Results
Included in this study were 71 diseased vascular tissue samples originally collected from human patients, representing malignant (seven angiosarcomas, two hemangiopericytomas), borderline (six hemangioendothelioma), and benign (five infantile hemangioma, one capillary hemangioma, 45 cavernous hemangiomas, three granulomatous hemangiomas, one venous hemangioma) vascular tumors and one thrombophlebitis. Known characteristics of patients grouped according to biopsy classification are reported in Table 1. As controls, we included two tissue sets in this analysis: 1) ten non-diseased blood vessel tissues and 2) a diverse panel of 58 human sarcoma tumors. The non-diseased blood vessel tissues were chosen to evaluate the expression of stem cell reprogramming factors in normal vasculature, while the various sarcomas were selected to compare the levels of stem cell reprogramming factors in borderline and malignant vascular sarcomas to that of other malignant mesenchymal tumors.
IHC staining for the stem cell reprogramming factors Oct4, Nanog, Myc, Klf4, and Sox2 was performed in the vascular tumor samples as well as the two control tissue sets. Representative images of each staining are depicted in Figs. 1, 2, 3, 4 and 5. With the exception of Myc, each of these proteins was detectable at variable levels across non-diseased vascular tissues, ranging from 50 % of normal tissues displaying Nanog and Klf4 immunoreactivity to 90 % of normal tissues displaying Oct4 immunoreactivity (Table 2). Expression of these "stem cell regulators" in non-diseased adult tissue is not surprising given that the HPA reports detection of Oct4, Nanog, Klf4, and Sox2 in approximately 70, 11, 29, and 51 % of normal human tissues, respectively. Though HPA reports Myc expression in 56 % of normal human tissues, we did not detect this protein in any non-diseased vascular tissues tested in this analysis. While immunostaining for these stem cell regulators was observed in non-diseased vasculature, the IHC score for these tissues was relatively low given that staining intensity for each stem cell marker was weak to moderate and often occurred in a very small fraction (<25 %) of the cells comprising the tissue (Fig. 6, Additional file 2: Table S1 https://www.biomedcentral.com/1472-6890/15/18/additional).
Figure 1.
Representative images of Oct4 staining in normal and vascular tumor tissues. Immunopositivity for Oct4 protein is represented by brown staining. Positive control (left panel) = human testicle; negative control (right panel) = human testicle with no added primary antibody. 400× total magnification for each image
Figure 2.
Representative images of Nanog staining in normal and vascular tumor tissues. Immunopositivity for Nanog protein is represented by brown staining. Positive control (left panel)=human testicle; negative control (right panel) = human testicle with no added primary antibody. 400× total magnification for each image
Figure 3.
Representative images of Myc staining in normal and vascular tumor tissues. Immunopositivity for Myc protein is represented by brown staining. Positive control (left panel) = human colon cancer; negative control (right panel) = human colon cancer with no added primary antibody. 400× total magnification for each image
Figure 4.
Representative images of Sox2 staining in normal and vascular tumor tissues. Immunopositivity for Sox2 protein is represented by brown staining. Positive control (left panel) = rat brain; negative control (right panel) = rat brain with no added primary antibody. 400× total magnification for each image
Figure 5.
Representastive images of Klf4 staining in normal and vascular tumor tissues. Immunopositivity for Klf4 protein is represented by brown staining. Positive control (left panel) = human intestine; negative control (right panel) = human intestine with no added primary antibody. 400× total magnification for each image
Figure 6.
Antigenicity for embryonic stem cell reprogramming factors in normal tissue and vascular tumors. Box and whisker plots depicting the IHC scores for Oct4, Nanog, Myc, Sox2, and Klf4 in normal vasculature, benign, borderline or malignant vascular tumors, or across a panel of various sarcomas. The Mann-Whitney rank sum test was used to determine statistical significance. Significance was determined if the two-sided P value of the test was < 0.05. Asterisks indicate level of significance relative to normal vasculature (* p <0.05, ** p < 0.05, *** p < 0.005, **** p < 0.0005)
In contrast to the non-diseased vascular rich tissue controls, benign vascular tumors and the single thrombophlebitis sample exhibited significantly increased staining (in both intensity and percentage of positive tissue) for Oct4, Nanog, Myc, and Sox2, with no statistically significant increase in antigenicity for Klf4 (Fig. 6, Additional file 2: Table S1 https://www.biomedcentral.com/1472-6890/15/18/additional). It is worth noting that unlike the absence of Myc expression in normal vasculature, 46 % of the benign tumors tested were positive for Myc protein. The data obtained from malignant and borderline vascular tumors were remarkably similar to that demonstrated from the benign vascular tumors. Malignant and borderline vascular sarcomas displayed 100 % immunoreactivity for Oct4, Nanog, and Sox2, while Myc protein was present in 50 % of malignant and borderline vascular tumors (Table 2). The IHC scores for all proteins tested except Klf4 were significantly increased in the malignant and borderline vascular tumors relative to the non-diseased controls, and were surprisingly very similar to the levels observed in benign vascular tumors. The elevated IHC scores observed for malignant and borderline vascular tumors correlated to the results obtained in a diverse panel of malignant sarcoma cells, revealing immunoreactivity for Oct4, Nanog, and Sox2 in 100 % of various sarcoma tissues and 72 % for Myc and Kfl4 (Additional file 2: Table S2 https://www.biomedcentral.com/1472-6890/15/18/additional). While Klf4 protein expression was not significantly different between any of the vascular tumors or vascular tissue controls, this protein did show a significantly increased mean IHC score in the sarcoma tissue control set (Fig. 6, Additional file 2: Table S2 https://www.biomedcentral.com/1472-6890/15/18/additional). The expression of Oct4, Nanog, Myc, Sox2, and Klf4 in the current study correlated well with data reported in the HPA which reveals expression of Oct4 in 88 % of cancers, Sox2 in 88 % of cancers, Myc in 78 % of cancers, Klf4 in 28 % of cancers, and Nanog in 7 % of cancers.
BMC Clin Pathol. 2015;15(18) © 2015 BioMed Central, Ltd.
