Immunhistochemical Staining

The protein marker expression was analyzed using tumor tissue sections and non-cancerous mucosa from the resection margins of all available histopathologic specimens (n = 156) (Table 2). Specimens without tumor were excluded from immunohistochemical analysis. Fresh 1.5 µm sections from formalin-fixed and paraffin-embedded samples were transferred to glass slides, dewaxed, and rehydrated. Antigen retrieval (microwave oven heating in citrate buffered saline for B1-R and B2-R and in EDTA buffered saline for VEGF-R2) was performed according to the manufacturers’ recommendations. After cooling, the slides were incubated with the primary antibody (Table 3, additional files). The reaction was developed with the labeled streptavidin–biotin-alkaline phosphatase system using DAB as a reaction indicator. After counterstaining with hematoxylin, the slides were dehydrated using ascending ethanol concentrations and mounted. Tissue samples known to express the respective antigens were used as a positive control. Antibodies of irrelevant specificity with an immunoglobulin isotype identical to that of the primary antibody were used as negative controls.

Overview of patient cohort of the immunohistochemical analysis

^†PCF pharyngocutaneous fistula

^‡Number of valid cases; Percentage indication refers to number of valid cases

Additional files: Characteristics of antibodies used for immunohistochemical staining

Thermo Fisher

Waltham. MA, USA

US Biological

Salem. MA, USA

Abcam

Cambridge. UK

The outcome of the antibody-expression was graded by the immunoreactive score displayed in Table 4 in the additional files. A graphic illustration of the respective scores is conveyed in Fig. 1, ​,2,2, ​,3.3. For B1-R and B2-R, the membrane-bound and cytoplasmic expression was analyzed. For VEGF-R2, membrane-bound expression was distinguished from endothelial vessel expression. The latter was evaluated by means of quantitative categorization of vascular density. In order to assess vascular density, immunohistochemical tissue samples were examined through a 10 × objective and the vessels visible in every field of vision (fov) were counted. Fewer than five vessels in every fov was categorized as low expression, whereas five and more vessels in at least one fov was categorized as high expression.

Additional files: Immunoreactive score

^†PP percentage points: percentage of stained tumor cells

^‡SI staining intensity

Exemplary demonstration of varied immunohistochemical staining scores for B1-R. from top to bottom: B1-R immunoreactive score negative; B1-R immunoreactive score = 2; B1-R immunoreactive score = 5

Exemplary demonstration of varied immunohistochemical staining scores for B2-R. from top to bottom: B2-R immunoreactive score negative; B2-R immunoreactive score = 2; B2-R immunoreactive score = 5

Exemplary demonstration of varied immunohistochemical staining scores for VEGF-R2. From top to bottom: VEGF-R2 immunoreactive score negative; VEGF-R2 immunoreactive score = 2; VEGF-R2 immunoreactive score = 5