# The Expression and Clinical Significance of Sphingosine Kinase 1 and Vascular Endothelial Growth Factor in Endometrial Carcinoma

Authors: Hong Yu; Dejizhuoga; Wei Huang; Donglian Wang; Gamaquzhen; Xiaomin Jia; Hao Feng
Journal: Emergency Medicine International
 (2022)

---

## Abstract

The aim of the study is to investigate the expression of sphingosine kinase 1 (SPHK1) and vascular endothelial growth factor (VEGF) in patients with endometrial carcinoma and its clinical significance. The tissues of 86 cases of patients with endometrial carcinoma and 54 cases of patients with endometrial atypical hyperplasia were collected. The expression of SPHK1 and VEGF in the tissue was detected by immunohistochemistry. The expression of SPHK1 in patients with endometrial carcinoma was compared with the clinicopathological data.Results. 69 cases (82.1%) of endometrial carcinoma were positive for SPHK1, which was higher than 2 cases (3.7%) of endometrial atypical hyperplasia (P<0.05). The VEGF expression in 54 patients (62.8%) with endometrial carcinoma was higher than that in 12 patients with endometrial atypical hyperplasia (22.2%) (P<0.05). There was a positive correlation between SPHK1 and VEGF expressions in endometrial carcinoma (c = 0.595). The expression of SPHK1 in endometrial cancer patients was different in different pathological types, FIGO stages, lymph node metastasis, ER, and PR positive or not, and the difference between the two groups was significant (P<0.05). There was no difference in age, degree of differentiation, and depth of myometrial infiltration (P<0.05). The expression of SPHK1 in patients with endometrial carcinoma is increased, which is helpful for early detection of patients with endometrial carcinoma, and may play a synergistic role with VEGF in the pathogenesis and development of endometrial carcinoma.


---

## Body

## 1. Introduction

Endometrial carcinoma is one of the most prevalent malignant tumors of the female reproductive system, which threatens more and more women [1, 2]. The pathogenesis of endometrial carcinoma is still unclear, which may be a multistep, multistage, and multifactor biological evolution process, involving genetic variation of various molecules [3]. The postoperative survival rate of endometrial carcinoma can reach 80%, but the incidence is still increasing gradually [4, 5]. Early diagnosis and timely intervention are essential to improve the prognosis of patients.At present, the role of sphingolipids in cancer biology is a new field of lipid research, mainly to study the roles of different sphingolipid-acting enzymes, sphingolipid-binding proteins, and transmembrane transporters in tumors [6, 7]. SPHK family members have attracted much attention because their catalytic activity is at the key intersection in regulating the metabolism of sphingolipids with biological activity [8, 9]. Sphingosine kinases 1 (SPHK1) are involved in the processes related to cancer progression, including cell transformation, survival and migration, metastasis, and neovascularization of the tumor microenvironment [10, 11].Angiogenesis is essential for physiological processes such as wound healing and tissue remodeling of ischemic tissue diseases, as well as embryo implantation and endometrial repair after menstruation [12]. The ability of a tumor to develop from a non-angiogenesis to angiogenesis phenotype is the core of cancer development, which is called the “angiogenesis switch” [13]. This phenomenon is a prerequisite for tumor growth and metastasis. Tumors can migrate from the primary site to the new site through direct metastasis, blood vessels, or the lymphatic system. It is considered that the growth of tumors larger than 1-2 mm is vascular-dependent. It has been previously reported that the epidermal growth factor receptor and vascular endothelial growth factor (VEGF) play an important role. Especially, the regulation of VEGF gene expression is related to differentiation, hormones, cytokines, oxygen partial pressure, and many other factors [14, 15]. VEGF, as the most effective promoter of vascular endothelial cell division, is the key to tumor occurrence, invasion, and metastasis. It can prevent the immune response of tumor cells by promoting tumor growth and hindering the maturation of host-specific antigen-presenting cells [16].However, there is little research on the effect of SPHK1 on endometrial carcinoma. Therefore, the purpose of this study is to explore the expression of sphingosine kinase 1 (SPHK1) and VEGF in patients with endometrial carcinoma and its clinical significance and provide a reference for further study.


## 2. Materials and Methods

### 2.1. Case Data

Eighty-six patients with endometrial carcinoma aged from 39 to 70 years, who were first seen in Hunan Provincial People’s Hospital, Hunan Maternal and Child Health Hospital, and Shenzhen Third People’s Hospital from June 2015 to December 2021 and all of who were diagnosed by pathological biopsy, were selected. According to FIGO surgical pathological staging, there were 63 cases in stages I-II and 23 cases in stages III-IV. Twenty-six cases were highly differentiated, 30 cases were moderately differentiated, and 30 cases were poorly differentiated. Also, 54 cases of patients with endometrial atypical hyperplasia aged from 36 to 68 years were selected. There was no significant difference in age and course of disease between the two groups (P<0.05). Patients with endometrial carcinoma did not receive radiotherapy or chemotherapy before the operation, and all patients in the control group had no other gynaecological diseases related to hormones. Patients’ tissues were routinely fixed with 10% formaldehyde, embedded in paraffin, and 3∼5 um sections were pasted on antidropping slides for later use. All patients signed the informed consent form, which was approved by the Ethics Committee of Hunan Provincial People’s Hospital.


### 2.2. Methods

#### 2.2.1. Reagents and Operations

The sphingosine kinase primary antibody (rabbit antihuman, concentrated) was purchased from Shanghai Yansheng Biochemical Reagents Co., Ltd., and the SP kit and DAB chromogenic kit were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Immunohistochemistry was performed by the SP method, and the operation method was carried out according to the instructions of the kit.


#### 2.2.2. The Detection of Expression of SPHK1 and VEGF in Tissues by the Immunohistochemical Method

The method of streptavidin peroxidase (SP) was used for the detection of expression of SPHK1 and VEGF in tissues, and the steps were as follows: Paraffin slices were soaked in fresh xylene for 10 min × 3; absolute ethanol for 3 min × 3, 95°C ethanol for 3 min × 2, 75% ethanol for 3 min × 2, washed for 1 min with distilled water, and put in PBS buffer. After antigen repair, an appropriate amount of endogenous peroxidase blocker was added, incubated at room temperature for 20 min, and rinsed with PBS. SPHK1(1 : 200) and VEGF (ready-to-use) primary antibodies were then added and incubated at 37°C for 60 min, and rinsed with PBS. A reaction enhancing solution was added, incubated at room temperature for 20 min, and washed with PBS. Then, goat anti-rabbit IgG polymer labeled with an enhance enzyme was dropwise added, incubated at room temperature for 20 min, and washed with PBS. Finally, freshly prepared DAB color solution was added, incubated at room temperature for 5–8 min, re-dyed with hematoxylin, dehydrated, and transparently sealed. PBS was used as a negative control.


#### 2.2.3. Results’ Judgement

10 high-power fields (×400) were randomly selected from each picture, and SPHK1-positive and VEGF-positive cells were brown-yellow particles stained by a cell membrane or cytoplasm. Referring to Ma X’s study [17], it can be divided into negative (−) <5%, positive (+) 6–25%, positive (++) 26–50%, and positive (++) > 50% according to the number of positive cells and the intensity of color development.


#### 2.2.4. Statistical Analysis

SPSS for windows 19.0 statistical software was used for analysis. The positive rate and correlation were compared by the Chi-square test, and the correlation column connection number (C) was calculated by the formulaC=x2/n+x2. P<0.05 means the difference was statistically significant.


## 2.1. Case Data

Eighty-six patients with endometrial carcinoma aged from 39 to 70 years, who were first seen in Hunan Provincial People’s Hospital, Hunan Maternal and Child Health Hospital, and Shenzhen Third People’s Hospital from June 2015 to December 2021 and all of who were diagnosed by pathological biopsy, were selected. According to FIGO surgical pathological staging, there were 63 cases in stages I-II and 23 cases in stages III-IV. Twenty-six cases were highly differentiated, 30 cases were moderately differentiated, and 30 cases were poorly differentiated. Also, 54 cases of patients with endometrial atypical hyperplasia aged from 36 to 68 years were selected. There was no significant difference in age and course of disease between the two groups (P<0.05). Patients with endometrial carcinoma did not receive radiotherapy or chemotherapy before the operation, and all patients in the control group had no other gynaecological diseases related to hormones. Patients’ tissues were routinely fixed with 10% formaldehyde, embedded in paraffin, and 3∼5 um sections were pasted on antidropping slides for later use. All patients signed the informed consent form, which was approved by the Ethics Committee of Hunan Provincial People’s Hospital.


## 2.2. Methods

### 2.2.1. Reagents and Operations

The sphingosine kinase primary antibody (rabbit antihuman, concentrated) was purchased from Shanghai Yansheng Biochemical Reagents Co., Ltd., and the SP kit and DAB chromogenic kit were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Immunohistochemistry was performed by the SP method, and the operation method was carried out according to the instructions of the kit.


### 2.2.2. The Detection of Expression of SPHK1 and VEGF in Tissues by the Immunohistochemical Method

The method of streptavidin peroxidase (SP) was used for the detection of expression of SPHK1 and VEGF in tissues, and the steps were as follows: Paraffin slices were soaked in fresh xylene for 10 min × 3; absolute ethanol for 3 min × 3, 95°C ethanol for 3 min × 2, 75% ethanol for 3 min × 2, washed for 1 min with distilled water, and put in PBS buffer. After antigen repair, an appropriate amount of endogenous peroxidase blocker was added, incubated at room temperature for 20 min, and rinsed with PBS. SPHK1(1 : 200) and VEGF (ready-to-use) primary antibodies were then added and incubated at 37°C for 60 min, and rinsed with PBS. A reaction enhancing solution was added, incubated at room temperature for 20 min, and washed with PBS. Then, goat anti-rabbit IgG polymer labeled with an enhance enzyme was dropwise added, incubated at room temperature for 20 min, and washed with PBS. Finally, freshly prepared DAB color solution was added, incubated at room temperature for 5–8 min, re-dyed with hematoxylin, dehydrated, and transparently sealed. PBS was used as a negative control.


### 2.2.3. Results’ Judgement

10 high-power fields (×400) were randomly selected from each picture, and SPHK1-positive and VEGF-positive cells were brown-yellow particles stained by a cell membrane or cytoplasm. Referring to Ma X’s study [17], it can be divided into negative (−) <5%, positive (+) 6–25%, positive (++) 26–50%, and positive (++) > 50% according to the number of positive cells and the intensity of color development.


### 2.2.4. Statistical Analysis

SPSS for windows 19.0 statistical software was used for analysis. The positive rate and correlation were compared by the Chi-square test, and the correlation column connection number (C) was calculated by the formulaC=x2/n+x2. P<0.05 means the difference was statistically significant.


## 2.2.1. Reagents and Operations

The sphingosine kinase primary antibody (rabbit antihuman, concentrated) was purchased from Shanghai Yansheng Biochemical Reagents Co., Ltd., and the SP kit and DAB chromogenic kit were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Immunohistochemistry was performed by the SP method, and the operation method was carried out according to the instructions of the kit.


## 2.2.2. The Detection of Expression of SPHK1 and VEGF in Tissues by the Immunohistochemical Method

The method of streptavidin peroxidase (SP) was used for the detection of expression of SPHK1 and VEGF in tissues, and the steps were as follows: Paraffin slices were soaked in fresh xylene for 10 min × 3; absolute ethanol for 3 min × 3, 95°C ethanol for 3 min × 2, 75% ethanol for 3 min × 2, washed for 1 min with distilled water, and put in PBS buffer. After antigen repair, an appropriate amount of endogenous peroxidase blocker was added, incubated at room temperature for 20 min, and rinsed with PBS. SPHK1(1 : 200) and VEGF (ready-to-use) primary antibodies were then added and incubated at 37°C for 60 min, and rinsed with PBS. A reaction enhancing solution was added, incubated at room temperature for 20 min, and washed with PBS. Then, goat anti-rabbit IgG polymer labeled with an enhance enzyme was dropwise added, incubated at room temperature for 20 min, and washed with PBS. Finally, freshly prepared DAB color solution was added, incubated at room temperature for 5–8 min, re-dyed with hematoxylin, dehydrated, and transparently sealed. PBS was used as a negative control.


## 2.2.3. Results’ Judgement

10 high-power fields (×400) were randomly selected from each picture, and SPHK1-positive and VEGF-positive cells were brown-yellow particles stained by a cell membrane or cytoplasm. Referring to Ma X’s study [17], it can be divided into negative (−) <5%, positive (+) 6–25%, positive (++) 26–50%, and positive (++) > 50% according to the number of positive cells and the intensity of color development.


## 2.2.4. Statistical Analysis

SPSS for windows 19.0 statistical software was used for analysis. The positive rate and correlation were compared by the Chi-square test, and the correlation column connection number (C) was calculated by the formulaC=x2/n+x2. P<0.05 means the difference was statistically significant.


## 3. Results

### 3.1. Comparison of the Expression of SPHK1 and VEGF in Endometrial Carcinoma and Endometrial Atypical Hyperplasia

Figure1 shows the expression of SPHK1 and VEGF in the tissues of patients in each group. As shown in Table 1, 69 cases (82.1%) of 86 patients with endometrial carcinoma were positive for SPHK1 and 2 cases (3.7%) of 54 patients with atypical hyperplasia of the endometrium were positive for SPHK1. The difference in the positive rate between the two groups was statistically significant (χ2 = 77.725, P<0.001). In 86 cases of endometrial carcinoma, 54 cases (62.8%) were positive for VEGF and 12 cases (22.2%) were positive for VEGF in patients with atypical hyperplasia of the endometrium. The difference in positive rates between the two groups was statistically significant (χ2 = 21.909, P<0.001).Figure 1
Expression of SPHK1 and VEGF in the tissues of patients in each group. Left-top: The expression of SPHK1 in endometrial carcinoma was positive (×200). Right-top: The expression of SPHK1 in endometrium (×200). Left-bottom: VEGF expression in endometrial atypical hyperplasia group (× 400). Right-bottom: The expression of VEGF in endometrium was negative (×400).Table 1
Comparison of the expression of SPHK1 and VEGF between the two groups.
GroupsNumberSPHK1 (n (%))VEGF (n (%))Endometrial carcinoma8669 (82.1)54 (62.8)Atypical hyperplasia of endometrium542 (3.7)12 (22.2)χ277.72521.909P<0.001<0.001


### 3.2. Correlation Analysis of SPHK1 and VEGF Expressions in Endometrial Carcinoma

A chi-square test was used to analyze the correlation between SPHK1 and VEGF expressions in endometrial carcinoma, and there was a positive correlation between them (χ2 = 6.857, P=0.009, column connection number (c = 0.595), as shown in Table 2.Table 2
Correlation analysis of SPHK1 and VEGF expressions in endometrial carcinoma.
SPHK1VEGFTotal number+−+482169−61117Total number543286


### 3.3. The Relationship between SPHK1 Expression and Clinicopathological Factors in Endometrial Carcinoma

As shown in Table3, among patients with endometrial carcinoma, SPHK1 in 18 cases (18/24) was positive in patients younger than 50 years old and 51 cases (51/62) in patients older than 50 years old. There was no significant difference in the positive rate between them (χ2 = 0.575, P=0.448). In degree of differentiation, SPHK1 of 22 cases (22/26) of highly differentiated patients was positive, SPHK1 of 23 cases (23/30) of moderately differentiated patients was positive, and SPHK1 of 24 cases (24/30) of poorly differentiated patients was positive. There was no statistical difference between the three groups (χ2 = 0.556, P=0.757). 43 cases (43/51) were positive for SPHK1 with myometrial infiltration <1/2 and 26 cases (26/35), with myometrial infiltration ≥1/2. There was no significant difference in the positive rate between them (χ2 = 1.316, P=0.251).Table 3
The relationship between SPHK1 expression and clinicopathological factors in endometrial carcinoma.
Clinicopathological factorsNumberSPHK1 expressionχ2P+−866917Age0.5750.448<50 years old24186≥50 years old625111Pathological types24.632<0.001Adenocarcinoma41401Serous type23194Clear cell type1367Other types945FIGO staging4.4630.035I-II63549III-IV23158Degree of differentiation0.5560.757Highly differentiated26224Moderately differentiated30237Poorly differentiated30246Muscle infiltration1.3160.251<1/251438≥1/235269Lymph node metastasis6.6570.010Yes58517No281810ER4.9860.026Positive30282Negative564115PR6.5610.010Positive39363Negative473314There were 40 cases (40/41) with positive SPHK1 in adenocarcinoma, 19 cases (19/23) with serous type, 6 cases (6/13) with clear cell type, and 4 cases (4/9) with other types. The difference between groups was statistically significant (χ2 = 24.632, P<0.001). SPHK1 was positive in 54 cases (54/63) in FIGO stage I-II and 15 cases (15/23) in stage III-IV. The difference between groups was statistically significant (χ2 = 4.463, P=0.035). There were 51 cases (51/58) with lymph node metastasis and 18 cases (18/28) without lymph node metastasis, and the difference between the two groups was statistically significant (χ2 = 6.657, P=0.010). Twenty-eight (28/30) of ER-positive patients were SPHK1-positive and 41 (41/56) of ER-negative patients were SPHK1-positive. The difference between the two groups was statistically significant (χ2 = 4.986, P=0.026). There were 36 cases (36/39) with positive PR and 33 cases (33/47) with negative PR, and the difference between the two groups was statistically significant (χ2 = 6.561, P=0.010).


## 3.1. Comparison of the Expression of SPHK1 and VEGF in Endometrial Carcinoma and Endometrial Atypical Hyperplasia

Figure1 shows the expression of SPHK1 and VEGF in the tissues of patients in each group. As shown in Table 1, 69 cases (82.1%) of 86 patients with endometrial carcinoma were positive for SPHK1 and 2 cases (3.7%) of 54 patients with atypical hyperplasia of the endometrium were positive for SPHK1. The difference in the positive rate between the two groups was statistically significant (χ2 = 77.725, P<0.001). In 86 cases of endometrial carcinoma, 54 cases (62.8%) were positive for VEGF and 12 cases (22.2%) were positive for VEGF in patients with atypical hyperplasia of the endometrium. The difference in positive rates between the two groups was statistically significant (χ2 = 21.909, P<0.001).Figure 1
Expression of SPHK1 and VEGF in the tissues of patients in each group. Left-top: The expression of SPHK1 in endometrial carcinoma was positive (×200). Right-top: The expression of SPHK1 in endometrium (×200). Left-bottom: VEGF expression in endometrial atypical hyperplasia group (× 400). Right-bottom: The expression of VEGF in endometrium was negative (×400).Table 1
Comparison of the expression of SPHK1 and VEGF between the two groups.
GroupsNumberSPHK1 (n (%))VEGF (n (%))Endometrial carcinoma8669 (82.1)54 (62.8)Atypical hyperplasia of endometrium542 (3.7)12 (22.2)χ277.72521.909P<0.001<0.001


## 3.2. Correlation Analysis of SPHK1 and VEGF Expressions in Endometrial Carcinoma

A chi-square test was used to analyze the correlation between SPHK1 and VEGF expressions in endometrial carcinoma, and there was a positive correlation between them (χ2 = 6.857, P=0.009, column connection number (c = 0.595), as shown in Table 2.Table 2
Correlation analysis of SPHK1 and VEGF expressions in endometrial carcinoma.
SPHK1VEGFTotal number+−+482169−61117Total number543286


## 3.3. The Relationship between SPHK1 Expression and Clinicopathological Factors in Endometrial Carcinoma

As shown in Table3, among patients with endometrial carcinoma, SPHK1 in 18 cases (18/24) was positive in patients younger than 50 years old and 51 cases (51/62) in patients older than 50 years old. There was no significant difference in the positive rate between them (χ2 = 0.575, P=0.448). In degree of differentiation, SPHK1 of 22 cases (22/26) of highly differentiated patients was positive, SPHK1 of 23 cases (23/30) of moderately differentiated patients was positive, and SPHK1 of 24 cases (24/30) of poorly differentiated patients was positive. There was no statistical difference between the three groups (χ2 = 0.556, P=0.757). 43 cases (43/51) were positive for SPHK1 with myometrial infiltration <1/2 and 26 cases (26/35), with myometrial infiltration ≥1/2. There was no significant difference in the positive rate between them (χ2 = 1.316, P=0.251).Table 3
The relationship between SPHK1 expression and clinicopathological factors in endometrial carcinoma.
Clinicopathological factorsNumberSPHK1 expressionχ2P+−866917Age0.5750.448<50 years old24186≥50 years old625111Pathological types24.632<0.001Adenocarcinoma41401Serous type23194Clear cell type1367Other types945FIGO staging4.4630.035I-II63549III-IV23158Degree of differentiation0.5560.757Highly differentiated26224Moderately differentiated30237Poorly differentiated30246Muscle infiltration1.3160.251<1/251438≥1/235269Lymph node metastasis6.6570.010Yes58517No281810ER4.9860.026Positive30282Negative564115PR6.5610.010Positive39363Negative473314There were 40 cases (40/41) with positive SPHK1 in adenocarcinoma, 19 cases (19/23) with serous type, 6 cases (6/13) with clear cell type, and 4 cases (4/9) with other types. The difference between groups was statistically significant (χ2 = 24.632, P<0.001). SPHK1 was positive in 54 cases (54/63) in FIGO stage I-II and 15 cases (15/23) in stage III-IV. The difference between groups was statistically significant (χ2 = 4.463, P=0.035). There were 51 cases (51/58) with lymph node metastasis and 18 cases (18/28) without lymph node metastasis, and the difference between the two groups was statistically significant (χ2 = 6.657, P=0.010). Twenty-eight (28/30) of ER-positive patients were SPHK1-positive and 41 (41/56) of ER-negative patients were SPHK1-positive. The difference between the two groups was statistically significant (χ2 = 4.986, P=0.026). There were 36 cases (36/39) with positive PR and 33 cases (33/47) with negative PR, and the difference between the two groups was statistically significant (χ2 = 6.561, P=0.010).


## 4. Discussion and Conclusion

From the perspective of molecular biology, endometrial carcinoma may be caused by abnormal activation of various oncogenes, overexpression of the encoded proteins, and uncontrollable malignant transformation induced by cell proliferation caused by deletion, mutation, and inactivation of nononcogenes [18]. SPHK, an important enzyme in cancer biology, has attracted much attention. It often exists in two subtypes, which are SPHK1 and SPHK2 [19]. Sphingosine-1-phosphate (S1P) is one of the metabolites produced by sphingosine kinase (SPHK1 and SPHK2) in cancer cells, which regulates many cellular processes, including inhibiting cell apoptosis and increasing cell proliferation and angiogenesis [20–22].In this study, 69 (82.1%) of 86 patients with endometrial carcinoma were positive for SPHK1, 2 (3.7%) of 54 patients with atypical hyperplasia of the endometrium. The positive rate between the two groups was different (P<0.05), indicating that the expression of SPHK1 was enhanced in endometrial carcinoma. More and more studies have also shown that SPHK1 is involved in the processes related to cancer progression, including cell transformation, survival and migration, metastasis, and neovascularization of the tumor microenvironment [23]. This study also found that there were differences in expression of SPHK1 in different pathological types, FIGO stages, lymph node metastasis, ER and PR positive or not (P<0.05), indicating that SPHK1 may be involved in the pathogenesis and development of endometrial carcinoma.VEGF is overexpressed in patients with anovulatory dysfunctional uterine bleeding. In gynecological tumors such as ovarian cancer, the expression of VEGF is related to the increased invasion of epithelial ovarian cancer cells in vivo and in vitro. In cervical cancer, VEGF is associated with a poor prognosis in young women. VEGF, as a marker of angiogenesis, is involved in endometrial remodeling after menstruation. During endometrial remodeling, the release of VEGF is thought to be caused by tissue hypoxia or ischemia. When tissues are hypoxic, hypoxia-inducible factors are stimulated in many ways, including the release of different growth factors including VEGF, which leads to the degradation of the extracellular matrix [24, 25]. These constant circulation changes of the endometrium, such as superficial shedding and neointimal reconstruction, are all related to angiogenesis and neovascularization [26].In this study, 54 out of 86 patients with endometrial carcinoma were positive for VEGF, which was higher than that of patients with atypical hyperplasia of the endometrium (22.2%) (P<0.05). This result was consistent with the results of previous research [27, 28]. In addition, there is a positive correlation between SPHK1 and VEGF expressions in endometrial carcinoma (P<0.05).The results of this study indicated that SPHK1 may be involved in the pathogenesis and development of endometrial carcinoma through its synergistic effect with VEGF. These findings are helpful for early detection of patients with endometrial carcinoma and also provide a clinical reference for further study on the influence of the expression of SPHK1 and VEGF in endometrial carcinoma.



---

*Source: 6716143-2022-09-21.xml*