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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 6  |  Page : 1373-1378

CD105 (endoglin) expression as a prognostic marker of angiogenesis in squamous cell cervical cancer treated with radical radiotherapy


1 Department of Radiation Oncology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, Turkey
2 Department of Pathology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, Turkey
3 Department of Obstetrics and Gynecology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, Turkey

Date of Web Publication28-Nov-2018

Correspondence Address:
Evrim Metcalfe
Department of Radiation Oncology, Eskisehir Osmangazi University Medical Faculty, Eskisehir
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.203602

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 > Abstract 


Introduction: Increased levels of endoglin may represent a new reagent of active neovascularization and angiogenesis process in various cancer types. The prognostic value of tumor CD105 (endoglin) expression in cervical squamous cell cancer (CSCC) patients treated with radical radiotherapy (RT) ± chemotherapy was investigated.
Materials and Methods: CD105 (endoglin) expression was assessed by immunohistochemical methods in seventy patients, who were treated with radical RT ± chemotherapy for CSCC. The prognostic effects of CD105 on patient and treatment characteristics, local-regional control, and survival were assessed.
Results: The median follow-up was 24 (5–99) months for the whole cohort. The median CD105 microvessel density was 55.5 (range; 12–136). Age (≤61 vs. >:61 years; P = 0.015), lymph node metastasis status (absent vs. present; P = 0.028), International Federation of Gynecology and Obstetrics stage (Ib–IIa vs. IIb–IVa; P = 0.036), cycles of concurrent chemotherapy (1–3 vs. 4–6 cycles; P = 0.001), and hemoglobin levels (≤10 g/dL vs. >:10 g/dL; P = 0.006) appeared to associate significantly with overall survival on univariate analysis.
Discussion: No correlation was identified between the tumor CD105 (endoglin) expression and survival in CSCC patients treated with radical RT ± chemotherapy.

Keywords: Angiogenesis, CD105/endoglin, cervical cancer, chemoradiotherapy, radiotherapy


How to cite this article:
Metcalfe E, Arik D, Oge T, Etiz D, Yalcin OT, Kabukcuoglu S, Pasaoglu O, Ozalp S S. CD105 (endoglin) expression as a prognostic marker of angiogenesis in squamous cell cervical cancer treated with radical radiotherapy. J Can Res Ther 2018;14:1373-8

How to cite this URL:
Metcalfe E, Arik D, Oge T, Etiz D, Yalcin OT, Kabukcuoglu S, Pasaoglu O, Ozalp S S. CD105 (endoglin) expression as a prognostic marker of angiogenesis in squamous cell cervical cancer treated with radical radiotherapy. J Can Res Ther [serial online] 2018 [cited 2020 Jul 12];14:1373-8. Available from: http://www.cancerjournal.net/text.asp?2018/14/6/1373/203602




 > Introduction Top


Cervical cancer is the third most common cancer worldwide but is also the second most frequent cause of death due to cancer among women.[1],[2] Concurrent chemoradiotherapy (C-CRT) is the standard of care in the management of locally advanced cervical cancer (LACC). Based on the results of Phase III randomized trials, pelvic radiotherapy (RT) and concurrent cisplatin-based chemotherapy are the standard of care treatment of LACC patients, which is also strongly recommended by the National Cancer Institute.[3],[4]

Due to the requirement for implementing personalized treatment and evaluating outcomes, biomarkers for predicting prognosis have emerged from recent studies.[5] In addition to squamous cell carcinoma (SCC), a tumor marker conventionally used for cervical cancer, several biomarkers have been identified that predict the response to anticancer therapy, including CD105 which may be a molecular target and predicts radiation sensitivity.[5],[6],[7]

Tumor growth beyond 1–2 mm is strictly dependent on neoangiogenesis (NA), the development of new blood vessels. Besides tumor growth, NA plays an essential role on tumor invasion and metastasis and is controlled by a complex and dynamic mechanism that involves the critical balance between multiple antiangiogenic and proangiogenic signals. In addition to conventional tumor-related factors, the markers of NA have also been investigated at various tumor sites for their prognostic significance.[7],[8] In these studies, levels of vascular endothelial growth factor and microvessel density (MVD) have been shown to be strongly associated with tumor behavior. For cervical carcinoma, the quantitative degree of NA, namely, high MVD levels, has been shown to indicate a poor prognosis.[8],[9]

Endoglin (CD105), a transforming growth factor-β (TGF-β) Type III auxiliary receptor (a 90 kDa cell membrane glycoprotein), is an endothelial marker of angiogenesis that is involved in the regulation of TGF-β1 signaling in endothelial cells. Endoglin is normally expressed within the vascular system, and its overexpression is associated with active endothelial cell proliferation. Therefore, increased levels of endoglin represent a new reagent of active neovascularization and angiogenesis process in various cancer types.[10],[11] However, despite this evidence, the potential prognostic value of endoglin has been addressed in only few tumor sites including the cervical cancer, with conflicting outcomes.[12],[13],[14],[15],[16],[17],[18] Regarding the cervical carcinoma, to the best of our knowledge, endoglin has been studied in surgically removed precancerous (cervical intraepithelial neoplasia [CIN]) and early cervical carcinomas, but the potential prognostic impact of endoglin levels has never been investigated in LACC patients treated with definitive C-CRT and brachytherapy (BRT) which is the current treatment recommendation for such patients group. Therefore, to throw light on the subject, in this retrospective cohort analysis, we investigated the potential prognostic value of endoglin levels in LACC patients treated with definitive C-CRT and BRT.


 > Materials and Methods Top


Study participants

The medical records of curatively treated seventy cervical squamous cell cancer (CSCC) patients who were treated with radical RT or cisplatin-based C-CRT between June 2004 and December 2012 were retrospectively analyzed. The Institutional Review Board exempt approval was obtained before collection of data. The patients' medical records, RT plans, chemotherapy protocols/administration schedules, and biopsy materials were utilized for this analysis.

Treatment

All patients were treated with three-dimensional conformal RT utilizing pelvic-box technique. Paraaortic irradiation was permitted only in cases with paraaortic lymph node involvement. Using four-field box technique and 18 MV linear accelerator (Elekta®), the standard prescribed total doses for external beam RT (EBRT) were 50.4 Gy and 45 Gy (1.8 Gy/fx) to high-risk and intermediate-risk planning target volumes, respectively. The prescribed concurrent chemotherapy was weekly 40 mg/m2 cisplatin for all patients. As an institutional standard, all patients were referred to nearby radiation oncology departments for high-dose-rate intracavitary BRT (HDR-ICBT) application at the end of EBRT.

Response assessment and follow-up

The tumor response assessment included the detailed clinical and gynecological examination, blood chemistry and  Pap smear More Details tests every 3 months for the first 2 years and every 6 months thereafter. Computerized tomography, 18-fluorodeoxyglucose-positron emission tomography, and/or magnetic resonance imaging were performed if local/regional recurrences or distant metastasis was suspected and confirmed with biopsies. Early and late adverse effects were graded according to the Radiation Therapy Oncology Group toxicity criteria grading system.

Immunohistochemical staining and evaluation of endoglin

Following review of hematoxylin and eosin-stained slides of paraffin blocks, cases with adequate cellular tissue were selected for immunohistochemical staining. Five-micrometer thick sections were deparaffinized and rehydrated stepwise in xylene and graded ethanol solutions, respectively. Antigen retrieval was performed at 95°C for 40 min in 0.01 M sodium citrate buffer (pH 6.0). All sections were incubated for 5 min in a protein-blocking solution (PBS) for suppression of nonspecific binding of subsequent reagents after the immersion in 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Sections were then incubated for 30 min with the anti-CD105 monoclonal antibody (Clone MRQ-14, Cell Marque, USA). The antibody was diluted in 1% bovine serum albumin in PBS. Renal cell carcinoma was used as a positive control, and the primary antibody was replaced with mouse immunoglobulin G for negative controls. Positive and negative controls were stained on each slide. Staining was completed by 5-min incubation of the specimens with 3,39-diaminobenzidine tetrahydrochloride (Cell Marque, USA) as chromogen, which resulted in a brown-colored precipitate. All slides were counterstained with Mayer's hematoxylin (Bio-optica, Milano, Italy) and mounted with mounting medium (Bio-optica, Milano, Italy). Immunohistochemically stained sections were evaluated with a light microscope. Initially, CD105-positive stromal vessels were quantified by searching for regions of hot spots (high density of vessels) at low magnification (×200). Then, the number of positive vessels was identified at high magnification (×400) in five of these hot spots as described by Cooper et al. previously,[11] and the final MVD value was obtained by calculating the average value of the vessel count for each case.

Statistical analysis

Correlation between immunohistochemical findings and clinical data was evaluated by Pearson Chi-square (Fischer's exact) test, Spearman's “ρ” correlation, and multivariate analysis. The mean differences between endoglin levels were compared with the use of paired t-tests. Receiver-operating characteristic (ROC) curve analysis was used for identification of significant cutoff values and stratification of patients accordingly for continuous variables. The primary end-point was overall survival (OS) which was calculated as the time between the dates of histological diagnosis and the last visit or death. Secondary end-points included the locoregional recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), and disease-free survival (DFS), calculated as the time between the dates of histological diagnosis and diagnosis of local and/or regional recurrences/death for LRFS, diagnosis of distant relapse (s) or death for DMFS, and any type of relapses or death for DFS, respectively. Kaplan–Meier curves and log-rank tests were used for survival estimates and comparisons. Multivariate analyses were performed for each end-point by including only the factors those revealed significance in univariate analysis. P ≤ 0.05 was considered statistically significant. The Institutional Review Board exempt approval was obtained from the Eskisehir Osmangazi University Ethical Committee.


 > Results Top


Seventy consecutive patients who had been treated with definitive C-CRT with CSCC at our institution between June 2004 and December 2012 were identified for this retrospective analysis. The median follow-up time was 29.5 (range; 5–99) and 43 (range; 13–99) months for the whole and surviving patient cohorts at the time of this analysis, respectively. Patients, disease, and treatment characteristics are summarized in [Table 1].
Table 1: Patient, tumor, and treatment characteristics of cervical squamous cell cancer patients treated with radiotherapy±cisplatin

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The median age was 61 (range; 40–86) years, and the median tumor size was 4 cm (range; 1–8). Sixty-two (89%) patients were able to complete the prescribed doses of pelvic and paraaortic EBRT, and 60 (85%) of them received HDR-ICBT at somewhere else. Revealed from the hospital records, the median total HDR-ICBT dose was 21 Gy (range; 10–28) given in median three fractions (range; 2–4). Forty-seven (67%) women were able to receive 4–6 cycles of weekly cisplatin concurrent with EBRT.

CD105-positive stromal vessels were seen in all cases. The median CD105 MVD was 55.5 (range; 12–136). The staining patterns of CD105 are demonstrated in [Figure 1]. We have evaluated both the presence of CD105-positive blood vessels within the epithelial cell clusters and the number of stromal CD105-positive blood vessels within the groups of age (≤61 vs. >:61 years), lymph node metastasis (absent vs. present), International Federation of Gynecology and Obstetrics (FIGO) stage (Ib–IIa vs. IIb–IVa), and hemoglobin levels (≤10 g/dL vs. >:10 g/dL). The cutoff values of age 61 years (ROC; 61.5) and hemoglobin levels 10 g/dL (ROC; 100.05) were the rounded forms of those values which revealed a significant association with OS. However, we could not identify any correlation between the CD105 and any of these factors which may be associated with its almost even distribution between groups. Similarly, ROC curve analysis did not reveal any significant cutoff for CD105 at any of the OS, locoregional progression-free survival (LRPFS), progression-free survival (PFS), and DMFS end-points. In addition, also the stratification of patients into two groups using median CD105 value did not translate into superior outcomes favoring one group over others at any time point.
Figure 1: (a) CD105 vascular staining of vessels of cervical squamous cell cancer patients treated with radiotherapy ± cisplatin. (a) CD105 vascular staining of vessels present in the tumor stroma (CD105, ×400). (b) An abundance of positive-staining vessels (CD105, ×400)

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At the time of this analysis, 50 (70.4%) patients were alive, and 30 (42.9%) were free of disease progression. The median OS time was not reached yet, while the median LRPFS, PFS, and DMFS were 47 (95% confidence interval [CI]: 29.1–64.9), 32 (95% CI: 24.2–39.8), and 35 (26.4–43.6) months, respectively [Figure 2]. Eight-year OS, LRPFS, PFS, and DMFS rates were 52.0%, 39.7% and 31.8%, and % 34.7%, respectively.
Figure 2: Survivals for all cohorts of cervical squamous cell cancer patients treated with radiotherapy ± cisplatin

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On univariate analysis among all covariates, age (≤61 vs. >:61 years; P = 0.015), lymph node metastasis status (absent vs. present; P = 0.028), FIGO stage (Ib–IIa vs. IIb–IVa; P = 0.036), cycles of concurrent chemotherapy (1–3 vs. 4–6 cycles; P = 0.001), and hemoglobin levels (≤10 g/dL vs. >:10 g/dL; P = 0.006) appeared to associate significantly with OS.

On multivariate analysis, lymph node status (P = 0.039), cycles of concurrent chemotherapy (P = 0.018), and hemoglobin levels (P = 0.032) retained their independent association with OS, while the age and FIGO stage lost their significance. As shown in [Figure 3], comparative survival analysis revealed significantly longer median survival times in patients with no involved lymph nodes (not reached yet vs. 31 months; P = 0.022), 4–6 cycles of concurrent chemotherapy (not reached yet vs. 48 months; P = 0.001), and hemoglobin levels >:10 g/dL (not reached yet vs. 47 months; P = 0.002), respectively. The median and 8-year estimates regarding the LRPFS, PFS, and DMFS were significantly superior in these patients groups compared to their counterparts with the unfavorable factors. As shown in [Figure 4], comparative survival analysis of CD105 groups for DMFS and PFS revealed not significant.
Figure 3: Multivariate analysis for significant factors of cervical squamous cell cancer patients treated with radiotherapy ± cisplatin

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Figure 4: Survival table for CD105 (endoglin) groups of cervical squamous cell cancer patients treated with radiotherapy ± cisplatin

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 > Discussion Top


Angiogenesis is the development of new blood vessels in areas of new tissue growth which is required for embryogenesis, tissue remodeling, tumor growth, invasion, and metastasis.[9],[10] The most of the studies shown a correlation between angiogenesis and treatment response rates in cervical cancer report conflicting outcomes, even though some have concluded that more extensive tumor angiogenesis is associated with higher rate of tumor recurrence and lower survival in cervical cancer.[6],[7],[8],[9],[10],[11]

The expression of CD105 (endoglin) in many solid tumors, including cervical, breast, and prostate cancer, makes CD105 a potential molecular target for cancer treatment.[13],[14],[15],[16],[17],[18],[19] Intratumor MVD, as determined by anti-endoglin staining and circulating levels of soluble endoglin, has prognostic significance in cancer patients.

In our study, all of seventy patients' specimens were identified positive CD105 value in different levels. The median CD105 MVD was 55.5 with a wide range between 12 and 136 MVD. Even we found no correlation between CD105 levels and prognostic factors effected any survival rate, we thought that because of most of the patients have local advanced disease (77%) and lower hemoglobin value (70%) at the diagnosis, no cutoff value of CD105 was found between prognostic groups in ROC analysis.

Regarding the cervical carcinoma, to the best of our knowledge, CD105 has been studied in surgically removed precancerous (CIN) and early cervical carcinomas, but the potential prognostic impact of endoglin levels has never been investigated in LACC patients treated with definitive C-CRT and BRT which is the current treatment recommendation for such patients group. The prognostic value and association between the number of CD105-positive vessels and lymph node metastasis in cervical carcinoma were observed in Zijlmans et al.'s study.[20] Only the presence of newly formed vessels within the epithelial cluster (CD105) has been correlated with poor DFS in 30 patients (67% of patients with early stage) who all underwent the operation.[20]

The limitation of our study is that it has been conducted with a limited number of patients. It is likely that repeated with larger series of patients and different patient groups in different stages, some near-significant prognostic factors may prove to be indeed significant. However, this study is important because so far there has not been any other study to specify a threshold value in mostly local advanced cervical cancer patient series.

The previous studies have reported an increased CD105 expression with advancing clinical stage in cervical cancer.[12] The groundwork for analysis of microvessels and standardization of many aspects of those techniques recently was suggested in an international consensus paper.[21],[22],[23],[24],[25] Various fields containing hot spots are counted, and an average or highest value is used.[13],[23] The use of diagnostic modalities based on CD105 may have an important role in clinical management. Areas with clinical potential include diagnosis, follow-up, prediction of response to treatment, and prognostic determination.[19],[20],[21]

As a result of our study, CD105 is thought to be a marker that is worth studying further prognostically through wider patient series in a multiple-center study. Even though no significant associations were found between CD105 and survival in this study, to clarifying which value of MVD reacts more, clinic studies will be assessed with a higher number of cervical cancer patients comparing different stage groups in near future.

Acknowledgments

The authors would like to thank Prof. Dr. Sinan Özalp and Prof. Dr. Erkan Topkan for their contribution in successfully bringing out this manuscript.

Financial support and sponsorship

Part of this study was supported by the supporting fund of Turkish Gynaecology and Obstetrics Association, Eskisehir, Turkey.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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