Growth Hormone in Vascular Pathology: Neovascularization and ...

ANTICANCER RESEARCH 27: 4201-4218 (2007)

Growth Hormone in Vascular Pathology: Neovascularization and Expression of Receptors is Associated with Cellular Proliferation D.T. LINCOLN1, P.K. SINGAL2 and A. AL-BANAW3 1Entity

Systems, Independent Research Foundation, 53 Ashburton Street, Chapel Hill QLD 4069, Australia; 2Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Center, Faculty of Medicine, University of Manitoba, Winnipeg, Canada; 3Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Kuwait University, Kuwait

Abstract. Vascular tumours are common lesions of the skin and subcutaneous tissue, but also occur in many other tissues and internal organs. The well-differentiated tumours consist of irregular anastomosing, blood-filled vascular channels that are lined by variably atypical endothelial cells. The less differentiated tumours may show solid strands and sheets, resembling carcinoma or lymphoma. Several growth factors, including basic fibroblast growth factor, transforming growth factors and vascular endothelial growth factor, play a role in tumour angiogenesis. Growth hormone (GH) is mitogenic for a variety of vascular tissue cells, including smooth muscle cells, fibroblasts and endothelial cells and exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific membrane-bound receptors, which trigger a phosphorylation cascade resulting in the modulation of numerous signalling pathways and of gene expression. Essential to the initiation of a cellular response to GH, the presence of receptors for this hormone may predict the adaptation of tumour cells resulting from GH exposure. To address the site/mode of action through which GH exerts its effects, a well characterized monoclonal antibody, obtained by hybridoma technology from Balb/c mice immunized with purified rabbit and rat liver GH-receptor (GHR) and directed against the hormone binding site of the receptor, was applied, using the ABC technique to determine GHR expression in a panel of vascular tumours. The GHR was cloned from a rabbit liver cDNA library with the aid of an oligonucleotide probe based on a 19 residue tryptic peptide sequence derived from 5900 fold purified rabbit liver receptor. A total of 64 benign and malignant vascular tumours were obtained from different human organ sites, including the chest wall, skin, axillary

Correspondence to: David T. Lincoln, Entity Systems, Independent Research Foundation, 53 Ashburton Street, Chapel Hill, QLD. 4069. Australia. Tel/Fax: +61 738782417, e-mail: [email protected] Key Words: Vascular tumours, growth hormone, growth hormone receptor, cellular proliferation.

0250-7005/2007 $2.00+.40

contents, duodenum, female breast, abdomen, stomach, colon, lymph node, bladder, body flank and neck regions. The tumours were of the following pathological entities: Haemangioma (n=12); haemangioendothelioma (n=10); Castleman’s disease (n=3), haemangiopericytoma (n=4); angiosarcoma, (n=11), Kaposi’s sarcoma with focal infiltration by lymphoma, HIV +ve (n=7), Kaposi’s sarcoma (n=17). The endothelial cell marker CD-31 was used to establish endothelial cell characteristics and microvascular density. To delineate tumour cell growth, immunohistochemical analysis of cycling nuclear protein and of proliferating cell nuclear antigen, using Ki-67 and PCNA polyclonal antibodies respectively, was used to demonstrate proliferative indexes. Results show that, compared to their normal tissue counterparts, nuclear and cytoplasmic expression of GHR consistently result in strong receptor immunoreactivity in the highly malignant angiosarcomas and Kaposi’s sarcomas and was localized in the cell membranes and cytoplasm, but strong nuclear immunoreactivity was also identified. The presence of intracellular GHR is the result of endoplasmic reticulum and Golgi localization. Nuclear localization is due to identical nuclear GHR-binding protein. Furthermore, there was a positive correlation of GHR immunoreactivity with neoplastic cellular proliferation and cycling, as measured by Ki-67 and PCNA. In conclusion, this study shows that GHR expression in vascular tumours is a function of malignancy and cancer progression. Malignant cells, which are highly expressive of the receptor, have a greater proliferation rate and thereby also higher survival rate compared to tumours expressing lower or minimal receptor level. The presence of GHR in endothelial cells of vascular neoplasm indicates that they are target cells and GH is of importance in the proliferation of vascular tumour angiogenesis. GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion and maintenance. The results support the hypothesis that GH is involved in the paracrine-autocrine mechanism, acting locally in regulating vascular tumour growth and will be useful for sitespecific studies of the evolution of vascular cancers. The use of anti-GHR antibodies to block tumour progression is an intriguing possibility.

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ANTICANCER RESEARCH 27: 4201-4218 (2007) Vascular tumours are common lesions of the skin and subcutaneous tissue, but also occur in many other tissues and internal organs. The well differentiated tumours show irregular anastomosing, blood-filled vascular channels, lined by variably atypical endothelial cells. The less differentiated tumours may show solid strands and sheets, resembling carcinoma or lymphoma. The tumours are a heterogenous group of neoplasms, ranging from very benign haemangiomas, to intermediate haemangioendothelioma lesions that are locally aggressive but infrequently metastasize, to highly malignant angiosarcomas and Kaposi’s sarcomas. In general, vascular neoplasms are divided into benign and malignant tumours on the basis of two major anatomical characteristics, namely the degree to which the neoplasm is composed of wellformed vascular channels, and secondly the abundance and regularity of the endothelial cell proliferation. Benign vascular tumours are made up largely of well-formed vessels with a significant amount of regular endothelial cell proliferation, whereas, on the opposite of the spectrum, the malignant tumours are solidly cellular and anaplastic, with scant numbers of only abortive vascular channels. The endothelial nature and the angiogenic profile of the neoplastic proliferations that do not form distinct vascular lumina, can be identified with the aid of immunocytochemical techniques using monoclonal antibodies (MAbs) against adhesion molecules such as CD31, associated with platelet adhesion in inflammation and wound healing. The ability of vascular cells to respond appropriately to extracellular stimuli is essential to their growth and survival in vivo, as well as to the growth, health, and survival of the organism. Growth factors and other soluble polypeptides bind to their respective receptors on the cell surface, triggering a variety of signal transduction pathways that often involve tyrosine phosphorylation of the receptor or other intracellular proteins. The activation of these signalling cascades then leads to the stimulation or repression of specific genes in the nucleus, thus linking external stimuli to the cell’s genetic machinery. A number of growth factors play important roles in the regulation of vascular tissue cell growth and differentiation. Thus, polypeptide epidermal growth factor (EGF) promotes the growth of epithelial cells, platelet-derived growth factor (PDGF) stimulates the growth of fibroblasts and smooth muscle cells and vascular endothelial growth factor (VEGF) stimulates the growth of endothelial cells and plays a mayor role in angiogenesis, the process by which a new capillary supply is formed in developing tissue. Furthermore, tumour cells, have been found to release angiogenic factors, including basic fibroblast growth factor (bFGF), transforming growth factor ‚ (TGF-‚) and VEGF, that may also play an important role in angiogenesis. Many angiogenic growth factors have multiple functions and are not only inducers of angiogenesis, but also act as growth

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factors and cytokines, stimulating non-endothelial cells. These molecules are produced by tumour cells or tumourinfiltrating lymphocytes and can also be secreted by the extracellular matrix. A tumour can directly stimulate endothelial cells by secreting inducers, such as VEGF or hepatocyte growth factor (HGF), that specifically bind to endothelial cell receptors. On the other hand, stimulation can occur indirectly and less specific in that the tumour secretes enzymes which release growth factors, including bFGF, that are stored in the extracellular matrix. A number of growth factors (GF), including insulin, VEGF, PDGF and EGF, acting by binding to the cell surface receptors, are high-affinity ligands for transmembrane receptors belonging to the family of receptor tyrosine kinases (RTKs) and play a role in human disease, including developmental disorders of cancer (1). Each type of ligand binds to the extracellular domain of its own specific receptor. Growth hormone (GH) is also mitogenic for a variety of vascular tissue cells, including smooth muscle cells, fibroblasts, adipocytes, macrophages, lymphocytes, endothelial cells and exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific membrane-bound receptors. It modulates the synthesis of multiple mRNA species, including that of insulinlike growth factor-1 (IGF-1), facilitating paracrine/autocrine interactions in mammalian tissues. The original somatomedin hypothesis suggested that the mitogenic growth promoting effects of GH were mediated by circulating serum factors known as somatomedins or insulin-like growth factors (IGFs), produced by the liver in response to GH, the latter being synthesized in the pituitary gland. However, evidence from animals and developing humans has clearly demonstrated that IGF-1 is also widely synthesized locally in many tissues and the concept, that it may be directly regulated by local action of GH to promote both stem cell differentiation and proliferation in vitro and in vivo became established. Green et al. (2) have proposed a "dual effector" hypothesis of GH action, in that GH promotes differentiation of stem cell precursors, whereas IGF-1 is required for their subsequent clonal expansion and the precise action of GH depends critically on the differentiation stage of the target cells (3). According to this model, the metabolic actions are predominant in differentiated cells. The differentiative action which renders cells competent to proliferate would be predominant in undifferentiated stem cells. Differentiated and undifferentiated target cells might differ with respect to receptor structure and or coupling of metabolic pathways to the receptor, just as target cells in different tissues might differ in these respects. However, IGF-1 is also produced at multiple extrahepatic sites and it was subsequently proposed that IGF1 might act locally in target tissues (4, 5). Both IGF-1 and IGF-1 mRNA (6) show GH dependence in many extrahepatic tissues. A local response to chemical mediators such as growth

Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours

factors or hormones and generally referred to as cytokines, requires the presence and subsequent binding to appropriately located receptors for cellular growth regulation, differentiation and specific functions by interacting with their cognate receptors. Growth hormone has a major effect on transcription, and this may be independent of the rapid chemical events triggered by ligand binding to plasma membrane-associated GHR (7). The multiplicity of actions of GH are mediated by an array of signals triggered by the activated GHR. These include among others control of postnatal growth, hepatic metabolism, fertility and immune functions. Postnatal growth is largely controlled by IGF-1, generated by activation of signal transducers and activators of transcription (STATs). The importance of the STAT family as mediators of cell signalling for a broad spectrum of cytokines and growth factors is becoming increasingly evident, as the ability of STATs to function as both cytosolic second messengers and nuclear transcription factors is better understood. As biologic proof of the importance of STATs in regulating cellular growth, survival and differentiation, inappropriate STAT activation is found in a growing number of haematological and epithelial cancers. The GHR was the first of the class 1 cytokine receptors cloned (8) and the immunocytochemical identification and localization of specific receptors for GH in human vascular tumours, which to our knowledge has hitherto not been demonstrated, would almost certainly indicate the likely target tissues for biological action and role of this receptor in vascular tumour growth. Essential to the initiation of a vascular response to GH, the presence of receptors for this hormone may predict the neoplastic adaptation of vascular endothelial cells resulting from GH exposure. To address the side/mode of action through which GH exerts its effects on vascular neoplasms, a well characterised monoclonal antibody (9-27), directed against the hormone binding site of the receptor (28) and cloned from a rabbit liver cDNA library with the aid of an oligonucleotide probe based on a 19 residue tryptic peptide sequence derived from 5900-fold purified rabbit liver receptor, was applied using immunohistochemical techniques to determine GHR expression in a panel of human vascular tumours, including haemangioma, haemangioendothelioma, Kaposi’s sarcoma, Kaposi’s sarcoma with focal infiltration by lymphoma (HIV +ve), Castleman’s disease, haemangiopericytoma and angiosarcoma.

Materials and Methods Tumour procurement and preparation. Vascular tumour tissues, collected at surgery, were obtained from the Mubarek Al-Kaber Hospital, Jabriya, Al-Amiri Hospital, Safat and the Hussain Makki Al-Jummaa Cancer Centre, Shuwaikh, Kuwait, Arabian Gulf. The age of the patients ranged from 16 to70 years with median age 47 years. Tissue biopsies from a total of 64 patients (44 males and 20

females) with benign and malignant vascular tumours, diagnosed in the period between January 1992 and December 2003, were obtained from different organ sites, including the chest wall, skin, axillary mass tissue, duodenum, female breast, abdomen, oral cavity, liver, stomach, colon, ano-rectum, lymph nodes, bladder, body flank, trunk and neck region. The tumours were of the following histological entities: haemangiomas; (n=12); haemangioendothelioma (n=10); Kaposi’s sarcoma, (n=17); Kaposi’s sarcoma with with focal infiltration by lymphoma, HIV +ve (n=7); Castleman’s disease (n=3); angiosarcomas (n=11); haemangiopericytoma (n=4). Laboratory data recorded included presence of lymphocytes, levels of interleukin 6, history of immunodeficiency (HTLV-1), human immunodeficiency virus status and hepatitis screening. Tissues were fixed in phosphate-buffered 4% paraformaldehyde (ph 7.4) overnight at 4ÆC, washed in several changes of Tris buffer at pH 7.4, routine processed and embedded in paraffin wax. Sections were cut at 5 Ìm thickness and mounted on 2% aminopyroethylsaline (APS)coated microscope slides. Routine haematoxylin-eosin stained sections were evaluated in all cases to assess general pathomorphological features. Positive controls were prepared using paraffin wax embedded tissues sections with proven good reactivity. Negative controls were performed by substituting the respective primary antibody by nonimmune serum. Clinicopathological evaluation of vascular tumours. Haemangiomas are benign vascular neoplasm that do not metastasize and often occur in the skin and mucosal surfaces of the body, but are also found in many viscera, particularly the liver, spleen, pancreas. The lesions were primarily composed of capillaries (capillary haemangioma) or widely dilated veins forming large cavernous vascular channel (cavernous haemangioma). Haemangioendothelioma is an endothelial cell neoplasm of intermediate (borderline) malignancy and follows the pattern of distribution of the haemangiomas. Three histological subgroups include epithelioid, spindle cell and malignant endovascular haemangioendothelioma. In this investigation, all cases presented with neoplasms of the skin. Most of the malignant primary angiosarcomas were multicentric and appeared as dark red nodular elevations of the skin and as axillary mass. Others were located in the breast of a 44 years old women, in the anorectal region and the liver of elderly patients. Clinically, the majority of the Kaposi’s sarcomas located in the skin were manifested by multiple purpuric plaques in the distal portions of the extremities, later becoming darker, more nodular, and eventually ulcerated. In addition to skin lesions, the tumours were also located in the stomach, duodenum and colon. Some patients with Kaposi’s sarcoma also develop lesions in lymph nodes. The four cases of haemangiopericytomas investigated were obtained from the abdomen, neck and trunk. Although haemangiopericytomas have a high incidence of recurrence and metastasis, all four cases were primary tumours. In Castleman’s disease, non-cancerous growths (tumours) develop in lymph node tissue the the body. The disease affects both males and females and may occur at any age, but typically it does not affect children. Three types of Castleman’s disease have been identified. These are hyalinevascular type, plasma cell type, and multicentric or generalized (MCD). The three cases investigated were all axillary lymph nodes cases involving a 55 year old female with plasma cell type and a 70 year old male having multicentric type of Castleman’s disease with a focus consistent with Kaposi’s sarcoma. The latter patient had an elevated level of immune factor interleukin-6 and exhibited an abnormally large liver.

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ANTICANCER RESEARCH 27: 4201-4218 (2007) Microvascular density. The optimal tissue block for analysis of vascular density was chosen by microscopic examination of all histological sections performed for pathologic assessment of the tumours. Serial sections from formalin-fixed and paraffin wax embedded vascular tumour tissues were cut at 4 Ìm, mounted on poly-L-lysine coated slides and dried overnight at 37ÆC. Sections were deparaffinised with three changes of xylene, treated in two changes of alcohol for 5 minutes, to be followed by treatment with 0.3% H2O2 in methanol for 20 minutes at room temperature to block endogenous peroxidase in tissue sections. Sections were washed in distilled water, followed by washing in three changes of Tris-buffer (pH 7.6) and subsequent treatment with blocking reagent (Tris-buffered saline (TBS) containing normal goat serum) for 30 minutes at room temperature (RT) to eliminate non-specific staining with the primary antibody, by inhibiting the non-specific binding. To improve immunostaining pattern, antigen retrieval was performed by treating tissue sections with 0.05% Trypsin for 15 minutes at 90ÆC and allowing to cool to RT in the same solution for 20 minutes, before incubation with the primary antibody against CD-31 (mouse monoclonal antibody clone F8/86, Dako, Denmark) at 1:50 dilution for 1 hour at RT in a humidified chamber. Sections were than incubated with biotinylated secondary antibody to mouse/rabbit immunoglobulins for 30 min at RT and subsequently incubated with streptavidin and biotinylated peroxidase complex (4 ml streptavidin buffer with 1 drop of streptavidin concentrate peroxidase conjugated) for 30 minutes at RT. Visualization was by incubating tissue sections at RT with 1 mM 3-amino-9-ethylcarbazole (AEC) chromagen containing 0.015% hydrogen peroxide in 0.1 M acetate buffer, pH 5.2 for 5 minutes. Between each incubation step, the sections were extensively washed with 0.1 M TBS. Immunostaining of vascular structures in lymph node tissue section was used as a positive control and a negative control was obtained by omission of the primary antibody in one section. With AEC as the chromogen, sections were washed with distilled water and mounted in aqueous mounting medium. Proliferative indices. Immunohistochemical analysis of nuclear antigen-associated cell proliferation was used to demonstrate levels of Ki-67 and of proliferating cell nuclear antigen (PCNA). Polyclonal anti-proliferating cell nuclear antigen (PCNA) antibody, clone 19A2 and of IgM isotype, and the polyclonal Ki-67 primary antibody in the form of ascites fluid were obtained from Dakopatts (Denmark). Prior to immunostaining, the Ki-67 specimen sections were subjected to microwave oven treatment. Tissue sections in citrate buffer (pH 6.0) were placed in the microwave oven for two cycles of 15 minutes each at 600 Watts. Sections were allowed to cool to RT and washed well. The presence of Ki-67 and PCNA antigen was demonstrated immunohistochemically by the peroxidase, anti-peroxidase (PAP) method and all incubation reactions were carried out at RT unless otherwise stated. Phosphate buffered formalin-fixed and paraffin wax embedded tissues sections of 4 Ìm in thickness were deparaffinised through xylene and hydrated by treatment in graded series of alcohols for 5 minutes each, followed by the removal of endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol for 30 min. Following extensive washes with phosphate-buffered saline (PBS), pH 7.2, a 2% normal goat serum was applied for 30 min and the sections were incubated overnight with primary anti Ki-67 and/or anti-PCNA antibody, diluted 1 in 50 in PBS at 4ÆC in humidified chambers. The

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sections were extensively washed and incubated with linked antibody solution for 60 minutes. Subsequent incubation with PAP reagent was carried out for 45 min, after extensive washes with PBS. Visualization of the peroxidase was by application of 0.05% 3,3 diaminobenzidine (DAB) tetrahydrochloride containing 0.09% hydrogen peroxide in Tris-buffer for 5-10 minutes. In some cases, visualization with 3-amino-9-ethylcarbazole (AEC) as chromogen (Dako, Denmark) in 0.1 M acetate buffer, pH 5.2 for six minutes or till a red nuclear reaction product had formed. The sections were rinsed in PBS, nuclei lightly counterstained in haematoxylin, blued in alkaline tap water and finally dehydrated in ascending series of alcohols, cleared in three changes of xylene and mounted with DePex (DAB) or sections were washed in tap water only before mounting in aqueous mounting medium (AEC). Production and characterization of monoclonal antibody to growth hormone receptor. MAb 263 (IgG1 k isotype) was prepared by immunization of mice with purified rat and rabbit GHR as the antigen. This MAb is directed against the hormone binding site of the human receptor, it recognizes a cross-species determinant with high affinity, also present on the human receptor (29). MAb 263 is a reactive agent against the GHR in a number of species with high affinity and does not react with insulin or prolactin receptors in a rabbit or rat liver. Under certain conditions MAb 263 precipitates rat and rabbit GHR, although it can also compete for hormone binding to subtypes of the GH receptor. This antibody inhibits 50% of human growth hormone binding to GHR in rat liver microsome preparations. For histochemical uses the antibody was purified from ascitic fluid using 1) ammonium sulphate precipitation and dialysis, 2) protein-A sepharose affinity chromatography or 3) protein-A chromatography followed by ammonium sulphate precipitation and then dialysis. Control MAb’s (anti-Brucella abortus and Heartworm) were of the same isotype. Growth hormone receptor immunohistochemistry. The presence of GHR was demonstrated immunohistochemically by the streptavidin-biotin horseradish peroxidase complex (ABC) technique. Endogenous peroxidase activity was eliminated by incubation with 0.5% hydrogen peroxide in absolute methanol for 15 minutes at room temperature (RT). Non-specific protein binding was blocked by incubation with 20% normal goat serum in PBS containing 1% bovine serum albumin (BSA) for 1 hour at RT. Deparaffinized tissue sections of 4 Ìm thickness were incubated 1) overnight at 4ÆC with primary antibody (MAb 263 at 12.5 Ìg/ml in PBS-1% BSA); 2) with biotinylated goat anti-mouse IgG for 1 hour at RT (1:200 in PBS-1% BSA); 3) with streptavidin-biotin horseradish peroxidase complex (1:250 in PBS-1% BSA) for 1 hour at RT. Between each step, sections were washed several times in PBS-1% BSA. Visualisation was with 1 mM 3-amino-9ethylcarbazole, containing 0.015% H2O2 in 0.1 M acetate buffer, pH 5.2 for 5 minutes. Controls were performed by a) replacing the anti-GHR MAbs with unrelated heartworm MAb 50.6 and/or Brucella abortus MAb at the same or greater concentration; b) substituting the anti-GHR MAbs with normal mouse serum at different concentrations; c) omission of the primary antibody; d) pre-incubation for 2 hours at 20ÆC with serial dilutions of recombinant GH (Genentech, USA) at 1, 5, 10, 20 and 30 Ìg/ml in PBS-10 mM MgCl2-1% BSA prior to incubation with primary monoclonal antibodies. All incubations were carried out in humidified chambers to prevent evaporation.


Quantitative analysis. The tumour sections were scanned in the entire area with a counting grid and for each field the number of points in coincidence with the area to be measured was recorded (Point count method). The quantitative analysis of PCNA, Ki-67, CD-31 and GHR on paraffin sections was done by calculating the percentage of stained cells in 10 low power magnification fields or 16 high power fields using a Zeiss microscope connected to a computer using image analysis software. To ensure reproducible and objective assessment of staining, 10 representative areas, each containing 1000 tumour cells, were observed under high-power field (objective lens, X 40) in a vertical section taken from the centre of the lesion. The proportion of positive cells was expressed as percentage of total cells counted. Estimation of PCNA, Ki-67 and GHR was as follows: + = labelling of great majority; +(–) = labelling of great majority, some negative; +/– = positive labelling is between 50-70%; –/+ = more than 50% are negative; –(+) = more than 90% are negative; – = no labelling. Intra-tumoural microvessel density was subjectively assessed by light microscopic analysis. Counting of microvessels was performed without knowledge of the stage, grade or clinical information. Each section was scanned at low magnification (x100) to identify the areas with the greatest density of microvessels ("hot spots"). A Chalkley point graticule was used for counting individual microvessels, as delineated by the stained endothelial cells. Microvessel count was then performed at 100x, 200x and 400x magnification (10x, 20x and 40x objective and 10x ocular) on 10 different fields (1.0 mm2). To facilitate the counting, a 1 mm2 gradicle divided into 100 squares and fitted into the eye piece, was used. The result was expressed as the highest number of microvessels identified within a single field. The mean of the Chalkley counts for each tumour was calculated and used in the statistical analysis. In accordance with published procedures, the maximum number of microvessels staining positive at x200 was graded using the following scoring system: up to 25 vessels = 1+, 26-50 vessels = 2+, 51-75 vessels = 3+, 76-100 vessels = 4+, while >100 vessels was graded 5+.

Results Histopathology of vascular tumours. Histologically, the haemangiomas investigated were well defined and not encapsulated. In patients who presented with lesions of the skin, the tumours were made up of closely packed aggregations of thin-walled capillaries (capillary haemangioma), at times partly filled with red blood cells and separated by scant connective tissue stroma. The lumina had signs of intra-vascular thrombosis or vessel rupture showing scarring and occasional haemosiderin pigment. The tumours obtained from the liver and oral cavity (Table I) consisted of spongy tissue mass, made up of large, cavernous, vascular spaces (cavernous haemangiomas) that were partly or completely filled with fluid and blood and separated by a scant connective tissue stroma. All haemangioendotheliomas obtained in this investigated were from skin tissue. The lesions consisted of vascular tumours composed largely of an irregular aggregate of vascular channels of varying size with an endothelial cell lining, in addition to the presence of dominant masses and sheets of spindle-shaped cells. The

endothelial cells ranged from bland to focally more plump, somewhat larger and atypical forms that were polygonal to spindle shaped, with individual cells showing mitotic figures and intracellular cytoplasmic vacuoles, producing a slight pleomorphism to the cell pattern. Varying degrees of intervening collagenous stromal fibrous tissue and focal area of myxoid stromal change was present. The immunohistochemical profile confirmed the endothelial and vascular nature of the lesion with both the intervening as well as vascular space lining cells were positive for the endothelial cell marker CD-31. The cases of haemangiopericytomas investigated displayed variation of cytological and histological features of branching networks of capillaries surrounded by clumps, trabeculae, or sheets of polygonal and spindle cells, thought to be derived from the vascular pericyte. The tumour obtained from the abdomen of a female patient (Table I) was composed of a variety of cells, some of which contained cytoplasmic fibrillar bundles similar to those found in smooth muscle and glomus tumour cells, pinocytic vesicles similar to those in endothelial and smooth muscle cells and long cytoplasmic projections as in the pericytes surrounding capillaries. Silver reticular fibres stain revealed that the tumour cells were surrounded by varying amounts of basement membrane material. All three axillary lymph node cases of Castleman’s disease investigated presented with marked follicular dendritic cell hyperplasia and prominent vascular proliferation. There was no clear difference in histological appearance in the case of the female patient to distinguish between solitary and multicentric types. The two axillary lymph node cases from a 70 year old male patient were of multicentric, plasma cell type, displaying characteristic ‘Kaposi-like lesions’ with mononuclear cells scattered in inter-follicular areas and follicular dendritic cell dysplasia. In addition, the adhesion molecule interleukin 6 and the Kaposi’s sarcoma herpes virus (KSHV) were also present. Patients who presented with angiosarcoma had localizations in a variety of organs (Table I). The skin (45%) was the site most frequently involved. Bulky disease was found in 27% of the patients. Histologically the tumours show a wide spectrum of differentiation, ranging from Grade 1 to Grade 3. The well differentiated tumours showed irregular anastomosing, blood-filled vascular channels, lined by variably atypical endothelial cells with display of papillary intra-luminal tufting. The less differentiated tumours had solid strands and sheets, resembling carcinoma or lymphoma. Immunohistochemically, the cells strongly expressed the endothelial marker CD-31. Of the 24 Kaposi’s sarcoma cases investigated, 29% were HIV +ve with focal infiltration of the skin lesions by lymphoma (Table I). Histologically, the early plaque stage lesions consisted of thin-walled, dilated vascular spaces in the epidermis, with interstitial inflammatory cells. The older nodular lesions consisted of

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ANTICANCER RESEARCH 27: 4201-4218 (2007) Table I. Expression of PCNA, Ki-67, GHR and MVD in vascular tumours. Tumour Haemangioma 03-617B 02-393C#3 02-465A#1 98-1069A 97-848#1 97-858C#1 96-1047 94-612A#1 93-435A#2 93-860 92-23A 92-125B#1 Haemangioendothelioma 02-2991 01-2564 99-887A 98-2111 97-2412B 96-4025A 96-4025B#1 93-2398B 92-600E#2 92-600E#4 Kaposi’s sarcoma 02-8577#1 02-8870#1 02-8870#2 01-7363 01-8193 97-2033 97-2657A 97-2657B 95-1797 95-2691B 94-209 94-368 93-245 92-1937 92-1960#1 92-1960#2 92-2072 Kaposi’s sarcoma with lymphoma 99-6549 97-7200 96-229#2 95-393 94-131 92-235A 92-235B Castleman’s disease 96-2790A#3 95-2691A#1 95-2691A#2

Tissue

Gender

Age

PCNA*

Ki-67*

GHR*

MVD**

Skin Skin Skin Skin Skin Oral cavity Liver Skin Skin Skin Skin Skin

F F F F M F M M F M M M

24 30 18 37 55 29 44 39 16 28 25 19

–/+ –/+ –(+) –/+ –(+) +/– +(–) –(+) –(+) –/+ –(+) –(+)

–(+) –(+) – –(+) – –(+) –/+ – –(+) –(+) – –(+)

–/+ –/+ –(+) –/+ –(+) +/– +/– –/+ –(+) –/+ –/+ –/+

3+ 2+ 2+ 2+ 2+ 2+ 3+ 3+ 2+ 3+ 3+ 2+

Skin Skin Skin Skin Skin Skin Skin Skin Skin Skin

M M M M F M M F F F

45 51 22 65 41 39 39 30 48 48

–/+ –/+ +/– –/+ +/– +/– +/– –(+) +/– +/–

–(+) –(+) –/+ –(+) –/+ –/+ –(+) – –/+ –/+

–/+ –/+ +/– –/+ –/+ +(–) +/– –/+ +/– +/–

2+ 2+ 3+ 3+ 2+ 4+ 3+ 2+ 3+ 3+

Skin nodule Skin Skin Skin nodule Skin Skin Skin Skin Skin Skin Skin Duodenum Skin plaque Skin Stomach Stomach Colon

F M M F M M M M M M F F M M M M M

41 40 40 46 38 60 63 63 52 70 45 45 65 43 56 56 37

–/+ +/– –/+ –(+) +/– +/– +/– +/– +/– –(+) –(+) +/– –(+) –/+ +(–) –(+) +/–

–(+) –/+ –(+) –(+) –/+ –(+) –/+ –/+ –/+ –(+) –(+) –/+ –(+) –(+) –/+ –(+) –/+

–/+ + –/+ –/+ +(–) + + + + –(+) –/+ + –/+ +/– +/– –/+ +

2+ 4+ 2+ 2+ 2+ 3+ 3+ 3+ 4+ 1+ 2+ 5+ 2+ 3+ 4+ 2+ 5+

Skin, HIV+ve Skin, HIV+ve Skin, HIV+ve Skin, HIV+ve Skin, HIV+ve Skin, HIV+ve Skin, HIV+ve

M M M M M M M

27 19 25 37 29 44 44

+(–) +/– +/– –/+ –/+ –/+ –/+

+/– –(+) –(+) –(+) –/+ –/+ –(+)

+(–) + + +(–) +(–) +(–) +/–

4+ 3+ 5+ 2+ 2+ 4+ 3+

Lymph node Lyymph node Lymph node

F M M

55 70 70

–/+ +/– +/–

–(+) –/+ –/+

+/– +/– +/–

3+ 3+ 3+ Table I. continued

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Table I. continued Tumour Angiosarcoma 01-5548A7 98-4700#1 97-539 97-6023 97-2412A#1 97-7736#1 93-120E#1–3 93-120E#1-5 93-120 E#10 93-337A#3-2 Haemangiopericytoma 98-7684A#1 98-7684B 97-814A#1 96-868

Tissue

Gender

Age

PCNA*

Ki-67*

GHR*

MVD**

Flank, Grade I-II Liver Breast Skin Bladder Anorectal Skin, Grade III Chest, GradeIII Skin, Grade III Axillary mass

M M F M F M M M M F

50 67 44 38 42 64 63 63 63 59

–/+ +/– +/– –/+ –/+ +/– –/+ +(–) +/– +/–

–(+) –/+ –/+ –/+ –/+ –/+ –/+ –/+ –(+) –/+

+/– + +(–) +/– +/– +/– +/– + +/– +(–)

3+ 4+ 4+ 3+ 5+ 5+ 3+ 4+ 4+ 3+

Neck mass Neck mass Abdomen Trunk mass

M M F M

56 56 29 63

–/+ +/– + +/–

–(+) –/(+) –(+) –/+

–/+ +(–) + +/–

5+ 5+ 5+ 4+

*Labelling of proliferating cell nuclear antigen (PCNA), cycling nuclear protein (Ki-67) and growth hormone-receptor (GHR): + = labelling of great majority; +(–) = labelling of great majority, some negative; +/– = positive labelling is between 50-70%; –/+ = more than 50% are negative; –(+) = more than 90% are negative; – = no labelling. **MDV-microvascular density (CD-31+ve), the maximum number of microvessels staining positive at X200 was graded using the following scoring system: up to 25 vessels = 1+, 26-50 vessels = 2+, 51-75 = 3+, 76-100 = 4+, while >100 vessels was graded 5+.

interconnecting, often deceptively benign channels lined by neoplastic, cytologically atypical endothelial and spindleshaped cells. The immunoperoxidase staining demonstrated strong expression of the endothelial cell marker CD-31 antigen in both the endothelial and spindle-shaped cells. The neoplastic channels had infiltrated the surrounding structures, mostly following perivascular and perineural spaces. In the skin lesions, the neoplastic vascular channel infiltrate was around skin adnexa and between individual collagen fibres.

magnification using the following scoring system: up to 25 vessels = 1+, 26-50 vessels = 2+, 51-75 vessels = 3+, 76-100 vessels = 4+, while >100 vessels was graded 5+. The median grade score of the vascular tumours was as follows: 2.4 for haemangiomas; 2.7 for haemangioendotheliomas; 2.9 for Kaposi’s sarcomas; 3.3 for Kaposi’s sarcomas with lymphomas; 3.0 for Castleman’s disease; 3.8 for angiosarcomas and 4.7 for haemangiopericytomas. MVD was significantly higher in HIV +ve patients with skin nodules of Kaposi’s sarcomas displaying focal infiltration by lymphoma.

Microvascular density. Table I shows the clinicopathological characteristics and results from the CD-31 immunostaining, providing a measure of microvascular density (MVD) and yielding an ‘angiogenic index’ of the vascular tumours. CD31 immunoreactivity was present in the vascular endothelial cells of the larger blood vessels (arteries and veins) and in the endothelium of the capillaries. All other components of the vascular tumour tissue, including connective tissue stromal cells, lymphocytes, neutrophils, plasma cells, adipocytes, smooth muscle cells and nerve cells were CD-31 negative. MVD was not affected by age, gender or by previous medical conditions and treatment, including radiotherapy and did not show a uniform increase with worsening pathological stage or grade, but varied significantly in different categories of benign and malignant vascular tumours. Grading of microvascular density was done according to the maximum number of microvessels staining CD-31 positive at X200

Expression of cycling nuclear protein (Ki-67). Table I shows the clinicopathological characteristics and cycling nuclear protein (Ki-67) index obtained from counting a total of 1000 cells in each of 10 representative fields from each vascular neoplasm specimen investigated. Immunoreactivity of Ki-67 as a measure of cycling nuclear protein was present to varying degree in all tumour specimens. Staining was mostly confined to the nuclei of the tumour cells. Cells in mitosis at times also displayed weak staining in their cytoplasm. There was no Ki-67 immunoreactivity present in the nuclei of stromal, vascular and nerve cells in the normal tissue. Depending on the type of vascular tumour, the number of cells with Ki-67-positive nuclei varied greatly between labelling more than 90% negative and labelling of great majority of nuclei with some being negative. Among the benign haemangiomas, in 58% of cases the tumour cell nuclei were more than 90% Ki-67 negative, 33% displayed

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Figure 1. Growth hormone receptor (GHR) expression in malignant vascular tumours, using MAb 263 and visualisation with 3-amino-9-ethylcarbazole (AEC). Red staining indicates GHR immunoreactivity. A) Angiosarcoma, bladder biopsy. Female; 42 years of age. Intense GHR expression in tumour cells. Magn. x400. B) Angiosarcoma, chest wall biopsy, Grade III. Male; 63 years of age. Strong GHR expression in tumour cells. Magn. x1000. C) Angiosarcoma, axillary tissue biopsy. Female; 59 years of age. Intense GHR expression in tumour cells. Magn. x400. D) Angiosarcoma, axillary tissue biopsy. Female; 59 years of age. Intense GHR expression in nuclei and cytoplasm of tumour cells. Magn. x1000.

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Figure 2. Growth hormone receptor (GHR) expression in malignant vascular tumours, using MAb 263 and visualisation with 3-amino-9-ethylcarbazole (AEC). Red staining indicates GHR immunoreactivity. A) Kaposi’s sarcoma, skin biopsy; atypical proliferative vascular lesion. Male; 63 years of age. Intense GHR expression in tumour cells. Note GHR immunoreactivity in vascular endothelial cell. Magn. x1000. B) Kaposi’s sarcoma, stomach; antral biopsy. Male; 56 years of age. Strong GHR expression in cell membranes/cytoplasm of tumour cells. Magn. x400. C) Kaposi’s sarcoma; skin biopsy. Female; 45 yearsof age. Intense GHR expression in cytoplasm and nuclei of tumour cells. Note strong GHRimmunoreactivity in vascular endothelial cells. Magn. x400. D) Kaposi’s sarcoma; skin biopsy. Male; 38 years of age. Intense GHR expression in tumour cells. Magn. x1000.

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Figure 3. Growth hormone receptor (GHR) expression in malignant vascular tumours, using MAb 263 and visualisation with 3-amino-9-ethylcarbazole (AEC). Red staining indicates GHR immunoreactivity. A) Kaposi’s sarcoma; skin biopsy; nodule with focal infiltration by lymphoma. Male; 19 years of age. HIV +ve. Intense GHR expression in nuclei and cytoplasm of tumour cells. Magn. x400. B) Kaposi’s sarcoma; skin biopsy; nodule with focal infiltration by lymphoma. Male; 29 years of age. Intense GHR expression in nuclei and cytoplasm of tumour cells. Magn. x1000. C) Kaposi’s sarcoma; skin nodule. Female; 41 years of age. Inrense GHR expression in nuclei and cytoplasm of tumour cells. Magn. x1000. D) Kaposi’s sarcoma/lymphoma. Male; 25 years of age; AIDS patient, HIV +ve;skin lesion biopsy. Intense GHR expression in cytoplasm of tumour cells. Note strong GHR immunoreactivity in vascular endothelial cells. Magn. x1000.

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no labelling and in one case of haemangioma of the liver, the nuclei were more than 50% immune-positive. Of the ten benign haemangioendothelioma tumours investigated, in half of the cases more than 50% of nuclei were Ki-67 negative, while in 4 cases more than 90% of the nuclei were negative and one case displayed no labelling. Of the Castleman’s disease investigated, in two cases more than 50% of the tumour cell nuclei were Ki-67 negative and in one case less than 10% were immune-positive. Of the 24 malignant Kaposis sarcoma investigated, the tumour cell nuclei were more than 90% Ki-67 immune-negative in 13 cases (54%, mostly skin lesions) and in ten cases (42%) more than 50% of nuclei were negative, while one of the seven HIV +ve cases had Ki-67 positive labelling between 50-70%. Of the haemangiopericytomas investigated, in the majority of cases the tumour cell nuclei were more than 90% negative and in one case less than 50% of the nuclei were stained positive. In the great majority (82%) of malignant angiosarcomas cases, positive Ki-67 nuclear staining was less than 50%, while more than 90% tumour cell nuclei were Ki-67 negative in 18% of the tumours. Expression of proliferating cell nuclear antigen (PCNA). Table I shows the clinicopathological characteristics and proliferating cell nuclear antigen (PCNA) index obtained from counting a total of 1000 cells in each of ten representative fields from each vascular neoplasm specimen investigated. Results from this investigation demonstrate that overnight application of primary anti-proliferating cell nuclear antigen (PCNA) at a dilution of 1:50 dilution at 4ÆC overnight in humidified chambers revealed distinct nuclear labelling and low background with diaminobenzedine (DAB) as the visualizing agent. Somewhat superior staining results were achieved with 3-amino-9-ethyl carbazole (AEC) as the visualising chromogen. Proliferating cell nuclear antigen immunoreactivity was present in all vascular tumour cases investigated and the expression of PCNA immunopositive tumour cell nuclei varied from being more than 90% negative to a positive labelling of the great majority, with a variable proportion of stained cells in different vascular tumour types. A distinct to strongly positive staining intensity was present in the nuclei of these cells. The nuclei of normal tissue cells, including fibroblasts, vascular endothelial cells, adipocytes, smooth muscle cells, lymphocytes, neutrophills, Mast cells and plasma cells were mostly negative. Of the twelve benign haemangioma tumours investigated, in one case of the liver (8.3%) PCNA tumour cell nuclei displayed labelling of great majority with some negative. In haemangioma of the oral cavity, positive labelling was between 50-70%, while nuclear labelling in lesions of the skin were less than 50% positive in four cases and more than 90% negative in half of all cases investigated. Of the ten benign haemangioendothelioma tumours

investigated, positive nuclear labelling of between 50-70% of tumour cells was observed in 6 cases, while in four cases less than 50% of nuclei were PCNA positive and in one case nuclear PCNA staining was more than 90% negative. Of the 24 malignant Kaposis sarcoma investigated, PCNA labelling of the great majority of nuclei with some negative was in two cases (stomach and HIV +ve skin). Positive nuclear labelling of between 50-70% was observed in ten tumours (41.7% cases) and in seven cases (29.2%) less than 50% tumour cell nuclei were immune-positive, while in the remaining tumours (20.6% cases) less than 10% of nuclei expressed PCNA. Of the Castleman’s disease investigated (lymph nodes), in two tumours (66.7% cases) positive PCNA labelling of tumour cell nuclei was between 50-70% and staining in the remaining case was less than 50% of the nuclei. In two cases of the haemangiopericytomas investigated, labelling was between 50-70% of tumour cell nuclei, while in one case (abdomen) labelling of great majority of nuclei was observed; in the remaining case less than 50% nuclei expressed PCNA immunoreactivity. Of the eleven cases of angiosarcomas investigated, positive nuclear PCNA labelling of between 50-70% was present in approximately half (54.5%) of the malignant tumours; in four cases (36.4%) less than 50% of the tumour cell nuclei were stained, while in one case (skin, grade III) labelling, was of the great majority of tumour cell nuclei, with some negative. Expression of growth hormone receptors (GHR). MAb 263 consistently showed immunoreactivity in paraffin wax embedded vascular tumour tissues. Table I shows the clinicopathological characteristics and GHR expression, obtained from counting a total of 1000 cells in each of 10 representative fields from each vascular neoplasm specimen investigated. Immunoreactivity of MAb 263 as a measure of GHR expression was present to varying degree in all vascular tumour specimens and consistently showed prominent immunoreactivity in malignant Kaposi’s sarcoma and angiosarcoma tumour cells and to a lesser extend in the cells of the benign vascular neoplasms. Expression of the GHR was well marked in cell membranes, was predominantly cytoplasmic, but strong nuclear immunoreactivity was also apparent in many instances (Figures 1A-3D). Nuclear staining was heterogeneous, sometimes with an open chromatin pattern being more dominant (Figures 1D, 2D, 3B, 3C). Not all cells, displaying localization of the chromagen, possessed nuclear GHR immunoreactivity. The presence of intracellular GHR is the result of endoplasmic reticulum and Golgi localisation. Nuclear localisation is due to identical nuclear GHR/binding protein. Localized immunoreactivity was also present in the vascular endothelial cells of tumours (Figures 2A, 2C, 3D) obtained from the different organ sites and this supports the concept of a direct role for GH in endothelial

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ANTICANCER RESEARCH 27: 4201-4218 (2007) cell proliferation and angiogenesis during carcinogenesis. Of the twelve benign haemangioma tumours investigated, GHR expression in tumour cells was more than 90% negative in 3 cases (25%). In the majority of lesions (58.3%), immunoreactivity was more than 50% negative, while GHR expression was positive between 50-70% of tumour cells in the haemangiomas of the liver and oral cavity (16.7%). Of the ten benign haemangioendothelioma tumours investigated, positive GHR labelling of between 50-70% of tumour cells was observed in 4 cases, while in one case the great majority of tumour cells were immunopositive, with some cells negative. In half of all haemangioendothelioma investigated, GHR expression was less than 50% of the tumour cells. Of the 24 malignant Kaposis sarcoma investigated, GHR labelling of the great majority of tumour cells was in nine tumours (37.5%), while in 5 cases (20.5%) receptor labelling was of the great majority of tumour cells, with some negative. Positive GHR labelling of between 50-70% was observed in three of the malignant tumours (12.5% cases) and in 6 cases (25%) less than 50% tumour cell nuclei were immune-positive, while in only one tumour less than 10% of the tumour cells were positive. Of the Castleman’s disease investigated, GHR expression of vascular tumour cells was between 50-70% in all cases. In the haemangiopericytomas specimen from the abdomen, GHR expression was in the great majority of vascular tumour cells; receptor labelling of tumour cells in the trunk mass specimen was between 50-70%, while in the other two haemangiopericytomas specimen of neck mass tumours investigated, GHR labelling was in the great majority, with some tumour cells negative in one specimen and was more than 50% negative in the second trunk mass specimen. Of the eleven cases of angiosarcomas investigated, GHR expression of between 50-70% of the tumour cells was in approximately half (54.5%) of the malignant tumours; while receptor labelling of the great majority of tumour cells with some negative (27.3%) was in breast and axillary mass tissue specimen. In angiosarcomas of the liver and abdomen, GHR expression was present in the great majority of the tumour cells. The strong immunoreactivity exhibited with MAb 263 is concordant with the specificity of this antibody. The intensity of GHR immunoreactivity, but not the location, was found to be dependent on the antibody preparation technique. The most intense immunoreactivity was observed with ascitic fluid precipitated with ammonium sulphate and dialysed. Protein-A purification resulted in a decrease in immunoreactivity, a degree of which was restored by ammonium sulphate precipitation and dialysis subsequent upon protein-A purification. Use of protein-A purified and proteinA/ammonium sulphate precipitated antibody at a four-fold concentration (5 mg/ml; 1:5 dilution) resulted in comparable staining to ascitic fluid/ammonium sulphate precipitated antibody. Tissue sections incubated with 20% normal serum, without the primary monoclonal antibody or with unrelated

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primary antibodies of identical IgGIK isotype (Brucella abortus, Heartworm MAb 50.6) at the same concentration as MAb 263, did not display detectable immunoreactivity. Pretreatment of tissue sections with recombinant human growth hormone (hGH) before application of the primary antibody (MAb 263) abolished staining both in the cell membrane/cytoplasm and the nucleus. On serial dilutions of GH at 1, 5, 10, 20 and 30 Ìg/ml, graded levels of immunoreactivity were observed. Immunoreactivity was reduced after pre-absorption at 5 Ìg/ml followed by incubation with MAb 263 and was absent with pre-incubation at 20 to 30 Ìg/ml. Immunoreactivity was more prominent in sections when pre-incubated with 5 Ìg/ml recombinant hGH followed by MAb 263, instead of the control monoclonal antibodies Brucella abortus or Heartworm 50.6.

Discussion Vascular tumours, composed of endothelial cells of blood and lymph vessels, are very common lesions of the skin and subcutaneous tissue, but they also occur in many other tissues and in internal organs. Although most vascular tumours are benign, low-grade malignant tumours such as haemangioendothelioma and highly malignant angiosarcoma and Kaposi’s sarcoma are common. Most benign vascular neoplasms (haemangiomas) arise in the skin and subcutaneous tissues, but no organ or tissue is exempt. Haemangiomas are common benign vascular neoplasm that do not metastasize, they occur in all age groups and may present in many clinical and pathological forms. Haemangioendothelioma is a true neoplasm of vascular origin, composed predominantly of masses of endothelial cells growing in and about vascular lumen (30) and represents an intergrade between the well-differentiated haemangiomas and the frankly anaplastic, totally cellular haemangiosarcoma (angiosarcoma). It follows the pattern of distribution of the haemangiomas and is most frequently encountered in the skin, but may affect the liver and the spleen. Haemangiopericytomas are primarily tumours of adults. In this investigation, the youngest patient was a 29 year old female and the oldest a male of 63 years. Large tumours (6.5 cm), tumours with foci of necrosis and haemorrhage, and those with increased mitotic rate, cellularity and anaplasia are more likely to exhibit malignant biological behavior The cytological variation of haemangiopericytomas is a reflection of the property of the pericyte to act as a precursor cell for the formation of fibroblasts, endothelial cells and histiocytes. Castleman’s disease represents an atypical lymphoproliferative disorder, highly associated with infection by human herpes virus 8 (HHV8), and patients have an increased risk for the development of other HHV8-associated neoplasms, or subsequent development of malignancy such as Kaposi’s sarcoma, malignant extra-nodal B-cell lymphoma


and plasmacytoma. Its clinicopathological features depend on various etiological factors such as Kaposi’s sarcoma herpes virus (KSHV), over-secretion of the cytokine interleukin 6 (IL-6) adhesion molecule (31) and follicular dendritic cell dysplasia (32, 33). Although the histogenesis of Kaposi’s sarcoma is a matter of some debate, the balance of evidence is that the cells are of endothelial origin. The pathogenesis of Kaposi’s sarcoma is unknown. It often occurs in association with other immune-deficient state of the patient. In 30 to 40% of AIDS patients, Kaposi’s sarcoma develops and this figure is even higher in patients who die of AIDS. The viral etiology is suggested by the epidemiologic features. Human immunodeficiency virus (HIV) itself is a cofactor in patients with AIDS. Growth factors released by retrovirus-infected CD4+ T-lymphocytes and from Kaposi’s sarcoma cells themselves seem to play a role in the vascular proliferation and growth of stromal cells that characterize histological lesions (Figures 2C, 3A). Another unfortunate aspect of Kaposi’s sarcoma is that about one third of all patients subsequently develop a second malignancy, usually lymphoma, leukemia or myeloma. In this investigation, seven patients out of a total of 24 Kaposi’s sarcoma cases were HIV positve with infiltrating lymphoma. Angiosarcoma is a highly malignant tumour and encompasses several clinical entities such as cutaneous tumours that occur without pre-existing lymphedema or that arise in lymphedematous extremities; angiosarcoma of the breast and of deep soft tissues. On crossexamination, tumours are often haemorrhagic and poorly demarcated from normal tissue. In this study, the immunohistochemical expression of growth hormone receptors (GHR), proliferative indices (Ki-67, PCNA) and vascular density was determined in a total of 61 vascular tumours, ranging from benign haemangioma to malignant Kaposi’s sarcomas. Many of the malignant tumours showed a high Ki-67 and PCNA proliferative activity, which is indicative of poor prognosis (34, 35). A positive correlation has been demonstrated between Ki-67 staining and both pathological and nuclear grade (35-37) and with mitotic index and over-expression of c-erbB-2 (38). The differentiation and proliferative activity of tumour cells are important predictors of the aggressiveness of a tumour (39, 40). Immunohistochemical analysis of nuclear antigen-associated cell proliferation has successfully been used to demonstrate levels of Ki-67 and proliferating cell nuclear antigen (PCNA) in fresh and fixed tissues (41). Estimation of Ki-67, which labels a nuclear protein expressed in cycling cells, correlates well with other proliferative markers such as thymine labelling (22), mitotic activity and sphase fraction, and when present in high levels is an independent prognostic indicator (42). Ki-67 monoclonal antibodies are specific for a nuclear antigen that is expressed only in proliferating cells in late G1, S, M and G2 phases of the cell cycle (43, 44). Ki-67 staining correlates directly with

tumour size, histological grade, vascular invasion and axillary lymph node status (45-47). In univariate analysis of survival data, Haersley et al. (48) showed that Ki-67 was a parameter of a poor overall survival in lymph node-positive and negative patients. In a multivariate analysis using a Cox model stratified by nodal status, Ki-67 failed to be of prognostic significance, whereas classical histopathological parameters such as tumour size and histological grade, are of independent prognostic significance in both lymph nodepositive and -negative patients. Compared to benign haemangiomas and haemangioendotheliomas, the results from this study show that proliferative activity is greatly increased in the highly malignant angiosarcoma and Kaposi’s sarcoma vascular tumours (Table I). PCNA may help to predict prognosis, since the measurement of PCNA immunolabelling has been shown to provide important prognostic information in T1-2 NOMO tumours (49). Although the proliferative activity can be variable in some vascular tumours, it nevertheless correlates well with the expression of GHR. Indeed, PCNA has a significantly higher expression in those malignant vascular tumours in which GHR immonostaining is pronounced (Table I). Since both the Ki-67 and/or PCNA proliferative activity are also significantly higher in vascular tumours with high GHR expression, the simultaneous validation of these markers may serve as a useful tool in the process of therapeutic strategy planning. Proliferating cell nuclear antigen is a nuclear protein associated with DNA polymerase, which is present throughout the cell cycle in proliferating cells. In immunohistochemical staining, PCNA is commonly found in G1- and G2-phases of the cell cycle. It is a 36 kD eukaryotic nuclear protein, primarily associated with the synthesis phase of the cell cycle and is involved in DNA repair, replication, post-replication modifications and chromatin assembly. Active PCNA is a trimeric protein forming a sliding clamp around DNA and interacts with eukaryotic DNA polymerase to form a replisome. The interaction of cell cycle regulatory proteins may be fundamental for cell survival, since it represents the link between the DNA damage response and the regulation of DNA replication and repair. When this mechanism is disrupted, mutations accumulate, genetic integrity is lost, and the cell cycle is deregulated (50). PCNA is highly representative of proliferating activity and is associated with poor prognostic factors such as high histological grade, numerous mitosis and presence of metastases (51, 52) The immunohistochemical evaluation of PCNA is considered to be a very useful index of tumour behaviour. In particular, it has been postulated that the PCNA index can be used as an objective and quantitative means for evaluation of the malignancy of a tumour (53) and PCNA expression was the only independent prognostic factor in lymph node negative cancer patients (54).

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ANTICANCER RESEARCH 27: 4201-4218 (2007) As a result of the availability of a specific monoclonal antibody to the GHR and probes for GHR mRNA, it has now been clearly established that GHR expression is far more widespread than previously realized and that GH is involved in many functions not involving local IGF-1 generation. Our results show that GHR immunoreactivity was up-regulated from benign to malignant vascular tumours respectively and strongly over-expressed in the majority of angisarcomas and Kaposi’s sarcoma (Table I). The results also show that vascular endothelial cells are targets for GH and thus indicate a direct role for GH in endothelial cell proliferation and in angiogenesis during tumour growth. Expression of GHR in vascular tumour cells was well marked in cell membranes (Figure 2B), was predominantly cytoplasmic (Figures 1A, 1B, 1C, 2A, 2C, 3A, 3D), but strong nuclear immunoreactivity was also apparent (Figure 1D, 2D, 3B, 3C). The presence of intracellular GHR is the result of endoplasmic reticulum and Golgi localisation. Nuclear localisation is due to identical nuclear GHR/binding protein. The human GHR specific monoclonal antibody (MAb 263) consistently resulted in strong GHR expression in both the aggressive malignant angiosarcomas and Kaposi’s sarcomas but to a lesser extend in benign vascular tumours. The strong immunoreactivity exhibited by MAb 263 is concordant with the specicifity of this monoclonal antibody. It recognizes the human GHR on IM-9 human lymphocyte tumour cells and blocks binding of about 50% of 125I hGH to these cells (55). The expression of GHR in the cell membrane, endoplasmic reticulum, perinuclear Golgi apparatus and in the nuclei of vascular neoplastic cells strongly supports the concept that GH also acts locally to stimulate both growth and cellular differentiation directly at the gene level and supports the hypothesis that GH is directly involved in paracrineautocrine mechanism acting locally in regulating vascular tumour growth. An interesting finding is the marked expression of GHR in the endothelium of the blood vessels (Figures 2A, 2C, 3D), which is especially pronounced in the newly forming capillaries. The receptor expression is especially prominent in the solid buds of newly forming capillaries of infiltrating vascular tumours (22). This indicates to a role of GHR in tumour angiogenesis, which may indirectly contribute to tumour growth. The contribution of neovascularisation to tumour growth lies not only in perfusion of the tumour, but also in the paracrine effect of vascular endothelial cells on neoplastic cells. The former can also release growth factors that stimulate tumour cells; thus, a bidirectional paracrine relationship emerges in which endothelial cells stimulate tumour cells and chemical signals from tumour cells shift resting vascular endothelial cells into a phase of rapid growth, leading to an angiogenic response. Nevertheless, the "switch" to the angiogenic phenotype involves more than

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simple up-regulation of angiogenic activity. Concomitant down-regulation of endothelial inhibitors, naturally present in cells before and after they become neoplastic, also seems to be necessary. Tumour angiogenesis is the growth of new vessel toward and within the tumour and is mediated by factors secreted by the tumour cells and/or tumour associated inflammatory cells. Micro-vessel density in the area of the most intense neo-vascularization in invasive tumours has been reported to correlate with the presence of tumour metastases, patient survival and vascular quantification was found to be an independent prognostic factor (56, 57). The established method of enumeration of micro-vessel density is to count the vessels using an ocular raster. It is critical to carefully select fields for measurement. In this study, the areas showing the highest estimated vascular density were chosen for the assay, using the sensitive endothelial cell marker CD-31. A total of 64 vascular tumours (Table I) were quantified for microvascular density (MVD) each with ten x20 fields. In other neoplasia, vascular surface area has been reported to correlate with stage of the disease, tumour size, nodal status and combined histological grade (25). The importance of endothelial cells to vascular tumour growth is emphasized by our finding of a positive correlation between vascular tumour cells having endothelial cell characteristics and both tumour vascularity and tumour growth rates, using CD31 and the proliferative index markers PCNA and Ki-67 respectively. Our findings also demonstrate that GH receptor are strongly expressed in vascular tumour cells and raises the possibility of a direct role for GH in endothelial cell proliferation during angiogenesis. This investigation on vascular tumour cells followed the discovery that dissociated cell suspensions from both experimental and human paediatric tumour biopsies could form two types of cellular colonies after plating in semisolid nutrient agar medium (22). The first type varied from tumour to tumour and generally consisted of tight colonies of round neoplastic cells. The second type consisted of loose colonies of larger, inter-connecting, elongated atypical "sprouting" or "variant" cells. The highest numbers of vascular tissue-derived endothelial colony forming progenitor cell (VECPC) colonies were obtained from highly vascular tumour tissue, whilst poorly vascularised, fibrotic and semi-necrotic tumour tissue yielded the lowest number of colonies (22), showing that this cell is of importance in vascularization and hence tumour growth. Both tritiated thymidine suiciding and gamma-radiation clearly showed that faster cycling cells are more vulnerable to therapeutic attack than non-cycling cells. Growth hormone and its receptor, similar to other growth factors and their cognate receptors, facilitates the uncontrolled proliferation of transformed cells through potential autocrine and/or paracrine pathways. Changes in


the level of their expression are important in the pathogenesis of vascular tumours. IGF-1, produced in response to GH by most tissues in the body, is involved in normal growth but also increases proliferation of cancer cells. The pathogenesis of GH-related disorders, including vascular neoplasms, is not mediated solely by IGF-1, although GH regulation of IGF-1 gene transcription is rapid. The expression of the alternative 5' untranslated region in the rat IGF-1 gene is differentially regulated by GH in various tissues and the administration of GH induces a rise of IGF-1 in GH-responsive patients. However, it is not necessary to invoke co-localization of IGF-1 and GHR expression because the role of GH in tumour cells may be to regulate the expression of mature cell function rather than to promote cellular proliferation through local IGF-1 synthesis. These two hormones do not always act in series, in some tissues IGF-1 is synthesized independently of GH, despite the fact that these tissues possess GHR, as is evidenced by potent mitogenesis independent of IGF-1 in response to GH. The GHR is a single-pass transmembrane protein without intrinsic tyrosine kinase activity. Using MAb to the GHR extracellular domain (ECD) to act as receptor agonists, Fuh et al. (58) proposed GHR activation by GHdependent dimerization and this observation supported the ligand-dependent receptor dimerization model for tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR). GH binding to a constitutive receptor dimer results in relative rotation of receptor subunits in the homodimer, producing realignment of JAK2 kinases bound to the membrane-proximal receptor sequence below the cell membrane, which in turn are than able to activate each other by transphosphorylation, initiating signalling cascades (59). This activation of GHR results in the initiation of a multiplicity of downstream signals, including STATs 1, 3 and 5 a/b, extracellularsignal-regulated kinase, stress-activated protein kinase pathways and increased cellular calcium. GHR signalling is essential for cellular growth and loss of STAT 5 signalling correlates progressively with loss of postnatal growth enhancement. Furthermore, Woelfe et al. (60, 61) identified a functional STAT5 element in the second intron of the insulin-like growth factor (IGF)-1 promoter. Specific STAT 5b gene mutation resulting in major retardation of postnatal growth in humans was also reported by Kofoed et al. (62). Using mice possessing homozygous mutations to the GHR cytoplasmic domain, Waters et al. (63) showed that trancation at residue 569 in the cytoplasmic domain, a loss of 70% of STAT 5 signalling results in approximately 50% loss of postnatal growth enhancement by GH, and around 75% loss of circulating IGF-1., while truncation at residue 391 removed all STAT5a/b signalling.

Signal transducers and activators of transcription (STATs) are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. These proteins directly link growth factor receptor activation to nuclear gene transcription by serving as both second messengers and nuclear transcription factors. STATs are found in the cytoplasm of resting cells, and become activated by the phosphorylation of a single conserved tyrosine residue (64). This phosphorylation can be catalysed by a variety of kinases, including Jak family tyrosine kinases associated with cytokine receptors, intrinsic tyrosine kinases of polypeptide growth factor receptors, and other cellular tyrosine kinases such as c-src. Once tyrosine phosphorylated, STATs form dimers through reciprocal phosphotyrosine-SH2 interactions, translocate to the nucleus, and bind to specific DNA elements, thereby modulating transcription of target genes. STAT activation has been identified in a variety of malignancies and in some tumours appears to occur via autocrine activation of cytokine receptor. For example, STAT 3 and STAT 5 activation are present in T cell lymphomas and may be related to autocrine activation of the interleukin (IL)receptor (65). There is growing evidence that STATs play an important role in the pathogenesis of haematological, epithelial and mesenchymal tumours (66), reflecting the importance of STATs in mediating cellular proliferation stimulated by a variety of growth factor receptors. The growing number of cancers that demonstrate inappropriate STAT activation suggests that this pathway may play an important role in the pathogenesis of malignancy. GHR immunoreactivity in the cytoplasm of vascular tumour cells is due to endoplasmic reticulum/Golgi localization as a result of receptor mediated intracellular GH transport. Once internalized, the hormone-receptor complex dissociates and the hormone is translocated by a receptor independent pathway, complexed to the growth hormone binding protein (GHBP). Growth hormone in serum circulates complexed with GHBP, which contains the extracellular portion of the GHR. The MAb used in this investigation recognizes epitopes shared by both the GHR and the GHBP. The identity of the extracellular domain of the GHR and the serum GHBP has been confirmed by sequencing. A consequence of the ability of GHR to modulate transcription is the localization of these receptors in the nucleus (Figures 1D, 2D, 3B, 3C), indicating that GH internalized to the nucleus binds to chromatin GHBP and modulates transcription of GH specific mRNA. Thus, nuclear GHR expression shows that GH, internalized to the nucleus, binds to chromatin GHBP and modulates transcription of GH specific mRNA. The nuclear function of GH seems to be dependent upon the presence in the nucleus of the receptor intracellular domain. The nuclear

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ANTICANCER RESEARCH 27: 4201-4218 (2007) chromatin shares two identical determinants with the membrane receptor and the nuclear chromatin associated GHR may be involved in transcriptional regulation. The heterogeneity of the GHR nuclear immunoreactivity observed in this study suggests a relationship of the nuclear receptor to specific cell events such as cell cycle and a functional significance of the nuclear GHR and nuclear translocation of GH is indicated. In seeking new approaches for the therapy of malignant diseases, understanding the roles played by not only the neoplastic but also the vascular tissue cells in tumour growth, is a prerequisite to any therapeutic approach which might attempt to control tumour growth by exerting control over the vascular tissue elements. Because of the specific action of GH on vascular endothelial cells, the identification and localization of GHR in vascular tumours may have considerable clinical implications in cancer treatment and GH and its receptor represent promising targets for antiangiogenesis agents. Since location of specific receptors at the cell surface is required for hormones and growth factors to interact with a cell, antibodies against receptors which block ligand binding or interfere with signal transmission after ligand binding occurs, offer a new therapeutic approach to hormone control of cancer cell growth. The use of anti-GHR antibodies to block tumour progression is an interesting possibility, as is the use of long acting somatostatin analogues, and is a possible alternative to tumour specific antibodies; the spread of proliferating tumour cells might be arrested or slowed down and this may be a more gentle way than existing therapies to shrink tumours. Monoclonal antibodies conjugated to radioisotopes, immunotoxins, pro-drug-activating enzymes, and chemotherapeutic agents are likely to be more effective cytotoxic agents than antibodies alone. Antibodies may be useful for tumours in which the GHR is highly expressed. Antibody-directed therapy is also likely to play a role in the treatment of minimal residual disease that persists after conventional treatments.

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Received June 26, 2007 Accepted October 8, 2007