Expression of Vascular Notch Ligand Delta-Like 4 and Inflammatory Markers in Breast Cancer

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.The growth of tumors requires angiogenesis,¹ which is the consequence of increased expression of proangiogenic factors (for example, vascular endothelial growth factor A [VEGF]^2,3). The expression of VEGF in cancer is controlled by oncogenic signaling,⁴ hypoxia,⁵ and inflammatory cells.⁶ Although there is redundancy among proangiogenic factors in advanced cancer,⁷ many in vivo early stage cancer models show VEGF dependence.^8,9This observation has been exploited clinically, where the addition of an anti-VEGF antibody (bevacizumab) to first line taxane-based chemotherapy in recurrent/metastatic breast cancer was associated with prolongation of progression free survival (from a median of 5.9 to 11.8 months, P < 0.001).¹⁰ Nevertheless, there was no statistically significant overall survival benefit, and all patients in this trial eventually progressed after 4 years.¹⁰ Furthermore, a trial evaluating the addition of bevacizumab to capecitabine in previously treated metastatic/advanced breast cancer demonstrated only a 10.7% improvement in response rate and no survival benefit.¹¹ To date, there are no validated clinical, radiological, or molecular biomarkers that can predict the survival benefit afforded by bevacizumab.^12,13,14,15 Clinical data suggest that antiangiogenic drugs are active in breast cancer,^10,16 and it may be necessary to identify biomarkers that predict their benefit.Additional agents that disrupt functional angiogenesis have been developed to target tumors resistant to anti-VEGF therapy.^17,18 Recent studies have focused on Delta-like ligand 4 (Dll4), a ligand for Notch receptors 1, 3, and 4^17,18,19 that is predominantly expressed by endothelial cells.^17,18,19 Transgenic mice in which Dll4 was replaced by a reporter gene showed that Dll4 expression is restricted to large arteries during development.^20,21 Furthermore, Dll4 heterozygous knockout mice are reported to have defective arterial development²² and venous malformations.²²Experimental systems^17,23,24 have shown that Dll4-Notch inhibition leads to increased sprouting and branching of vessels in association with gradients of VEGF. Conversely, VEGF blockade causes a reduction in Dll4 expression and vessel sprouting.^{17,18,23,24,25,26,27} In addition, endothelial cells transfected with Dll4 down-regulated VEGF receptors KDR and neuropilin1 and showed reduced proliferative and migratory responses to VEGF.²⁸ The implication of this research is that Dll4-Notch signaling regulates endothelial sprouting and branching to form functional vascular beds, under the control of VEGF and by autoregulation of VEGF signaling.²³Disruption of Dll4 signaling by overexpression or inhibition of Dll4 may impair angiogenesis,^17,18 and blockade of Dll4-Notch signaling results in an increased density of nonfunctional vasculature and is associated with a reduction in the growth of human tumor xenografts.^17,18 Indeed, certain xenografts that are resistant to anti-VEGF therapy are reported to be sensitive to anti-Dll4,^17,18,29 and combination treatment with anti-VEGF and anti-Dll4 has additive inhibitory effects on tumor growth.¹⁸ Together these data provide a rationale to target Dll4 in cancer and suggest that Dll4 may have a role in mediating resistance to anti-VEGF therapies.Besides direct vascular effects, Fung et al³⁰ showed that Dll4-Notch signaling in macrophages stimulates a proinflammatory response, which may be proangiogenic.⁶ Moreover, Shojaei et al^31,32 have reported that bevacizumab resistance in certain preclinical in vivo cancer models is causally associated with tumor infiltration by myeloid cells.The characterization of Dll4 protein expression in human cancer is important for the rational design of clinical trials to test the safety and activity of anti-Dll4 therapy. Defining the pattern of Dll4 expression, in association with markers of inflammation, may identify subgroups with distinct clinical behavior and responses to treatment. The aims of this study were to characterize the in situ expression of Dll4 in breast cancer, to assess the association between Dll4 and established markers of inflammation (CD68, CD14, neutrophil elastase, CD123, and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin [DC-SIGN]) and hypoxia (carbonic anhydrase 9 [CA9]), and to determine the prognostic significance of these markers.     

Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis, and microvessel density.

Carbonic anhydrase IX (CA IX) is a transmembrane glycoprotein with an active extracellular enzyme site. We have shown previously that it was hypoxia inducible and may therefore be an endogenous marker of hypoxia. It is overexpressed in some tumors, particularly renal cell carcinoma. The aim of this study was to examine the expression and localization of CA IX in head and neck squamous cell carcinoma (HNSCC) and relate this to the location of tumor microvessels, angiogenesis, necrosis, and stage. Expression of CA IX was determined by immunoblotting in three HNSCC cell lines grown in normoxia and hypoxia (pO(2) 0.1%) and three paired tumor and normal tissue samples of HNSCC. Archived paraffin sections (79) of HNSCC were immunostained with antibodies to CA IX and CD34 to determine microvessel density (MVD). By double staining sections with CA IX and CD34, the distance between blood vessels and the start of CA IX expression and necrosis was calculated. CA IX was induced by hypoxia in all three HNSCC cell lines and overexpressed in HNSCC tumor tissue. Overexpression was localized to the perinecrotic area of the tumor on immunostaining, and the percentage area of the tumor expressing CA IX was significantly higher with more tumor necrosis (P = 0.001), a high MVD (P = 0.02), and advanced stage (P = 0.033) on univariate analysis and necrosis (P = 0.0003) and MVD (P = 0.0019) on multivariate analysis. The median distance between a blood vessel and the start of CA IX expression was 80 microm (range, 40-140 microm). CA IX is overexpressed in HNSCC because of hypoxia and is a potential biomarker for hypoxia in this tumor. Overexpression may help to maintain the intracellular pH, giving tumor cells a survival advantage and enhancing resistance to radiotherapy and chemotherapy. CA IX is a potential target for future therapy in HNSCC.     

Carcinoma of the cervix uteri: an assessment of tumour proliferation using the monoclonal antibody Ki67

Thirty-one cervical biopsies of invasive carcinoma have been studied by immunohistochemical means using the monoclonal antibody Ki67 to determine tumour cell proliferation rates. A wide range (10-50%) in the extent of Ki67 staining (expressed as the percentage of labelled tumour cells) was observed indicating considerable variation on tumour growth rates. There was no significant relationship between the percentage of positive cells and conventional histological parameters such as cell type or tumour differentiation. Immunostaining with monoclonal antibody Ki67 therefore provides a new approach to the assessment of cervical tumour biopsies which will require long term clinical follow-up to establish its prognostic significance.     

Reed-Sternberg and Hodgkin cells in lymphocyte-predominant Hodgkin's disease of nodular subtype contain J chain

To throw light on the question of whether B-cell-derived forms of Hodgkin's disease exist, more than 100 cases of Hodgkin's disease (including all four major histologic categories) were investigated for the presence of J chain and were also immunostained for epithelial membrane antigen and the granulocyte-associated antigen X hapten. Reed-Sternberg and Hodgkin cells (RS & H) expressed J chain in 22 cases, 8 of which also expressed epithelial membrane antigen (EMA). X hapten was found in 62 cases, but all of these were J chain negative. J chain-positive RS & H cells were restricted to cases of lymphocyte-predominant disease, while X hapten-positive tumor cells were found frequently in nodular sclerosis, mixed cellularity, and lymphocyte-depletion Hodgkin's disease, but only occasionally in cases of lymphocyte-predominant disease. These findings indicate that nodular lymphocyte-predominant Hodgkin's disease differs from the other subtypes of Hodgkin's disease and that the neoplastic cells are of B-lymphoid origin.     

Pulsed field gel electrophoresis on frozen tumour tissue sections.

The application of pulsed field gel electrophoresis (PFGE) to the molecular genetic analysis of solid tumours has been restricted by the requirement for whole single cells as a DNA source. A simple technique which allows for the direct analysis of histologically characterised solid tumour material by pulsed field gel electrophoresis was developed. Single frozen tissue sections obtained from colonic carcinoma specimens were embedded without further manipulation in molten, low melting temperature agarose. The tumour DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is yielded by this method to obtain a hybridisation signal with a single copy probe. Histological examination of adjacent tissue sections may also be carried out, permitting correlation between molecular analysis and tumour histology.     

Immunohistochemistry in apparently normal bone marrow trephine specimens from patients with nodal follicular lymphoma.

AIM--To establish the role of immunohistochemistry (using a limited panel of antibodies) in detecting minimal involvement by follicular lymphoma in routinely processed bone marrow trephine specimens, which show no obvious morphological (light microscopic) evidence of lymphoma; to determine whether bcl-2 immunostaining in bone marrow distinguishes between benign and malignant infiltrates in a patient with nodal follicular lymphoma. METHODS--Twenty seven consecutively selected paraffin wax embedded, formalin fixed bone marrow trephine specimens were stained with the following antibodies: anti-bcl-2, anti-CD79a, anti-CD3, and kappa and lambda light chains, using the Streptavidin biotin complex technique. RESULTS--Five of the 27 cases, which showed no evidence of involvement by follicular lymphoma on routine stains, showed monotypic B cells on immunohistochemistry. Two of the cases were diffuse, while the remaining three showed mini-aggregates around bony trabeculae. In all five cases the lymphomatous infiltrates were strongly bcl-2 positive. Reactive B lymphoid nodules did not show the same degree of bcl-2 positivity, and negative cells could be discerned within the reactive nodules. CONCLUSIONS--There is merit in studying so-called negative bone marrows immunohistochemically in order to detect minimal involvement by follicular lymphoma. A limited panel of antibodies including anti-bcl-2, anti-CD79a and anti-CD3 is usually adequate to accomplish this. Strongly bcl-2 positive lymphoid aggregates in the bone marrow of patients with nodal follicular lymphoma are indicative of lymphoma.     

Angiogenesis in pituitary adenomas and the normal pituitary gland

Angiogenesis is essential for tumor growth beyond a few millimeters in diameter, and the intratumoral microvessel count that represents a measure of angiogenesis has been correlated with tumor behavior in a variety of different tumor types. To date no systematic study has assessed pituitary tumors of different secretory types, correlating vascular count with tumor size. The vascular densities of pituitary tumors and normal anterior pituitary were therefore assessed by counting vessels labeled using the vascular markers CD31 and ulex europaeus agglutinin I. One hundred and twelve surgically removed pituitary adenomas (30 GH-secreting, 25 prolactinomas, 15 ACTH-secreting, and 42 nonfunctioning tumors) were compared with 13 specimens of normal anterior pituitary gland. The vascular counts in the normal anterior pituitary gland were significantly higher (P < 0.05) than those in the tumors using both CD31 and ulex europaeus agglutinin I. In addition, microprolactinomas were significantly less vascular (P < 0.05) than macroprolactinomas, although there was no such difference between vascular densities of microadenomas and macroadenomas producing GH. ACTH-secreting tumors were, like microprolactinomas, of much lower vascular density than the normal pituitary and other secreting and nonsecreting tumor types. In marked contrast to other tumors, pituitary adenomas are less vascular than the normal pituitary gland, suggesting that there may be inhibitors of angiogenesis that play an important role in the behavior of these tumors.