Roselle J. Eisma

Vascular endothelial growth factor expression in head and neck squamous cell carcinoma

BACKGROUND Angiogenesis is an essential process required for growth and metastasis in cancer. In breast, gastric, and prostate cancer, vascular endothelial growth factor (VEGF) has been implicated in angiogenesis; however, little is known about VEGF in HNSCC. In this study, we hypothesize that VEGF is present in elevated levels in HNSCC and may therefore play a role in promoting angiogenesis. METHODS We obtained tumor tissue from 63 HNSCC patients undergoing primary resection. All tissue samples were analyzed by immunohistochemistry (IHC) techniques for the presence and localization of VEGF; however, only 36 had sufficient amounts of tissue for quantitative analysis of VEGF by ELISA. Nine control specimens taken from patients undergoing uvulopalatopharyngoplasty were also analyzed. RESULTS In all 63 of our patient samples we found VEGF to be present and localized to the cancer cells and endothelial cells. The poorly differentiated cancer cells stained more intensely in comparison with the well-differentiated ones. There was a 20-fold increase in the patient levels when compared with controls levels (P > or =0.05). Analysis by enzyme-linked immunosorbent assay revealed elevated mean levels of VEGF (241 +/- 326 pg/mg total protein [TP]) with a range of 2 to 1484 pg/mg TP. The control specimens had mean levels of 13 +/- 11 pg/mg TP and a range of 1 to 78 pg/mg TP. Patients who exhibited higher levels of VEGF tended to have a higher rate of disease recurrence (P < or =0.048) and shorter disease-free interval (P < or =0.05). CONCLUSIONS The expression of VEGF in elevated levels in the HNSCC tumor microenvironment appears to be associated with more aggressive disease. Based on our results, VEGF may be an important angiogenic factor associated with cancer cells and endothelial cells in HNSCC. Further studies are needed to better define the role of VEGF in HNSCC and its role as a potential target for therapeutic intervention.

Coexpression of interleukin-8 receptors in head and neck squamous cell carcinoma

BACKGROUND Interleukin 8 (IL-8) is an important cytokine involved in tumor growth and angiogenesis in a variety of malignancies. We hypothesize that IL-8 plays an important role in the cellular proliferation and angiogenesis seen in head and neck squamous cell carcinoma (HNSCC) and set out to identify its receptors, IL-8RA and IL-8RB. METHODS Immunohistochemical analysis was performed on specimens from 38 HNSCC patients with stage I to IV disease and control tissues. RESULTS All of cancer specimens demonstrated positive staining for IL-8RA. The IL-8RA staining of microvessel endothelial cells was seen in 51%. The IL-8RB pattern was similar to the IL-8RA pattern in that 97% of cancer sections demonstrated positive cancer cell staining, and 74% of the specimens demonstrated positive staining for microvessel endothelial cells. CONCLUSION Our studies demonstrate that IL-8 receptors are expressed by cancer cells and microvessel endothelial cells in HNSCC, suggesting that IL-8 may act in an autocrine/paracrine fashion to stimulate cellular proliferation and angiogenesis.

Role of angiogenic factors : Coexpression of interleukin-8 and vascular endothelial growth factor in patients with head and neck squamous carcinoma

Objective/Hypothesis: Angiogenesis has been used as a prognostic indicator in a variety of cancers and is believed to be controlled by angiogenic factors, including the cytokines interleukin‐8 (IL‐8) and vascular endothelial growth factor (VEGF). We hypothesized that the in vivo coexpression of both IL‐8 and VEGF in head and neck tumors contributes to perpetuating tumor growth and metastasis by enhancing angiogenesis. Methods: Immunohistochemical analysis for IL‐8 and VEGF was performed using specimens from 33 cancer patients and 6 control patients. We quantitatively evaluated levels of IL‐8 and VEGF in tumor tissue homogenates from those same patients using enzyme‐linked immunosorbent assay and radioimmunoassay. Comprehensive histories of each patient were taken and later analyzed for clinical correlations with IL‐8 or VEGF levels. Results: IL‐8 and VEGF were found to be colocalized within the head and neck squamous cell carcinoma (HNSCCA) tumor cells. In the head and neck tumor specimens, IL‐8 levels ([38,152 ± 1.8] × 10 5 pg/mg total protein [TP]) were 22‐fold greater than controls (1,721 ± 2,122 pg/mg TP). The tumor levels of VEGF (1,304 ± 6,037 pg/mg TP) were nearly fourfold higher than the controls (317 ± 400 pg/mg TP. Interleukin‐8 and VEGF levels were found to have a positive correlation (P ⩽ .0001). Patients exhibiting high levels in picograms per milligram of TP and/or number of moles of IL‐8 and VEGF were found to clinically have more aggressive disease manifested by higher TNM stage, more recurrences, and shorter disease‐free intervals (P ⩽ .03) Conclusions: Marked increase in HNSCCA of IL‐8 and VEGF underscores the importance of these angiogenic factors in this disease. Understanding the roles and interplay of angiogenic factors such as IL‐8 and VEGF may have value in the treatment of HNSCCA.

Expression of basic fibroblast growth factor and its receptors by head and neck squamous carcinoma tumor and vascular endothelial cells

BACKGROUND Basic fibroblast growth factor (bFGF) is a potent angiogenic factor implicated in tumor growth and metastasis. To determine if bFGF and basic fibroblast growth factor receptor 1 (bFGFR1) and 2 (bFGFR2) are upregulated in head and neck squamous cell carcinoma (HNSCC), we measured the distribution and levels of each in HNSCC specimens and control specimens. METHODS Head and neck squamous cell carcinoma and control tissue specimens were analyzed qualitatively (40 patients, 10 controls) using immunohistochemistry, and quantitatively (26 patients, 8 controls) using immunoassays and graded immunohistochemistry. Control tissue consisted of palatal tissue obtained during uvulopalatopharyngoplasty (UPPP). RESULTS Immunohistochemical analysis revealed that bFGF, bFGFR1, and bFGFR2 antigens were strongly associated with cancer and vascular endothelial cells in HNSCC. Control tissue had moderate staining of vascular endothelium and no stromal staining. Quantitative analysis of bFGF in tissue homogenates indicated that bFGF levels in cancer specimens were significantly elevated compared with control tissues (420.3 +/- 360.9 ng/mg total protein versus 49.2 +/- 48.7, respectively, P < or =0.05). When analyzed by clinical stage, bFGF levels were significantly higher in stage III patients as compared with stage IV patients (P < or =0.01). When immunohistochemistry results were correlated with clinical stage, bFGF (P < or =0.01), bFGFR1 (P < or =0.001), and bFGFR2 (P < or =0.0001) staining was significantly more intense in the cancer cells of stage III versus stage IV patients. CONCLUSION Enhanced expression of bFGF and bFGF receptors by cancer and vascular endothelial cells is present in HNSCC, and may contribute to tumor growth and metastasis in HNSCC by mediating angiogenesis. Strategies aimed at decreasing the expression of bFGF and its receptors may be of therapeutic benefit in HNSCC, particularly at an early stage of disease.

Characterization of the Eosinophil Chemokine Rantes in Nasal Polyps

Previous studies have demonstrated that the cytokine RANTES (Regulated And Normal T cell Expressed and Secreted) has been shown to be a potent mediator of eosinophil Chemotaxis in vitro and of leukocyte recruitment. Because eosinophils are the hallmark cells in nasal polyposis, we hypothesize that RANTES is locally produced within the nasal polyp microenvironment and is responsible for the eosinophil recruitment seen in nasal polyposis. To begin to test this hypothesis, we evaluated nasal polyps from 17 patients and 3 control specimens for distribution and content of RANTES using immunohistochemical techniques and enzyme-linked immunosorbent assay technology. Our immunohistochemical studies demonstrated that in nasal polyposis, RANTES antigen staining occurred predominantly within eosinophils and epithelial cells. To quantify the relative levels of RANTES in normal and nasal polyp specimens, tissue homogenates were prepared, quantified, and normalized to protein levels. We detected RANTES in all 17 nasal polyp tissue homogenates (566 ± 16 pg/mg total protein). The RANTES levels in nasal polyp homogenates were nearly 40-fold higher man the RANTES levels in normal tissue (15.7 ± 28.2 pg/mg total protein). Thus, it appears that increased expression of RANTES by eosinophils and epithelial cells within the nasal polyp microenvironment promotes eosinophil recruitment and activation within nasal polyps. We hypothesize that RANTES induces increased recruitment and activation of eosinophils, presumably contributing to the increased tissue changes associated with nasal polyposis.

Interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor expression in nasal polyps☆

PURPOSE Nasal polyps (NP) are grape-like clusters of chronically inflamed tissue. Little is known about the underlying cells and cytokines involved in nasal polyposis. For the present study, we hypothesize that elevated tissue levels of interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulation factor (GM-CSF) contribute to eosinophil recruitment and activation in NP. MATERIALS AND METHODS To begin to test this hypothesis, we evaluated IL-3, IL-5, and GM-CSF levels and distributions in nasal polyp specimens obtained intraoperatively from 13 patients and two normal controls. For these studies, nasal polyp levels were determined by enzyme-linked immunosorbent assay (ELISA), and IL-3, IL-5, and GM-CSF distribution was determined by immunohistochemistry. RESULTS Immunohistochemical staining of the NP indicated that in all 13 patient samples, IL-3, IL-5, and GM-CSF were associated with infiltrating cells, primarily eosinophils, in the NP. Quantitation of IL-3, IL-5, and GM-CSF in NP tissue homogenates indicated that IL-3, IL-5, and GM-CSF levels were evaluated in the NP tissues when compared with control tissues. Additionally, elevation of individual cytokines correlated with previous polypectomy (IL-3), steroid use (IL-3, IL-5, and GM-CSF), asthma (IL-5), and age (GM-CSF). CONCLUSION These data support our hypothesis that IL-3, IL-5, and GM-CSF are likely to play a key role in eosinophil recruitment/activation and NP formation and support recently advanced theories that cytokines play a key role in the pathogenesis of this disease.

Decreased Expression of Transforming Growth Factor Beta Receptors on Head and Neck Squamous Cell Carcinoma Tumor Cells

BACKGROUND Transforming growth factor beta (TGF-beta) has antiproliferative effects on normal and neoplastic cells that express specific TGF-beta receptors. We hypothesize that diminished expression of TGF-beta and/or its receptors may contribute to the uncontrolled proliferation of head and neck squamous cell carcinoma (HNSCCA) cancer cells. METHODS Using immunohistochemical techniques, we characterized the expression of TGF-beta isoforms and TGF-beta receptors, TGF-beta(RI) and TGF-beta(RII), in HNSCCA. Tumor production of TGF-beta was evaluated in culture supernatants from a cytokine-stimulated HNSCCA tumor line (HTB-43). RESULTS All control specimens displayed strong cell-associated staining of TGF-beta as well as both receptors. Forty-seven of 47 cancer specimens exhibited positive staining for TGF-beta in the tumor matrix. Forty of the 47 cancer specimens demonstrated no expression of TGF-beta(RI), and 43 of the 47 expressed no TGF-beta(RII). Only interleukin 1 alpha (IL-1 alpha) and IL-1 beta induced significant TGF-beta expression from the HTB-43 cells. CONCLUSIONS Decreased expression of TGF-beta receptors may play a significant role in the pathogenesis of HNSCCA by allowing uncontrolled cell proliferation.

Interleukin-8 Expression in Human Nasal Polyps

The cytokine interleukin 8 (IL-8) has been shown to be a potent mediator of leukocyte recruitment and neovascularization in inflammatory and neoplastic diseases. In this study we hypothesize that IL-8 produced in the nasal polyp microenvironment is responsible for the leukocyte recruitment seen in nasal polyposis. To test this hypothesis we evaluated nasal polyps for distribution and content of IL-8 antigen with immunohistochemical techniques and radioimmunoassay to determine tissue levels of IL-8. The immunohistochemical results demonstrated that IL-8 antigen staining occurred predominantly within inflammatory cells and epithelium. IL-8 was detected in all nasal polyp tissue homogenates (a mean value of 1767 +/- 1633 pg/mg total protein (TP) with a range of 134 to 3668 pg/mg TP vs control specimens with a mean value of 77 pg/mg TP with a range of 0.09 to 255 pg/mg TP). These data demonstrate the presence and distribution and levels of IL-8 antigen in nasal polyps in vivo, supporting our hypothesis that local production of IL-8 could be an important factor in the sustained recruitment of leukocytes in nasal polyposis. Thus IL-8 likely plays a significant role in the pathogenesis of this disease process and therefore is a potential target for therapeutic intervention.

Transforming growth factor-β expression in otitis media with effusion†

Objective: To characterize the existence and role of transforming growth factor‐β (TGF‐β) in otitis media with effusion (OME). Study Design: Retrospective. Methods: The levels of two major TGF‐β isoforms, TGF‐β1 and TGF‐β2, in the middle ear effusions (MEEs) of 44 children were evaluated using enzyme‐linked immunospecific assays (ELISAs). Forty‐eight MEEs were separated into three clinically relevant groups (i.e., serous, mucoid, and purulent), and TGF‐β levels were correlated with clinical parameters of disease for these MEEs. Results: Both TGF‐β1 and TGF‐β2 were present in the samples. Mean levels of TGF‐β1 (920.36 ± 437.75 pg/mg total protein) were generally 100‐fold greater than those of TGF‐β2 (9.65 ± 11.19 pg/mg total protein). TGF‐β1 levels were elevated in association with a history of previous tympanostomy tube placements (TTPs) (P = .029) and mucoid effusions (P = .042). TGF‐β2 levels were elevated in association with a history of previous TTPs (P = .100) and chronic (i.e., serous or mucoid) effusions (P = .003). Conclusions: TGF‐β1 is present in the MEEs of children with OME. Furthermore, TGF‐β1 and TGF‐β2 levels were elevated differentially in the presence of chronic disease indicators in OME, suggesting that these isoforms may have differing roles in the inflammatory processes that characterize OME.

Eosinophil expression of transforming growth factor-beta and its receptors in nasal polyposis: Role of the cytokines in this disease process

PURPOSE Nasal polyposis (NP) is characterized by an increase in inflammatory processes including fibrosis. Because transforming growth factor beta (TGF-beta) has been proven to induce fibrosis, we hypothesize that TGF-beta and its receptors are present in NP and influence polyp development. MATERIALS AND METHODS To test this hypothesis, we evaluated distribution (immunohistochemistry) of TGF-beta isoforms (TGF-beta 1, TGF-beta 2, and TGF-beta 3) and its receptors [(TGF-beta(RI) & TGF-beta(RII)] in NP from 36 NP patients and in five normal sinus tissue specimens obtained from septoplasty/inferior turbinectomy. Tissue levels of TGF-beta 1 and TGF-beta 2 levels were determined by enzyme-linked immunosorbent assay (ELISA) and protein content was determined by Bio Rad assay (Bio Rad, Richmond, CA). All tissue levels of TGF-beta were normalized and expressed as pg of TGF-beta per mg of total protein (pg/mg TP). RESULTS Immunohistochemical studies showed eosinophils as the major cells positively staining for TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta(RI), and TGF-beta. In fibrotic sections, increased staining of eosinophils, fibroblast, and mononuclear cells was found for all three isoforms and both receptors. Evaluation of tissue levels indicated mean levels of TGF-beta 1 in the NP were 11.64 +/- 22.12 pg/mg TP versus normal control mean 44.36 +/- 22.12 pg/mg TP.TGF-beta 2 mean levels were 11.46 +/- 23.73 pg/mg TP versus normal control mean of 2.03 +/- 1.13 pg/mg TP. NP showed decreased expression of TGF-beta 1 and enhanced expression of TGF-beta 2 isoforms with presence of their receptors. Higher levels of TGF-beta 2 correlated with an increase in previous polypectomies perhaps indicative of severity of disease (P < or = .0001). CONCLUSION Our studies show the presence of the TGF-beta isoforms and receptors in NP tissue. The results support our hypothesis that the eosinophil continues to be a pivotal inflammatory cell in NP, a differential regulation may govern the activity of TGF-beta in NP, and hence, the TGF-beta family of cytokines and receptors likely play a key role in controlling NP formation.