corresponding author:
Eny Soesilowati
Department of Urology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
inclusion and exclusion criteria. The inclusion criteria for this study are as follows: paraffin blocks from biopsy preparations diagnosed with high-grade sarcoma (FNCLCC grades 2 and 3) histopathologically that had performed radiotherapy [16]; patients receiving complete radiotherapy with a dose of 60–70 Gy; availability of radiotherapy response data (based on clinical observation/clinical response) in the patient’s medical record; availability of paraffin blocks. The exclusion criteria for this study were paraffin blocks with a depleted and damaged tumor mass. The characteristic data included age, gender, histopathological type, histopathologic grade, and tumor size.
Furthermore, the samples were stained with Immunohistochemical staining, carried out manually by labeling the complex streptavidin-biotin immunoperoxidase using the One-Step Neopoly Detection Kit (Biogear Scientific, BioVentures, Inc., Iowa, USA). The sample was cut 4μm, deparaffinated using xylene, and rehydrated using an alcohol solution. The antigen collection used a decloaking tool for 45–60 minutes at 98˚C. The primary antibody was VEGF A PA1080 using rabbit monoclonal antibody from Boster with a dilution of 1:200. The second primary antibody was the polyclonal mouse anti-mTOR GTX 63019 (GeneTex, USA) with a 1:200 dilution. VEGF and mTOR immunoexpression in the cytoplasm were assessed using semi-quantitative scores based on the intensity and distribution of positive cells. VEGF was considered positive if the membrane and cytoplasm of the tumor cell were brown. The mTOR-positive cytoplasm of the tumor cell was brown. The intensity scores were 0 (negative), + 1 (weak), +2 (moderate), and +3 (strong).
The distribution of positive cells on VEGF immunoexpression was scored as negative (0%), 1 (1–
25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The distribution of positive cells on mTOR immunoexpression was scored as 0 (< 5%), 1 (6–20%), 2 (21–50%), and 3 (> 51%). Histoscore was determined from the multiplication of intensity and staining distribution scoring. The immunoexpression histoscore was later categorized as low (< 6) and high (≥ 6). These analyses were performed independently by two experienced pathologists using an Olympus CX 21 light microscope with magnifications of 4x, 10x, and 40x.
Multivariate analysis with multiple logistic regression was used. The significance of the data was obtained when p-value < 0.05. The data were processed using Statistical Package for Social Science (SPSS) version 24.0 for Windows [15].
Ethical Committee conducted the ethical test, and this study was declared ethical with certificate number LB.02.01/X.6.5/57/2020. Researchers have described the research objectives, benefits, procedures, risks, and confidentiality as ethical needs in research.
so that it is useful for planning more effective and selective therapy [8]. The molecular markers related to radiotherapy and being developed are VEGF and mTOR.
The association of VEGF immunoexpression with radiotherapy response has been investigated in various studies. However, the result is still conflicting [8,9]. In the study of Wibisono et al. [9] on cervical adenocarcinoma, it was shown that increased VEGF immunoexpression was correlated with a poor response to radiotherapy of cervical adenocarcinoma. In another study by Nurhidayat et al. [8] on nasopharyngeal carcinoma, there was no relationship between VEGF immunoexpression and radiotherapy response. The same association conflict is also seen in various studies of mTOR immunoexpression with response radiotherapy [10,11,12]. Kim et al. [10] conducted a study on 119 cervical cancer patients who had received radiotherapy, 21 of whom experienced recurrence or treatment failure, and patients with radiotherapy failure showed high mTOR expression. Nassim et al. [11] conducted a study on bladder cancer, showing an increase in the mTOR expression in cells that do not respond to radiotherapy.
In a study conducted by Kurnia et al. [12] on 21 samples of cervical carcinoma that underwent radiotherapy associated with mTOR immunoexpression, the results concluded that mTOR expression before radiotherapy could not be used as a predictor of radiotherapy response.
Therefore, this study aimed to analyze the association of VEGF and mTOR immunoexpression with radiotherapy response in high-grade sarcomas, so VEGF and mTOR examination before radiotherapy can be recommended to predict radiotherapy response of patients with high- grade sarcoma.
METHODS
This research was an observational analytic study with a cross-sectional design. This study’s population was patients with sarcoma who had a biopsy in Surgical Oncology Department, and the specimens were received by the Anatomical Pathology Department for histopathological examination, and the patients had complete radiotherapy at a dose of 60–70 Gy in the Radiotherapy Department from January 1, 2014, to December 31, 2018, at Dr. Hasan Sadikin Hospital Bandung, Indonesia.
All study materials were paraffin blocks of patients who had a biopsy before radiotherapy and had been diagnosed histopathologically as high-grade sarcoma (FNCLCC grade 2 and 3). The research used the minimum sample based on Lameshow’s formula [13]. 40 samples were consisting of 20 samples of radioresistance and 20 samples of radiosensitive. The research samples were taken consecutively [14,15]. The samples had met the
RESULT
Clinicopathological characteristics of patients
This study included 40 patients with an age range between 6 and 73 years and a mean age of 40 years.Patient characteristics are presented in Table 1. Fifteen patients (37.50%) were females, and 25 patients (62.50%) were males. Grading system was adopted from FNCLCC (French Federation Nationale dea Centres de Lutte Contre le Cancer). Most histopathologic type is rhabdomyosarcoma (11 patients {27.50%}). The highest degree of differentiation is grade III (24 patients {60%}).
Tumor size ≥ 10 cm (22 patients {55%}) was the largest in this study.
Association between patient characteristic and radiotherapy response in high-grade sarcoma is shown in Table 2. Based on P value and OR in Table 2, there was no significant association between patients characteristic (age, gender, histopathology type, histopathological grade and tumor size) with radiotherapy response.
Association of VEGF Immunoexpression with Radiotherapy Response in High-grade Sarcomas
The P-value of the VEGF immunoexpression variable less than 0.05 (0.00**) showed a statistically significant difference between the VEGF immunoexpression variable in radioresistant high-grade sarcoma patients and radiosensitive high-grade sarcoma patients. Based on the odds ratio, it could be concluded that the radioresistance risk of high-grade sarcoma patients with high VEGF immunoexpression was 17 times higher than low VEGF immunoexpression (confidence interval of 3.46–83.33) (Table 3).
Association of mTOR Immunoexpression with Radiotherapy Response in High-grade Sarcomas
The P-value in the mTOR immunoexpression variable was less than 0.05 (0.00**), showing a statistically significant difference between the mTOR immunoexpression variable in radioresistant high-grade sarcoma patients and radiosensitive high-grade sarcoma patients. Based on the odds ratio, it could be concluded that the radioresistance risk of high-grade sarcoma patients with high mTOR immunoexpression was 16 times higher than low mTOR immunoexpression. (confidence interval of 3.40–76.92) (Table 4).
Multivariate Analysis of the Association of VEGF and mTOR Immunoexpression with Radiotherapy Response in High-Grade Sarcomas
The result of the multivariate analysis shows that all p-values of all variables were less than 0.05 (p < 0.05), meaning that VEGF and mTOR immunoexpression is associated with radiotherapy response in high-grade sarcomas simultaneously. VEGF immunoexpression
simultaneously had more radiotherapy response (OR:
26.74) than mTOR immunoexpression (OR: 25.15) (Table 5).
Immunoexpression of VEGF and mTOR
IHC staining on VEGF and mTOR was performed on each sample. The staining results were evaluated according to the intensity and staining distribution of positive cell tumors (Figures 1 and 2).
DISCUSSION
Various studies are analyzing the association of VEGF or mTOR immunoexpression with radiotherapy response in carcinoma, but in this study, we analyzed the association of both VEGF and mTOR immunoexpression in high-grade sarcoma. We demonstrated that VEGF and mTOR immunoexpression was significantly higher in high-grade sarcoma patients with radioresistance. The stepwise logistic regression multivariate analysis revealed that both VEGF and mTOR immunoexpression influenced radiotherapy response simultaneously. This result indicated that both VEGF and mTOR expression were upregulated in the radioresistance of high-grade sarcoma and might contribute to radioresistance in high-grade sarcoma. The result of this study is consistent with the study performed by Wibisono et al. [9] and Armanza et al. [17] that describe a significant correlation of VEGF overexpression with radioresistance in cervical carcinoma [9]. Similarly, the result of this study was also observed in the study performed by Kim et al. [10] that described a significant correlation of mTOR overexpression with radioresistance in cervical carcinoma.
In many cancers, including sarcoma, VEGF and mTOR play a role in radiosensitivity. Radiosensitivity is influenced by several factors, including the tumor environment such as hypoxia [18] Hypoxia in high-grade sarcomas may occur due to rapid tumor growth and damage to blood vessel endothelial cells by radiation [19]. Hypoxia induces HIF-1α and stimulates VEGF release. VEGF circulates and binds to VEGF receptors (VEGFR) on endothelial cells, triggering the tyrosine kinase pathway that leads to angiogenesis [20-22].
Angiogenesis is a process that involves the formation of new blood vessels to obtain the nutrients and oxygen needed for growth and survival [23,24]. This angiogenesis causes abnormal neovascularization. The new blood vessels are typically highly permeable and curved in random patterns, interconnecting, and branching due to the excessive recruitment and proliferation of the disorganized and irregular endothelial cells (caused by excessive secretion of VEGFA by the tumor cells) [9,25,26]. It causes a hypoxia tumor environment. The decreased oxygen levels observed in hypoxia cells lead to resistance to radiotherapy due to the reduced availability of oxygen which is needed to stabilize the
Variable High-Grade Sarcoma Group
P value OR (Cl 95%) Radiosensitive
N=20 Radioresistance N=20
Age (year)
< 40 40–60
> 60
10 (50%) 9 (45%) 1 (5%)
8 (40%) 10 (50%) 2 (10%)
0.23 5.02 (0.35–72.75)
Gender
MaleFemale 13 (65%)
7 (35%) 12 (60%) 8 (40%)
0.72 0.55 (0.02–13.88) Histopathology Type
Myxoid liposarcoma Pleomorphic Liposarcoma Leiomyosarcoma
Rhabdomyosarcoma Fibrosarcoma Myxofibrosarcoma
Undifferentiated Pleomorphic Sarcoma Synovial Sarcoma
Malignant Peripheral Nerve Sheath tumor Osteosarcoma
0 1 (5%) 0 6 (30%) 6 (30%) 1 (5%) 4 (20%) 0 2 (10%) 0
4 (20%) 0 1 (5%) 5 (25%) 2 (10%) 2 (10%) 2 (10%) 1 (5%) 1 (5%) 2 (20%)
0.50 0.82 (0.48–1.44)
Grading (FNCLCC)
IIIII 6 (30%)
14 (70%) 10 (50%) 10 (50%)
0.06 237.36 (0.76–73897.66) Tumor size
< 5cm 5–10 cm
> 10cm
4 (20%) 7 (35%) 9 (45%)
2 (10%) 5 (25%) 13 (65%)
0.40 2.06 (0.37–11.53) Table 2.
Association between patients Characteristics and Radiotherapy response in High-Grade Sarcoma
Variable N= 40 (%)
Age (year) Mean ± SD Median
Range (min-max)
< 40 40–60
> 60
39.90 ± 16.97 446–73
18 (45%) 19 (47.50%) 3 (7.50%) Gender
MaleFemale 25 (62.50%)
15 (37.50%) Histopathology Type
Myxoid liposarcoma Pleomorphic Liposarcoma Leiomyosarcoma
Rhabdomyosarcoma Fibrosarcoma Myxofibrosarcoma
Undifferentiated Pleomorphic Sarcoma Synovial Sarcoma
Malignant Peripheral Nerve Sheath Tumor Osteosarcoma
4 (10%) 1 (2.50%) 1 (2.50%) 11 (27.50%) 8 (20%) 3 (7.50%) 5 (12.50%) 1 (2.50%) 3 (7.50%) 2 (5%) Grading (FNCLCC)
IIIII 16 (40%)
24 (60%) Tumor size
< 5cm 5–10 cm
> 10cm
6 (15%) 12 (30%) 22 (55%) Table 1. Characteristics
of Research Subjects
SD, standard deviation; min, minumum; ma, maximum; FNCLCC, Fédération Nationale des Centres de Lutte Contre le Cancer
Figure 2.
Immunoexpression of mTOR. A. Strong staining intensity (+3);
B. Moderate staining intensity (+2); C. Weak staining intensity (+1).
(Magnification 200x) Figure 1.
Immunoexpression of VEGF. A. Strong staining intensity (+3);
B. Moderate staining intensity (+2); C. Weak staining intensity (+1).
(Magnification 200x)
variable High-grade sarcoma group
OR (CI 95%) P value Radiosensitive N = 20 Radioresistance N=20
mTORLow
High 16 (80%)
4 (20%) 4 (20%)
16 (80%)
15.87 ≈ 16 (3.40-76.92)
0.00**
OR, odds ratio; Cl, confidence interval. mTOR, mammalian target of rapamycin The value of significance based on the value of p ≤ 0.05
The ** sign indicates p value ≤0.05 means significant or statistically significant Table 4. Relationship of
mTOR immunoexpression and radiotherapy response in high-grade sarcomas
Variable High-grade sarcoma group
OR (CI 95%) P value Radiosensitive N = 20 Radioresistance N = 20
VEGFLow
High 15 (75%)
5 (25%) 3 (15%)
17 (85%)
16.94 ≈ 17
(3.46–83.33) 0.00**
OR, odds ratio; Cl, confidence interval. VEGF, vascular endothelial growth factor; mTOR, mammalian target of rapamycin
The value of significance based on the value of p ≤ 0.05
The ** sign indicates p value ≤0.05 means significant or statistically significant Table 3. Association of VEGF
Immunoexpression with Radiotherapy response on High-grade sarcoma
Variable B P value OR OR CI 95%
Lower Upper
VEGF 3.28 .00** 26.74 2.69 265.23
mTOR 3.22 .00** 25.15 2.61 241.95
OR, odds ratio; Cl, confidence interval. VEGF, vascular endothelial growth factor; mTOR, mammalian target of rapamycin
The value of significance based on the value of p ≤ 0.05
The ** sign indicates p value ≤0.05 means significant or statistically significant Table 5. Multivariate analysis
of the association between VEGF and mTOR
Immunoexpression and Radiotherapy Response in High-Grade Sarcomas
Research Funding
This study was funded by Residence Funding from Ministry of Defense and Security of the Republic of Indonesia. (KEP/01/I/2016).
Acknowledgment
This study supported and granted by Ministry of Defense and Security of the Republic of Indonesia. We would like to show our gratitude to all staf Radiotherapy Departement and Anatomical Pathology Departement of Hasan Sadikin General Hospital, Bandung, for their support in this study.
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DNA strand breaks caused by radiotherapy [26]. Hypoxia tumor cells are three times more radioresistant than cells with normoxia [9]. Radiosensitivity is also affected by cell cycle regulation. mTOR plays a role in cell cycle regulation through activation of the PI3k/AKT pathway for tumor cell proliferation and the repair of radiation- damaged cells. This causes radioresistance [18,26]. VEGF and mTOR also cooperate in angiogenesis and cell cycle regulation through the PI3K/AKT/mTOR pathway [26].
This theory supports our result that VEGF and mTOR immunoexpression influence radiotherapy response in high-grade sarcoma.
This study showed that the risk of high-grade sarcoma patients with high VEGF and mTOR immunoexpression radioresistance was 17 and 16 times higher than patients with low VEGF and mTOR immunoexpression, respectively.
Interestingly, the risk of high-grade sarcoma patients with both VEGF and mTOR immunoexpression for radioresistance increased to 26.74 and 25.15 times higher, respectively.
The variable that simultaneously had the most influence on radioresistance was VEGF immunoexpression (OR:
26.74), instead of mTOR immunoexpression (OR: 25.15).
The limitations of this study are the difficulty of finding medical record data, other than because sarcoma cases are rare, and the damaged medical record data and paraffin block samples, especially those stored for more than 3 years. Therefore, the sample used in this study was the minimum sample. We need a similar study using larger samples and variables to further elucidate the mechanism by which VEGF and mTOR affect radiotherapy response simultaneously.
CONCLUSIONS
Our study using a minimum sample showed that VEGF and mTOR immunoexpression in high-grade sarcomas had a significant association with radiotherapy response. It can be concluded that the higher the immunoexpression of VEGF and mTOR, the higher the radioresistance in high-grade sarcomas. The results of this study are expected to be a consideration for clinical doctors in treating patients and increase knowledge in other similar studies.
DECLARATIONS Ethics Approval
The research has received ethical approval from the Ethical committee of of Hasan Sadikin General Hospital with certificate number: LB.02.01/X.6.5/57/2020.
Competing of Interest
The authors declare no competing interest in this study.
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