Skip to main content
  • Research article
  • Open access
  • Published:

Evaluation of Endoglin (CD105) expression in pediatric rhabdomyosarcoma



The Intratumoral Microvessel Density (IMVD) is commonly used to quantify tumoral vascularization and is usually assessed by pan-endothelial markers, such as CD31. Endoglin (CD105) is a protein predominantly expressed in proliferating endothelium and the IMVD determined by this marker measures specifically the neovascularization. In this study, we investigated the CD105 expression in pediatric rhabdomyosarcoma and assessed the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31.


Paraffin-embedded archival tumor specimens were selected from 65 pediatric patients affected by rhabdomyosarcoma. The expression levels of CD105, CD31 and Vascular Endothelial Growth Factor (VEGF) were investigated in 30 cases (18 embryonal and 12 alveolar) available for this study. The IMVD-CD105 to IMVD-CD31 expression ratio was correlated with clinical and pathologic features of these patients.


We found a specific expression of endoglin (CD105) in endothelial cells of all the rhabdomyosarcoma specimens analyzed. We observed a significant positive correlation between the IMVD individually measured by CD105 and CD31. The CD105/CD31 expression ratio was significantly higher in patients with lower survival and embryonal histology. Indeed, patients with a CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI, 60%–97%) compared to patients with higher values (40%, 95%CI, 12%–67%). We did not find any statistical correlation among VEGF and EFS, OS and CD105/CD31 expression ratio.


CD105 is expressed on endothelial cells of rhabdomyosarcoma and represent a useful tool to quantify neovascularization in this tumor. If confirmed by further studies, these results will indicate that CD105 is a potential target for combined therapies in rhabdomyosarcoma.

Peer Review reports


Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma (STS) in children and young adults, accounting for up to 5% of all childhood cancers and for about 40% of pediatric STS [1]. Embryonal (ERMS) and alveolar (ARMS) RMS are the two major histologic subtypes. ARMS is associated with PAX3/7-FOXO1 gene fusions and with a poor prognosis, often being metastatic at diagnosis [2]. Although during the last three decades, multimodal treatment strategies have substantially improved the prognosis of localized RMS, for metastatic disease the prognosis remains dismal [3]. Therefore, new targets and tailored therapies directed against the metastatic process are needed for these patients. The formation of new blood vessels is a requirement for tumor growth and metastatic spread and many regulators of tumor angiogenesis have been identified in different types of cancer [4]. Studies on inhibitors of angiogenesis have shown antitumor activity in pediatric sarcoma models, including RMS, mostly in combination with other drugs [5,6,7], and several trials showed promising results for selected clinical indications [8,9,10]. The quantification of tumor vasculature is a useful indicator of angiogenesis, by helping patients stratification prior to anti-angiogenic therapy and monitoring patient response. One often-quantified aspect of tumor vasculature is the Intratumoral Microvessel Density (IMVD). IMVD is commonly used as a surrogate marker to quantify angiogenic activity and is usually assessed by pan-endothelial markers, such as CD34 and CD31 [10,11,12,13]. However, these markers are not tumor endothelial-specific, as they are also expressed on pre-existing/mature vasculature and on large vessels [14, 15]. Recent studies have shown that IMVD assessed by detection of Endoglin (CD105) is more specifically associated with tumor neovascularization [16,17,18,19,20] and represents a significant prognostic marker in several tumors [19,20,21,22,23,24]. CD105 is a transforming growth factor β (TGF-β) transmembrane co-receptor required for angiogenesis [25] and is highly expressed on the surface of actively proliferating microvascular endothelial cells, forming immature, highly permeable tumor neovessels [26]. In line with its supportive role in tumor neoangiogenesis, CD105 is up-regulated by hypoxia [27,28,29]. The expression of CD105 has been reported on the tumor vasculature of several sarcomas, including Kaposi sarcoma, angiosarcoma, leiomyosarcoma, chondrosarcoma and gastrointestinal stromal tumor and correlated with worse survival for some of these tumors [21, 30,31,32,33]. In this study, we aimed to investigate if CD105 was expressed in pediatric RMS and assess the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31. For this purpose, we evaluated the immunohistochemical expression of CD105, CD31 and VEGF in a retrospective series of pediatric patients with RMS. In order to define the proliferation fraction of the endothelium we compared the CD105 microvessels count with CD31 immunoexpression obtaining the CD105/CD31 expression ratio. In the cases where the CD105/CD31 expression ratio is higher, the angiogenesis is increased because CD105 marks the neoformed vessels [26] whereas CD31 is also expressed in mature vessels [18]. This ratio has been reported to have a prognostic value and be a potential predictor of response to anti-VEGF therapy [34,35,36].


Study population

Tumor tissue specimens from 65 patients with RMS who underwent surgical resection or biopsy of their primary tumor at the Bambino Gesù Children’s Hospital from 2005 to 2016 were retrospectively reviewed. Among these, we selected 30 appropriate paraffin embedded tissue blocks. The criteria for selecting the patients were based on the availability of an adequate tumor specimens obtained before any treatment and detailed clinical information. Patients’ clinical details, information on therapy and follow-up were collected retrospectively from the medical files. The median age at diagnosis was 48.5 months (range 1–199) with a sex ratio of 1. The most frequent primary site was head and neck (6 parameningeal and 2 non parameningeal patients respectively), followed by orbit (5 patients), pelvis (4 patients), genitourinary non-bladder or prostate (3 patients), extremity (2 patients), genitourinary bladder or prostate (1 patient), and other localizations (7 patients). This series include 18 patients with ERMS and 12 with ARMS. The study was approved according to local institutional guidelines.

Patient variables analyzed

Patient- and tumor-related prognostic factors considered were: age at diagnosis (favorable if ≥ 12 or < 120 months and unfavorable if <12 or ≥ 120 months), primary tumor size (≤ 5 cm versus > 5 cm), tumor site favorable (orbit, genitourinary non bladder/prostate, head and neck non parameningeal) and unfavorable (parameningeal, extremities, genitourinary bladder-prostate and “other site”), histology (embryonal versus alveolar) and COG risk stratification [37].

Immunohistochemistry methods

The tissues were fixed with 10% formalin and embedded in paraffin. Consecutive 2.5 μm-thick serial sections were cut, deparaffinised in xylene, rehydrated and washed using double distilled water. These sections were used for immunohistochemical staining for CD105, VEGF and CD31 and human tonsils were used as positive controls for CD105, CD31 and VEGF. For staining with VEGF and CD31, sections were pretreated with DAKO PT link (PT200) in low pH solution (cod. K8005 DAKO North America, CA) for antigen retrieval. As for CD105, sections were pretreated with Proteinase K (cod. S3020 DAKO North America, CA) for 10 min at room temperature. The immunostaining was done at 4 °C overnight using the following monoclonal mouse anti-human antibodies as primary antibody: anti-CD105 (clone SN6h, 1:10, DAKO North America, CA), anti-VEGF (MS-1467-P, 1:200, Thermo Fisher, Fremont, CA), anti-CD31 (IR610, Ready-to-Use, DAKO North America, CA). As the secondary antibody, we used En Vision Flex/HRP (cod. K8024, Ready-to-Use, DAKO North America, CA). The sections were then reacted in chromogen 3,3’-diaminobenzidine to detect the peroxidase activity, counterstained with hematoxylin and mounted.

Measurement of IMVD

Hematoxylin-Eosin staining has been used by an experienced pathologist (RB) in order to select the area of the tumor, the necrotic areas were excluded. The sections were examined using a double-headed light microscope (Leica DM4 B) by two independent operators (RB and VDP), who were not aware of the clinical status of the patients. IMVD was assessed by immunostaining for either CD31 (IMVD-CD31) or CD105 (IMVD-CD105) according to the procedure described by Weidner et al., [11, 38]. The most vascularized area (hot-spots) was identified at low magnification (40X) and then three fields were counted at high magnification (20X). We considered as a countable single microvessel any endothelial cell or endothelial-cell cluster stained and clearly separated from the adjacent microvessels, tumor cells and other connective-tissue elements. The mean of the vessels in three fields was used as CD105 IMVD or CD31 IMVD. CD105 IMVD and CD31 IMVD have been evaluated in two different serial slides, within the same “hot spot” area. In order to define the proliferation fraction of the endothelium, we calculated the CD105/CD31 ratio dividing the IMVD of CD105 by the IMVD of CD31, as previously described [34,35,36]. Indeed, since CD31 is a pan-endothelial marker and CD105 is primarily expressed by proliferating endothelial cells, this ratio specifically measures the fraction of proliferating endothelial cells.

Evaluation of VEGF

The VEGF expression was estimated according to the percentage of immunoreactive cells in a total of 1000 cells. The tumors were classified into 4 categories based on VEGF staining: negative (0), weak (1+), moderate (2+) and strong (3+). The percentage of positive cells was defined as sporadic (positive cells ≤ 1% and < 10%), focal (positive cells ≤ 10% and < 50%) or diffused (positive cells ≥ 50%). The immuno-histochemical scores were recorded as score 0 (no immunoreactivity), score 1 (1+ with sporadic or focal distribution), score 2 (1+ with diffused distribution or 2+ or 3+ with sporadic distribution), score 3 (2+ with focal or diffused distribution), score 4 (3+ with focal or diffused distribution) [39].

Statistical analysis

Categorical data was represented as counts and proportions, and continuous data as mean and standard deviation or median and range. We analyzed the overall survival (OS) and event-free survival (EFS) defined as the time from diagnosis until the date of death and the date of disease relapse/progression, respectively. The follow-up period was calculated from the date of diagnosis until the last follow-up visit. Correlation between CD105 and CD31 IMVD was examined using the Spearman’s Rho. The ROC (Receiving operation curve) analysis was used in order to find an appropriate cut-off of CD105/CD31 ratio discriminating between death and survival, and event (disease relapse/progression) and non event in terms of sensibility and specificity.

Univariable analysis of time to event data (OS and EFS) was performed through the Kaplan Meier method, Log-rank test and Cox proportional hazard model. Relationships between the CD105/CD31 ratio and clinico-pathological data were assessed using univariable quantile regression analysis. P values less than 0.05 were considered to be statistically significant. Data was analyzed using the STATA software version 13.1.


Clinico-pathological features of RMS patients

Patients’ characteristics are detailed in Table 1.

Table 1 Characteristics of the 30 patients with Rhabdomyosarcoma

Patients were staged according to COG-STS risk stratification [37]. PAX3/PAX7-FKHR fusion gene transcripts were evaluated in 9 cases of 12 ARMS and 9 cases of 18 ERMS. PAX3-FKHR fusion gene was positive in 8 ARMS cases, and PAX7-FKHR in 1 ARMS case. None of PAX3/PAX7-FKHR fusion gene was detected in the ERMS examined. The median follow-up of patients was 5 years (range 0.28–11.12 years). Eight patients died of disease (median time from diagnosis 16.5 months, range 5–64). Patients #6, #17 and #24 presented a short follow-up since they died after 5, 7 and 10 months respectively due to rapid disease progression. The immunostaining was performed on pretreatment tumor biopsy specimens. The expression of CD31 and CD105 was localized in endothelial cells in all the specimens analyzed and not expressed by tumor cells. In the tumor CD105 was specifically associated with immature vessels which showed a stronger positivity compared to the large vessels (Additional file 1: Figure S1). VEGF expression was detected mainly in the cytoplasm of the tumor cells or endothelium (Fig. 1). Nineteen tumors (63.3%) showed a VEGF staining score of 1–2, while 11 (36.7%) showed a score of 3–4.

Fig. 1
figure 1

Representative immunostaining for CD105, CD31and VEGF of ARMS and ERMS. Magnification × 200

The ratio of IMVD-CD105 to IMVD-CD31 in RMS primary samples

Analysis of CD105 and CD31 expression demonstrated that the average of CD105-IMVD was not statistically, significantly higher than CD31-IMVD in RMS tissue (P = 0.122 Wilcoxon signed-rank test). We observed a statistically significant positive correlation between the IMVD individually measured by the two markers (Spearman’s rho = 0.86, P = 0.05), (Fig. 2). CD105/CD31 expression ratio in the tumor specimens ranged from 0.32% to 2.35%, with a median value of 1% and a mean of 1.15% (Table 1). The ROC curve analysis was used to determine the optimal cut-off of CD105/CD31 ratio (Fig. 3). EFS showed a cut-off point value of 0.9 with a 90.9% sensitivity and 52.6% specificity (Fig. 3a). OS had a cut-off point value of 1.3 with a 71.4% sensitivity and 78.2% specificity (Fig. 3b). Our analysis demonstrated that ten patients with a CD105/CD31 expression ratio equal or higher than 0.9 (50% of patients in this group) had relapse or disease progression. Only one patient (#6) with a ratio lower than 0.9 (10%) experienced disease progression, but had metastatic disease, which is per se a poor prognostic factor. These results suggest that the CD105/CD31 expression ratio in the primary tumor could be associated with disease aggressiveness.

Fig. 2
figure 2

Correlation between CD105-IMVD and CD31-IMVD in rhabdomyosarcoma tumor samples (R2 = 0.83, P < 0.001)

Fig. 3
figure 3

ROC analysis of CD31/CD105 ratio regarding Event-Free survival (a) and Overall survival (b)

Correlation between prognostic factors and outcome in RMS patients

We then investigated the relationship amongst EFS or OS, selected prognostic clinico-pathological parameters (age at diagnosis, tumor size, primary site, histology, COG risk stratification) and the angiogenic CD105/CD31 ratio. As summarized in Table 2, the EFS and OS of patients with high risk RMS, according to COG stratification, resulted dismal, as it was previously reported [37].

Table 2 Univariable Cox proportional hazards regression for Event Free Survival and Overall Survival

Furthermore, in the univariable Cox proportional hazard regression the CD105/CD31 expression ratio resulted to be related with decreased OS (P = 0.03) [38].

Relationship of CD105/CD31 expression ratio with clinico-pathological characteristics and outcome

Based on ROC curves cut-off values, Kaplan-Meier analysis showed that patients with a value of the CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI = 60%–97%) compared to patients with higher values (40%, 95%CI = 12%–67%; P = 0.013 by the log-rank test), (Fig. 4b). The estimated 5-year EFS was 91% (95%CI = 51%–98%) for patients with a CD105/CD31 ratio lower than 0.9 compared with 45% (95%CI = 22%–65%) for those with a ratio higher or equal to 0.9 (P = 0.054 by the log-rank test) (Fig. 4a). We further evaluated VEGF expression in order to correlate this marker, which is upregulated in RMS [39,40,41,42,43], with the neo-angiogenic ratio. Determinants of CD105/CD31 expression ratio were assessed by univariable quantile regression analysis (Table 3). This ratio was significantly associated with the patients’ survival (P = 0.016) and the embryonal histology (P = 0.019).

Fig. 4
figure 4

Kaplan-Meier curve for Event-Free survival (a) and Overall survival (b) according to CD105/CD31 ratio groups

Table 3 Factors associated with CD105/CD31 expression ratio – univariable analysis


Neo-angiogenesis has long been implicated in generating a microenvironment suitable for tumor growth and metastatic spread [44]. Several pro-angiogenic factors have been described and among them VEGF appears to play a central role in the activation of angiogenesis in various cancer [45, 46]. Several efforts have been made to develop therapies focused on the inhibition of the VEGF signaling pathway also in RMS [47, 48]. However these drugs led to transient responses and the complementary/dual inhibition of non-VEGF angiogenic pathways might represent a way to improve anti-angiogenic therapy. A phase I study using an anti-endoglin monoclonal antibody (TRC105) in combination with bevacizumab in adults with advanced cancers showed good tolerance and clinical activity in a VEGF inhibitor-refractory population [49]. A trial testing TRC105 in combination with pazopanib in patients with STS (≥12 years old) is currently ongoing [50]. In this context, methods which enable to quantify tumor angiogenesis are useful surrogate markers of angiogenic activity and response to therapy, and might help stratify patients with RMS for treatment. The IMVD is the most commonly used parameter to quantify intra-tumoral neovascularization and is measured by pan-endothelial markers, such as CD31. CD105 presents a higher specificity for new developing vessels and recent studies have shown that IMVD as determined by this marker has a higher prognostic impact than CD31 in several tumors [21,22,23]. In particular, IMVD ratio of CD105/CD31 expression, had been used to specifically assess neovascularization showing a prognostic value [34,35,36]. In the present study, we analyzed for the first time, the CD105 immunoexpression in pediatric RMS and quantified the presence of proliferating endothelial cells by using the CD105/CD31 expression ratio. CD105 was detected in small tumor capillary-like vessels, whereas CD31 presented a more diffused expression in endothelial cells. The significant positive correlation found between the IMVD measured by the two markers is coherent with the association between CD105 expression and other endothelial markers, such as CD31 and CD34, already described in other tumors [51, 52]. We also evaluated whether a correlation between this neoangiogenic ratio and clinic-pathological variables exists. Several prognostic factors, such as the age at diagnosis, primary tumor size, primary site, histology, post-surgical stage and presence or absence of distant metastases are currently used for risk-adapted treatment approaches in clinical trials of RMS patients [53]. Using these parameters, in the univariable survival analysis, we found that the advanced disease, classified according to the COG risk stratification, was a significant predictor of worse OS and EFS. In line with previously reported works, our study confirms that metastatic disease is the main prognostic factor in RMS [3]. The ROC curve for the OS indicated a cutoff point of 1.3, which was used to separate patients with good and poor prognosis. A value of the CD105/CD31 expression ratio < 1.3 was associated with a significantly better patients’ OS. These findings suggest that neovascularization could be an indicator of prognosis in patients with RMS and are supported by the correlation described between the CD105/CD31 expression ratio and aggressive phenotypes in other tumors [35]. Indeed, it has been already reported that IMVD quantified by CD105 correlate with poor survival in patients with breast carcinoma, non-small cell lung cancer and hepatocellular carcinoma [13, 54, 55]. Interestingly, when the histotype was specifically considered, we found that the ERMS correlated significantly with neo-angiogenesis. Kuda et al., previously described that IMVD, assessed by CD31, was higher in ERMS than ARMS [56]. We speculate that this association could be related to the different growth rate displayed by these two RMS histotypes. Indeed, although angiogenesis is a key process activated during cancer invasion and metastasis, highly aggressive histotypes are also able to support their growth through a process known as vasculogenic mimicry (VM) [57]. The generation of non-endothelialized vessel-like channels allows the perfusion of a variety of tumors, enabling them to aggressively proliferate and metastasize [58]. The VM channels are not lined by endothelial cells, but by tumor cells instead, and therefore are not stained by endothelial markers, including CD31 [59]. A higher incidence of VM has been described in tumors presenting necrosis, as well as in ARMS, and has been associated with poor prognosis [60, 61]. The faster growth of ARMS compared to ERMS may explain the different pattern of neovessels in the two variants. No statistically significant differences in CD105/CD31 expression ratio were encountered with respect to age, tumor size, primary tumor location, COG risk groups and VEGF. Despite VEGF overexpression has been reported to be associated with prognosis in RMS patients [42], data regarding the correlation amongst IMVD, VEGF expression and prognosis has shown conflicting results in several tumors including STS and RMS [39, 56, 62,63,64].

In conclusion, this small proof-of-concept study suggests that CD105 is expressed in endothelial cells of pediatric RMS and that CD105/CD31 expression ratio might be useful to measure the proportion of proliferating endothelial cells in this tumor. Despite the small cohort of patients studied, these data indicate that a high value of CD105/CD31 expression ratio could be related with a “pro-angiogenic” RMS subset of patients with low OS.


If further studies confirm these results in larger cohorts of patients, CD105 may also represent a potential therapeutic target as part of combined therapy in RMS. In particular, an inter-institutional cooperative study would be advisable considering the low frequency of this tumor in the pediatric population. This type of large study could also be a tool to elucidate if the CD105/CD31 expression ratio may be useful for patient’s stratification and/or evaluate response to therapy.



Italian Association of Pediatric Hematology/Oncology


Alveolar Rhabdomyosarcoma




Children’s Oncology Group


Event-Free Survival


European Pediatric Soft Tissue Sarcoma Study Group


Embryonal Rhabdomyosarcoma


Intratumoral Microvessel Density


Overall Survival




Soft Tissue Sarcoma


Transforming Growth Factor beta


Vascular Endothelial Growth Factor


Vasculogenic Mimicry


  1. Gurney JG, Young JL Jr, Roffers SD, Smith MA, Bunin GR. Soft tissue sarcomas. In: LAG R, Smith MA, Gurney JG, et al., editors. Cancer incidence and survival among children and adolescents: United States SEER program 1975–1995. Bethesda: National Cancer Institute, SEER Program; 1999. p. 111–23. NIH Pub. No. 99-4649.

    Google Scholar 

  2. Sorensen PH, Lynch JC, Qualman SJ, et al. PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children’s oncology group. J Clin Oncol. 2002;20:2672–9.

    Article  CAS  PubMed  Google Scholar 

  3. McDowell HP, Foot AB, Ellershaw C, Machin D, Giraud C, Bergeron C. Outcomes in paediatric metastatic rhabdomyosarcoma: results of the International Society of Paediatric Oncology (SIOP) study MMT-98. Eur J Cancer. 2010;46(9):1588–95.

    Article  PubMed  Google Scholar 

  4. de Castro JG, Puglisi F, de Azambuja E, El Saghir NS, Awada A. Angiogenesis and cancer: a cross-talk between basic science and clinical trials (the “do ut des” paradigm). Crit Rev Oncol Hematol. 2006;59(1):40–50.

    Article  Google Scholar 

  5. Maris JM, Courtright J, Houghton PJ, Morton CL, Kolb EA, Lock R, Tajbakhsh M, Reynolds CP, Keir ST, Wu J, Smith MA. Initial testing (stage 1) of sunitinib by the pediatric preclinical testing program. Pediatr Blood Cancer. 2008;51(1):42–8.

    Article  PubMed  Google Scholar 

  6. Kumar S, Mokhtari RB, Sheikh R, Wu B, Zhang L, Xu P, Man S, Oliveira ID, Yeger H, Kerbel RS, Baruchel S. Metronomic oral topotecan with pazopanib is an active antiangiogenic regimen in mouse models of aggressive pediatric solid tumor. Clin Cancer Res. 2011;17(17):5656–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Keir ST, Morton CL, Wu J, Kurmasheva RT, Houghton PJ, Smith MA. Initial testing of the multitargeted kinase inhibitor pazopanib by the pediatric preclinical testing program. Pediatr Blood Cancer. 2012;59(3):586–8.

    Article  PubMed  Google Scholar 

  8. Navid F, Baker SD, McCarville MB, Stewart CF, Billups CA, Wu J, Davidoff AM, Spunt SL, Furman WL, McGregor LM, Hu S, Panetta JC, Turner D, Fofana D, Reddick WE, Leung W, Santana VM. Phase I and clinical pharmacology study of bevacizumab, sorafenib, and low-dose cyclophosphamide in children and young adults with refractory/recurrent solid tumors. Clin Cancer Res. 2013;19(1):236–46.

    Article  CAS  PubMed  Google Scholar 

  9. Glade Bender JL, Lee A, Reid JM, Baruchel S, Roberts T, Voss SD, Wu B, Ahern CH, Ingle AM, Harris P, Weigel BJ, Blaney SM. Phase I pharmacokinetic and pharmacodynamic study of pazopanib in children with soft tissue sarcoma and other refractory solid tumors: a children's oncology group phase I consortium report. J Clin Oncol. 2013;31(24):3034–43.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Giatromanolaki A, Koukourakis MI, Theodossiou D, Barbatis K, O'Byrne K, Harris AL, Gatter KC. Comparative evaluation of angiogenesis assessment with anti-factor-VIII and anti-CD31 immunostaining in non-small cell lung cancer. Clin Cancer Res. 1997;3(12 Pt 1):2485–92.

    CAS  PubMed  Google Scholar 

  11. Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat. 1995;36(2):169–80. Review

    Article  CAS  PubMed  Google Scholar 

  12. Offersen BV, Pfeiffer P, Hamilton-Dutoit S, Overgaard J. Patterns of angiogenesis in nonsmall-cell lung carcinoma. Cancer. 2001;91(8):1500–9.

    Article  CAS  PubMed  Google Scholar 

  13. Tanaka F, Otake Y, Yanagihara K, Kawano Y, Miyahara R, Li M, Yamada T, Hanaoka N, Inui K, Wada H. Evaluation of angiogenesis in non-small cell lung cancer: comparison between anti-CD34 antibody and anti-CD105 antibody. Clin Cancer Res. 2001;7(11):3410–5.

    CAS  PubMed  Google Scholar 

  14. de la Taille A, Katz AE, Bagiella E, et al. Microvessel density as a predictor of PSA recurrence after radical prostatectomy. A comparison of CD34 and CD31. Am. J Clin Pathol. 2000;113:555–62.

    Article  CAS  Google Scholar 

  15. Tomisaki S, Ohno S, Ichiyoshi Y, Kuwano H, Machra Y, Sugimachi K. Microvessel quantification and its possible relation with liver metastasis in colorectal cancer. Cancer. 1996;77:1772–8.

    Article  Google Scholar 

  16. Behrem S, Zarkovic K, Eskinja N, Jonjic N. Endoglin is a better marker than CD31 in evaluation f angiogenesis in glioblastoma. Croat Med J. 2005;46(3):417–22.

    PubMed  Google Scholar 

  17. Yang LY, Lu WQ, Huang GW, Wang W. Correlation between CD105 expression and postoperative recurrence and metastasis of hepatocellular carcinoma. BMC Cancer. 2006;6:110.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Li SL, Gao DL, Zhao ZH, et al. Correlation of matrix metalloproteinase suppressor genes RECK, VEGF, and CD105 with angiogenesis and biological behavior in esophageal squamous cell carcinoma. World J Gastroenterol. 2007;13:6076–81.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Miyata Y, Sagara Y, Watanabe S, et al. CD105 is a more appropriate marker for evaluating angiogenesis in urothelial cancer of the upper urinary tract than CD31 or CD34. Virchows Arch. 2013;46:673–9.

    Article  Google Scholar 

  20. Ding S, Li C, Lin S, Yang Y, Liu D, Han Y, Zhang Y, Li L, Zhou L, Kumar S. Comparative evaluation of microvessel density determined by CD34 or CD105 in benign and malignant gastric lesions. Hum Pathol. 2006;37(7):861–6.

    Article  CAS  PubMed  Google Scholar 

  21. Basilio-de-Oliveira RP, Pannain VL. Prognostic angiogenic markers (endoglin, VEGF, CD31) and tumor cell proliferation (Ki67) for gastrointestinal stromal tumors. World J Gastroenterol. 2015;21(22):6924–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cavar S, Jelašic D, Seiwerth S, Miloševic M, Hutinec Z, Mišic M. Endoglin (CD 105) as a potential prognostic factor in neuroblastoma. Pediatr Blood Cancer. 2015;62(5):770–5.

    Article  CAS  PubMed  Google Scholar 

  23. Sugita Y, Takase Y, Mori D, Tokunaga O, Nakashima A, Shigemori M. Endoglin (CD 105) is expressed on endothelial cells in the primary central nervous system lymphomas and correlates with survival. J Neuro-Oncol. 2007;82(3):249–56.

    Article  CAS  Google Scholar 

  24. Zhou D, Cheng SQ, Ji HF, Wang JS, Xu HT, Zhang GQ, Pang D. Evaluation of protein pigment epithelium-derived factor (PEDF) and microvessel density (MVD) as prognostic indicators in breast cancer. J Cancer Res Clin Oncol. 2010;136(11):1719–27.

    Article  CAS  PubMed  Google Scholar 

  25. Li DY, Sorensen LK, Brooke BS, Urness LD, Davis EC, Taylor DG, Boak BB, Wendel DP. Defective angiogenesis in mice lacking endoglin. Science. 1999;284(5419):1534–7.

    Article  CAS  PubMed  Google Scholar 

  26. Seon BK, Haba A, Matsuno F, Takahashi N, Tsujie M, She X, Harada N, Uneda S, Tsujie T, Toi H, Tsai H, Haruta Y. Endoglin-targeted cancer therapy. Curr Drug Deliv. 2011;8(1):135–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bockhorn M, Tsuzuki Y, Xu L, Frilling A, Broelsch CE, Fukumura D. Differential vascular and transcriptional responses to anti-vascular endothelial growth factor antibody in orthotopic human pancreatic cancer xenografts. Clin Cancer Res. 2003;9(11):4221–6.

    CAS  PubMed  Google Scholar 

  28. Davis TW, O'Neal JM, Pagel MD, Zweifel BS, Mehta PP, Heuvelman DM, Masferrer JL. Synergy between celecoxib and radiotherapy results from inhibition of cyclooxygenase-2-derived prostaglandin E2, a survival factor for tumor and associated vasculature. Cancer Res. 2004;64(1):279–85.

    Article  CAS  PubMed  Google Scholar 

  29. Anderberg C, Cunha SI, Zhai Z, Cortez E, Pardali E, Johnson JR, Franco M, Páez-Ribes M, Cordiner R, Fuxe J, Johansson BR, Goumans MJ, Casanovas O, ten Dijke P, Arthur HM, Pietras K. Deficiency for endoglin in tumor vasculature weakens the endothelial barrier to metastatic dissemination. J Exp Med. 2013;210(3):563–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ciernik IF, Krayenbühl Ciernik BH, Cockerell CJ, Minna JD, Gazdar AF, Carbone DP. Expression of transforming growth factor beta and transforming growth factor beta receptors on AIDS-associated Kaposi's sarcoma. Clin Cancer Res. 1995;1(10):1119–24.

    CAS  PubMed  Google Scholar 

  31. Hara H. Endoglin (CD105) and claudin-5 expression in cutaneous angiosarcoma. Am J Dermatopathol. 2012;34(7):779–82.

    Article  PubMed  Google Scholar 

  32. Mitsui H, Shibata K, Mano Y, Suzuki S, Umezu T, Mizuno M, Yamamoto E, Kajiyama H, Kotani T, Senga T, Kikkawa F. The expression and characterization of endoglin in uterine leiomyosarcoma. Clin Exp Metastasis. 2013;30(6):731–40.

    Article  CAS  PubMed  Google Scholar 

  33. Boeuf S, Bovée JV, Lehner B, van den Akker B, van Ruler M, Cleton-Jansen AM, Richter W. BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors. BMC Cancer. 2012;12:488.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Takase Y, Kai K, Masuda M, Akashi M, Tokunaga O. Endoglin (CD105) expression and angiogenesis status in small cell lung cancer. Pathol Res Pract. 2010;206(11):725–30.

    Article  CAS  PubMed  Google Scholar 

  35. Bauman TM, Huang W, Lee MH, Abel EJ. Neovascularity as a prognostic marker in renal cell carcinoma. Hum Pathol. 2016;57:98–105.

    Article  CAS  PubMed  Google Scholar 

  36. Tachezy M, Reichelt U, Melenberg T, Gebauer F, Izbicki JR, Kaifi JT. Angiogenesis index CD105 (endoglin)/CD31 (PECAM-1) as a predictive factor for invasion and proliferation in intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Histol Histopathol. 2010;25(10):1239–46.

    PubMed  Google Scholar 

  37. Malempati S, Hawkins DS. Rhabdomyosarcoma: review of the Children’s oncology group (COG) soft-tissue sarcoma committee experience and rationale for current COG studies. Pediatr Blood Cancer. 2012;59(1):5–10.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Weidner N. Chapter 14. Measuring intratumoral microvessel density. Methods Enzymol. 2008;444:305–23.

    Article  PubMed  Google Scholar 

  39. Miyoshi K, Kohashi K, Fushimi F, Yamamoto H, Kishimoto J, Taguchi T, Iwamoto Y, Oda Y. Close correlation between CXCR4 and VEGF expression and frequent CXCR7 expression in rhabdomyosarcoma. Hum Pathol. 2014;45(9):1900–9.

    Article  CAS  PubMed  Google Scholar 

  40. Onisto M, Slongo ML, Gregnanin L, Gastaldi T, Carli M, Rosolen A. Expression and activity of vascular endothelial growth factor and metalloproteinases in alveolar and embryonal rhabdomyosarcoma cell lines. Int J Oncol. 2005;27(3):791–8.

    CAS  PubMed  Google Scholar 

  41. Gee MF, Tsuchida R, Eichler-Jonsson C, Das B, Baruchel S, Malkin D. Vascular endothelial growth factor acts in an autocrine manner in rhabdomyosarcoma cell lines and can be inhibited with all-trans-retinoic acid. Oncogene. 2005;24(54):8025–37.

    Article  CAS  PubMed  Google Scholar 

  42. Barber TD, Barber MC, Tomescu O, Barr FG, Ruben S, Friedman TB. Identification of target genes regulated by PAX3 and PAX3-FKHR in embryogenesis and alveolar rhabdomyosarcoma. Genomics. 2002;79(3):278–84.

    Article  CAS  PubMed  Google Scholar 

  43. Wang W, Slevin M, Kumar S, Kumar P. The cooperative transforming effects of PAX3-FKHR and IGF-II on mouse myoblasts. Int J Oncol. 2005;27(4):1087–96.

    CAS  PubMed  Google Scholar 

  44. Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature. 1989;339:58–61.

    Article  CAS  PubMed  Google Scholar 

  45. Liu CD, Tilch L, Kwan D, McFadden DW. Vascular endothelial growth factor is increased in ascites from metastatic pancreatic cancer. J Surg Res. 2002;102(1):31–4.

    Article  CAS  PubMed  Google Scholar 

  46. Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol. 2001;19:1207–25.

    Article  CAS  PubMed  Google Scholar 

  47. Chisholm JC., Merks JH., Casanova M., Bisogno G., Orbach D., Gentet JC., Thomassin Defachelles AS, Chastagner PB., Lowis S., Ronghe M., McHugh K., van Rijn R., Hilton M., Bachir J., Fürst-Recktenwald S., Geoerger B., Oberlin O.; BERNIE: Open-label, randomized, phase II study of bevacizumab plus chemotherapy in pediatric metastatic rhabdomyosarcoma (RMS) and non-rhabdomyosarcoma soft tissue sarcoma (NRSTS). J Clin Oncol, 2016; 34 (suppl; abstr 11054).

  48. Mascarenhas L, Meyers WH, Lyden E, Rodeberg DA. Randomized phase II trial of bevacizumab and temsirolimus in combination with vinorelbine (V) and cyclophosphamide (C) for first relapse/disease progression of rhabdomyosarcoma (RMS): a report from the Children’s Oncology Group (COG). J Clin Oncol. 2014; 32: 5s (suppl; abstract 10003).

  49. Gordon MS, Robert F, Matei D, Mendelson DS, Goldman JW, Chiorean EG, Strother RM, Seon BK, Figg WD, Peer CJ, Alvarez D, Adams BJ, Theuer CP, Rosen LS. An open-label phase Ib dose-escalation study of TRC105 (anti-endoglin antibody) with bevacizumab in patients with advanced cancer. Clin Cancer Res. 2014 Dec 1;20(23):5918–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Attia S, Kumar S, Riedel RF., Robinson SI, Conry RM, McKay Boland P, Barve MA, Fritchie K, Seon BK, Alvarez D, Adams BJ, Shazer RL, Theuer CP, Maki RG. A phase 1B/ phase 2A study of TRC105 (Endoglin Antibody) in combination with pazopanib (P) in patients (pts) with advanced soft tissue sarcoma (STS). J Clin Oncol. 2016;34 (suppl; abstr 11016).

  51. Afshar Moghaddam N, Mahsuni P, Taheri D. Evaluation of Endoglin as an angiogenesis marker in Glioblastoma. Iran J Pathol. 2015 Spring;10(2):89–96.

    PubMed  PubMed Central  Google Scholar 

  52. Barresi V, Cerasoli S, Vitarelli E, Tuccari G. Density of microvessels positive for CD105 (endoglin) is related to prognosis in meningiomas. Acta Neuropathol. 2007;114(2):147–56. Epub 2007 Jun 27

    Article  CAS  PubMed  Google Scholar 

  53. Wexler LH, Meyer WH, Helman LJ. Rhabdomyosarcoma. In: Pizzo PA, Poplack DG, editors. Principles and Practice of Pediatric Oncology, vol. 6. Philadelphia: Wolter Kluwer–Lippincott Williams & Wilkins. 2011. pp. 923–53.

  54. Kumar S, Ghellal A, Li C, Byrne G, Haboubi N, Wang JM, Bundred N. Breast carcinoma: vascular density determined using CD105 antibody correlates with tumor prognosis. Cancer Res. 1999;59:856–61.

    CAS  PubMed  Google Scholar 

  55. Yao Y, Pan Y, Chen J, Sun X, Qiu Y, Ding Y. Endoglin (CD105) expression in angiogenesis of primary hepatocellular carcinomas: analysis using tissue microarrays and comparisons with CD34 and VEGF. Ann Clin Lab Sci. 2007;37:39–48.

    CAS  PubMed  Google Scholar 

  56. Kuda M, Kohashi K, Yamada Y, Maekawa A, Kinoshita Y, Nakatsura T, Iwamoto Y, Taguchi T, Oda Y. FOXM1 expression in rhabdomyosarcoma: a novel prognostic factor and therapeutic target. Tumour Biol. 2016;37(4):5213–23.

    Article  CAS  PubMed  Google Scholar 

  57. Folberg R, Hendrix MJ, Maniotis AJ. Vasculogenic mimicry and tumor angiogenesis. Am J Pathol. 2000;156(2):361–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Cao Z, Bao M, Miele L, Sarkar FH, Wang Z, Zhou Q. Tumour vasculogenic mimicry is associated with poor prognosis of human cancer patients: a systemic review and meta-analysis. Eur J Cancer. 2013;49(18):3914–23.

    Article  PubMed  Google Scholar 

  59. Chen X, Maniotis AJ, Majumdar D, Pe'er J, Folberg R. Uveal melanoma cell staining for CD34 and assessment of tumor vascularity. Invest Ophthalmol Vis Sci. 2002;43(8):2533–9.

    PubMed  Google Scholar 

  60. Chen L, He Y, Sun S, Sun B, Tang X. Vasculogenic mimicry is a major feature and novel predictor of poor prognosis in patients with orbital rhabdomyosarcoma. Oncol Lett. 2015;10(3):1635–41. Epub 2015 Jul 8

    PubMed  PubMed Central  Google Scholar 

  61. Sun B, Zhang S, Zhao X, Zhang W, Hao X. Vasculogenic mimicry is associated with poor survival in patients with mesothelial sarcomas and alveolar rhabdomyosarcomas. Int J Oncol. 2004;25(6):1609–14.

    PubMed  Google Scholar 

  62. Yudoh K, Kanamori M, Ohmori K, Yasuda T, Aoki M, Kimura T. Concentration of vascular endothelial growth factor in the tumour tissue as a prognostic factor of soft tissue sarcomas. Br J Cancer. 2001;84(12):1610–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Chao C, Al-Saleem T, Brooks JJ, Rogatko A, Kraybill WG, Eisenberg B. Vascular endothelial growth factor and soft tissue sarcomas: tumor expression correlates with grade. Ann Surg Oncol. 2001;8(3):260–7.

    Article  CAS  PubMed  Google Scholar 

  64. West CC, Brown NJ, Mangham DC, Grimer RJ, Reed MW. Microvessel density does not predict outcome in high grade soft tissue sarcoma. Eur J Surg Oncol. 2005;31(10):1198–205.

    Article  CAS  PubMed  Google Scholar 

Download references


We thank Professor Franco Locatelli for critical reading this paper and for his suggestions. We would also like to thank the children’s parents, who gave their informed consent for publication and “Il cuore grande di Flavio” Onlus. Dr. Marta Colletti is a post-doctoral fellow of the Umberto Veronesi Foundation. To Valentina Polcini for proofreading.


Not applicable

Availability of data and materials

All data generated and analyzed during this study are included in this published article.

Author information

Authors and Affiliations



VDP help in the histological revision, analyzed the results and helped to draft the manuscript, IR collected the data of patients, RB performed histological diagnosis, LR performed the statistical analysis, MP and MCB cut the paraffin blocs and performed immunohistochemistry, AG helped to perform the figures in the manuscript, MC helped to draft the manuscript, RR reviewed the manuscript, DO reviewed the manuscript, AC helped to select the cases, HP reviewed the manuscript, GMM selected the cases and helped to draft the manuscript, ADG designed the study, interpreted the results and drafted the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Giuseppe Maria Milano or Angela Di Giannatale.

Ethics declarations

Ethics approval and consent to participate

Informed consent to participate at the study was obtained from parent or legal guardian of the patient.

Consent for publication

Written informed consent for publication of their clinical details and clinical images was obtained from the parent or guardian of the patient.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Additional file

Additional file 1: Figure S1.

Immunostaining of CD105 for ERMS and ARMS. Magnification × 200. (PDF 266 kb)

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Di Paolo, V., Russo, I., Boldrini, R. et al. Evaluation of Endoglin (CD105) expression in pediatric rhabdomyosarcoma. BMC Cancer 18, 31 (2018).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: