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 contents, duodenum, female breast, abdomen, stomach, colon, and subcutaneous tissue, but also occur in many other tissues lymph node, bladder, body flank and neck regions. The tumours and internal organs. The well-differentiated tumours consist of were of the following pathological entities: Haemangioma irregular anastomosing, blood-filled vascular channels that are (n=12); haemangioendothelioma (n=10); Castleman’s disease lined by variably atypical endothelial cells. The less differentiated (n=3), haemangiopericytoma (n=4); angiosarcoma, (n=11), tumours may show solid strands and sheets, resembling Kaposi’s sarcoma with focal infiltration by lymphoma, HIV +ve carcinoma or lymphoma. Several growth factors, including basic (n=7), Kaposi’s sarcoma (n=17). The endothelial cell marker fibroblast growth factor, transforming growth factors and CD-31 was used to establish endothelial cell characteristics and vascular endothelial growth factor, play a role in tumour microvascular density. To delineate tumour cell growth, angiogenesis. (GH) is mitogenic for a variety immunohistochemical analysis of cycling nuclear and of of vascular tissue cells, including smooth muscle cells, fibroblasts proliferating cell nuclear antigen, using Ki-67 and PCNA and endothelial cells and exerts its regulatory functions in polyclonal antibodies respectively, was used to demonstrate controlling metabolism, balanced growth and differentiated cell proliferative indexes. Results show that, compared to their expression by acting on specific membrane-bound receptors, normal tissue counterparts, nuclear and cytoplasmic expression which trigger a phosphorylation cascade resulting in the of GHR consistently result in strong immunoreactivity modulation of numerous signalling pathways and of in the highly malignant angiosarcomas and Kaposi’s sarcomas expression. Essential to the initiation of a cellular response to and was localized in the cell membranes and cytoplasm, but GH, the presence of receptors for this hormone may predict the strong nuclear immunoreactivity was also identified. The adaptation of tumour cells resulting from GH exposure. To presence of intracellular GHR is the result of endoplasmic address the site/mode of action through which GH exerts its reticulum and Golgi localization. Nuclear localization is due to effects, a well characterized monoclonal antibody, obtained by identical nuclear GHR-binding protein. Furthermore, there was hybridoma technology from Balb/c mice immunized with a positive correlation of GHR immunoreactivity with neoplastic purified rabbit and rat liver GH-receptor (GHR) and directed cellular proliferation and cycling, as measured by Ki-67 and against the hormone binding site of the receptor, was applied, PCNA. In conclusion, this study shows that GHR expression in using the ABC technique to determine GHR expression in a vascular tumours is a function of malignancy and cancer panel of vascular tumours. The GHR was cloned from a rabbit progression. Malignant cells, which are highly expressive of the liver cDNA library with the aid of an oligonucleotide probe receptor, have a greater proliferation rate and thereby also higher based on a 19 residue tryptic sequence derived from survival rate compared to tumours expressing lower or minimal 5900 fold purified rabbit liver receptor. A total of 64 benign and receptor level. The presence of GHR in endothelial cells of malignant vascular tumours were obtained from different vascular neoplasm indicates that they are target cells and GH is human organ sites, including the chest wall, skin, axillary 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 Correspondence to: David T. Lincoln, Entity Systems, Independent involved in the paracrine-autocrine mechanism, acting locally Research Foundation, 53 Ashburton Street, Chapel Hill, QLD. 4069. in regulating vascular tumour growth and will be useful for site- Australia. Tel/Fax: +61 738782417, e-mail: [email protected] specific studies of the evolution of vascular cancers. The use of Key Words: Vascular tumours, growth hormone, growth hormone anti-GHR antibodies to block tumour progression is an receptor, cellular proliferation. intriguing possibility.

0250-7005/2007 $2.00+.40 4201 ANTICANCER RESEARCH 27: 4201-4218 (2007)

Vascular tumours are common lesions of the skin and factors and cytokines, stimulating non-endothelial cells. subcutaneous tissue, but also occur in many other tissues and These molecules are produced by tumour cells or tumour- internal organs. The well differentiated tumours show infiltrating lymphocytes and can also be secreted by the irregular anastomosing, blood-filled vascular channels, lined extracellular matrix. A tumour can directly stimulate by variably atypical endothelial cells. The less differentiated endothelial cells by secreting inducers, such as VEGF or tumours may show solid strands and sheets, resembling hepatocyte growth factor (HGF), that specifically bind to carcinoma or lymphoma. The tumours are a heterogenous endothelial cell receptors. On the other hand, stimulation group of neoplasms, ranging from very benign haemangiomas, can occur indirectly and less specific in that the tumour to intermediate haemangioendothelioma lesions that are secretes enzymes which release growth factors, including locally aggressive but infrequently metastasize, to highly bFGF, that are stored in the extracellular matrix. A number malignant angiosarcomas and Kaposi’s sarcomas. In general, of growth factors (GF), including , VEGF, PDGF vascular neoplasms are divided into benign and malignant and EGF, acting by binding to the cell surface receptors, are tumours on the basis of two major anatomical characteristics, high-affinity ligands for transmembrane receptors belonging namely the degree to which the neoplasm is composed of well- to the family of receptor tyrosine kinases (RTKs) and play a formed vascular channels, and secondly the abundance and role in human disease, including developmental disorders of regularity of the endothelial cell proliferation. Benign vascular cancer (1). Each type of ligand binds to the extracellular tumours are made up largely of well-formed vessels with a domain of its own specific receptor. significant amount of regular endothelial cell proliferation, Growth hormone (GH) is also mitogenic for a variety of whereas, on the opposite of the spectrum, the malignant vascular tissue cells, including smooth muscle cells, fibroblasts, tumours are solidly cellular and anaplastic, with scant numbers adipocytes, macrophages, lymphocytes, endothelial cells and of only abortive vascular channels. The endothelial nature and exerts its regulatory functions in controlling metabolism, the angiogenic profile of the neoplastic proliferations that do balanced growth and differentiated cell expression by acting not form distinct vascular lumina, can be identified with the on specific membrane-bound receptors. It modulates the aid of immunocytochemical techniques using monoclonal synthesis of multiple mRNA species, including that of insulin- antibodies (MAbs) against adhesion molecules such as CD- like growth factor-1 (IGF-1), facilitating paracrine/autocrine 31, associated with platelet adhesion in inflammation and interactions in mammalian tissues. The original wound healing. hypothesis suggested that the mitogenic growth promoting The ability of vascular cells to respond appropriately to effects of GH were mediated by circulating serum factors extracellular stimuli is essential to their growth and survival known as or insulin-like growth factors (IGFs), in vivo, as well as to the growth, health, and survival of the produced by the liver in response to GH, the latter being organism. Growth factors and other soluble polypeptides synthesized in the . However, evidence from bind to their respective receptors on the cell surface, animals and developing humans has clearly demonstrated that triggering a variety of signal transduction pathways that IGF-1 is also widely synthesized locally in many tissues and often involve tyrosine phosphorylation of the receptor or the concept, that it may be directly regulated by local action of other intracellular . The activation of these GH to promote both stem cell differentiation and signalling cascades then leads to the stimulation or proliferation in vitro and in vivo became established. Green et repression of specific in the nucleus, thus linking al. (2) have proposed a "dual effector" hypothesis of GH external stimuli to the cell’s genetic machinery. A number action, in that GH promotes differentiation of stem cell of growth factors play important roles in the regulation of precursors, whereas IGF-1 is required for their subsequent vascular tissue cell growth and differentiation. Thus, clonal expansion and the precise action of GH depends polypeptide epidermal growth factor (EGF) promotes the critically on the differentiation stage of the target cells (3). growth of epithelial cells, platelet-derived growth factor According to this model, the metabolic actions are (PDGF) stimulates the growth of fibroblasts and smooth predominant in differentiated cells. The differentiative action muscle cells and vascular endothelial growth factor (VEGF) which renders cells competent to proliferate would be stimulates the growth of endothelial cells and plays a mayor predominant in undifferentiated stem cells. Differentiated and role in angiogenesis, the process by which a new capillary undifferentiated target cells might differ with respect to supply is formed in developing tissue. Furthermore, tumour receptor structure and or coupling of metabolic pathways to cells, have been found to release angiogenic factors, the receptor, just as target cells in different tissues might differ including basic fibroblast growth factor (bFGF), in these respects. However, IGF-1 is also produced at multiple transforming growth factor ‚ (TGF-‚) and VEGF, that may extrahepatic sites and it was subsequently proposed that IGF- also play an important role in angiogenesis. Many 1 might act locally in target tissues (4, 5). Both IGF-1 and angiogenic growth factors have multiple functions and are IGF-1 mRNA (6) show GH dependence in many extrahepatic not only inducers of angiogenesis, but also act as growth tissues. A local response to chemical mediators such as growth

4202 Lincoln et al: Growth Expression in Vascular Tumours factors or hormones and generally referred to as cytokines, females) with benign and malignant vascular tumours, diagnosed in requires the presence and subsequent binding to appropriately the period between January 1992 and December 2003, were obtained located receptors for cellular growth regulation, from different organ sites, including the chest wall, skin, axillary mass differentiation and specific functions by interacting with their tissue, duodenum, female breast, abdomen, oral cavity, liver, stomach, colon, ano-rectum, lymph nodes, bladder, body flank, trunk cognate receptors. Growth hormone has a major effect on and neck region. The tumours were of the following histological transcription, and this may be independent of the rapid entities: haemangiomas; (n=12); haemangioendothelioma (n=10); chemical events triggered by ligand binding to plasma Kaposi’s sarcoma, (n=17); Kaposi’s sarcoma with with focal membrane-associated GHR (7). The multiplicity of actions of infiltration by lymphoma, HIV +ve (n=7); Castleman’s disease GH are mediated by an array of signals triggered by the (n=3); angiosarcomas (n=11); haemangiopericytoma (n=4). activated GHR. These include among others control of Laboratory data recorded included presence of lymphocytes, levels postnatal growth, hepatic metabolism, fertility and immune of interleukin 6, history of immunodeficiency (HTLV-1), human immunodeficiency virus status and hepatitis screening. Tissues were functions. Postnatal growth is largely controlled by IGF-1, fixed in phosphate-buffered 4% paraformaldehyde (ph 7.4) generated by activation of signal transducers and activators of overnight at 4ÆC, washed in several changes of Tris buffer at pH 7.4, transcription (STATs). The importance of the STAT family routine processed and embedded in paraffin wax. Sections were cut as mediators of cell signalling for a broad spectrum of at 5 Ìm thickness and mounted on 2% aminopyroethylsaline (APS)- cytokines and growth factors is becoming increasingly evident, coated microscope slides. Routine haematoxylin-eosin stained as the ability of STATs to function as both cytosolic second sections were evaluated in all cases to assess general messengers and nuclear transcription factors is better pathomorphological features. Positive controls were prepared using paraffin wax embedded tissues sections with proven good reactivity. understood. As biologic proof of the importance of STATs in Negative controls were performed by substituting the respective regulating cellular growth, survival and differentiation, primary antibody by nonimmune serum. inappropriate STAT activation is found in a growing number of haematological and epithelial cancers. Clinicopathological evaluation of vascular tumours. Haemangiomas are The GHR was the first of the class 1 cytokine receptors benign vascular neoplasm that do not metastasize and often occur in cloned (8) and the immunocytochemical identification and the skin and mucosal surfaces of the body, but are also found in many localization of specific receptors for GH in human vascular viscera, particularly the liver, spleen, pancreas. The lesions were tumours, which to our knowledge has hitherto not been primarily composed of capillaries (capillary haemangioma) or widely demonstrated, would almost certainly indicate the likely dilated veins forming large cavernous vascular channel (cavernous haemangioma). Haemangioendothelioma is an endothelial cell target tissues for biological action and role of this receptor in neoplasm of intermediate (borderline) malignancy and follows the vascular tumour growth. Essential to the initiation of a pattern of distribution of the haemangiomas. Three histological vascular response to GH, the presence of receptors for this subgroups include epithelioid, spindle cell and malignant hormone may predict the neoplastic adaptation of vascular endovascular haemangioendothelioma. In this investigation, all cases endothelial cells resulting from GH exposure. To address the presented with neoplasms of the skin. Most of the malignant primary side/mode of action through which GH exerts its effects on angiosarcomas were multicentric and appeared as dark red nodular vascular neoplasms, a well characterised monoclonal antibody 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 (9-27), directed against the hormone binding site of the of elderly patients. Clinically, the majority of the Kaposi’s sarcomas receptor (28) and cloned from a rabbit liver cDNA library located in the skin were manifested by multiple purpuric plaques in with the aid of an oligonucleotide probe based on a 19 the distal portions of the extremities, later becoming darker, more residue tryptic peptide sequence derived from 5900-fold nodular, and eventually ulcerated. In addition to skin lesions, the purified rabbit liver receptor, was applied using tumours were also located in the stomach, duodenum and colon. immunohistochemical techniques to determine GHR Some patients with Kaposi’s sarcoma also develop lesions in lymph expression in a panel of human vascular tumours, including nodes. The four cases of haemangiopericytomas investigated were obtained from the abdomen, neck and trunk. Although haemangioma, haemangioendothelioma, Kaposi’s sarcoma, haemangiopericytomas have a high incidence of recurrence and Kaposi’s sarcoma with focal infiltration by lymphoma (HIV metastasis, all four cases were primary tumours. In Castleman’s +ve), Castleman’s disease, haemangiopericytoma and disease, non-cancerous growths (tumours) develop in lymph node angiosarcoma. 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 Materials and Methods types of Castleman’s disease have been identified. These are hyaline- vascular type, plasma cell type, and multicentric or generalized Tumour procurement and preparation. Vascular tumour tissues, (MCD). The three cases investigated were all axillary lymph nodes collected at surgery, were obtained from the Mubarek Al-Kaber cases involving a 55 year old female with plasma cell type and a 70 Hospital, Jabriya, Al-Amiri Hospital, Safat and the Hussain Makki year old male having multicentric type of Castleman’s disease with a Al-Jummaa Cancer Centre, Shuwaikh, Kuwait, Arabian Gulf. The focus consistent with Kaposi’s sarcoma. The latter patient had an age of the patients ranged from 16 to70 years with median age 47 elevated level of immune factor interleukin-6 and exhibited an years. Tissue biopsies from a total of 64 patients (44 males and 20 abnormally large liver.

4203 ANTICANCER RESEARCH 27: 4201-4218 (2007)

Microvascular density. The optimal tissue block for analysis of sections were extensively washed and incubated with linked vascular density was chosen by microscopic examination of all antibody solution for 60 minutes. Subsequent incubation with PAP histological sections performed for pathologic assessment of the reagent was carried out for 45 min, after extensive washes with PBS. tumours. Serial sections from formalin-fixed and paraffin wax Visualization of the peroxidase was by application of 0.05% 3,3 embedded vascular tumour tissues were cut at 4 Ìm, mounted on diaminobenzidine (DAB) tetrahydrochloride containing 0.09% poly-L-lysine coated slides and dried overnight at 37ÆC. Sections hydrogen peroxide in Tris-buffer for 5-10 minutes. In some cases, were deparaffinised with three changes of xylene, treated in two visualization with 3-amino-9-ethylcarbazole (AEC) as chromogen changes of alcohol for 5 minutes, to be followed by treatment (Dako, Denmark) in 0.1 M acetate buffer, pH 5.2 for six minutes or with 0.3% H2O2 in methanol for 20 minutes at room temperature till a red nuclear reaction product had formed. The sections were to block endogenous peroxidase in tissue sections. Sections were rinsed in PBS, nuclei lightly counterstained in haematoxylin, blued washed in distilled water, followed by washing in three changes in alkaline tap water and finally dehydrated in ascending series of of Tris-buffer (pH 7.6) and subsequent treatment with blocking alcohols, cleared in three changes of xylene and mounted with reagent (Tris-buffered saline (TBS) containing normal goat DePex (DAB) or sections were washed in tap water only before serum) for 30 minutes at room temperature (RT) to eliminate mounting in aqueous mounting medium (AEC). non-specific staining with the primary antibody, by inhibiting the non-specific binding. To improve immunostaining pattern, Production and characterization of monoclonal antibody to growth antigen retrieval was performed by treating tissue sections with hormone receptor. MAb 263 (IgG1 k isotype) was prepared by 0.05% Trypsin for 15 minutes at 90ÆC and allowing to cool to RT immunization of mice with purified rat and rabbit GHR as the in the same solution for 20 minutes, before incubation with the antigen. This MAb is directed against the hormone binding site of primary antibody against CD-31 (mouse monoclonal antibody the human receptor, it recognizes a cross-species determinant with clone F8/86, Dako, Denmark) at 1:50 dilution for 1 hour at RT high affinity, also present on the human receptor (29). MAb 263 is in a humidified chamber. Sections were than incubated with a reactive agent against the GHR in a number of species with high biotinylated secondary antibody to mouse/rabbit immunoglobulins affinity and does not react with insulin or receptors in a for 30 min at RT and subsequently incubated with streptavidin rabbit or rat liver. Under certain conditions MAb 263 precipitates and biotinylated peroxidase complex (4 ml streptavidin buffer rat and rabbit GHR, although it can also compete for hormone with 1 drop of streptavidin concentrate peroxidase conjugated) binding to subtypes of the GH receptor. This antibody inhibits 50% for 30 minutes at RT. Visualization was by incubating tissue of human growth hormone binding to GHR in rat liver microsome sections at RT with 1 mM 3-amino-9-ethylcarbazole (AEC) preparations. For histochemical uses the antibody was purified chromagen containing 0.015% hydrogen peroxide in 0.1 M from ascitic fluid using 1) ammonium sulphate precipitation and acetate buffer, pH 5.2 for 5 minutes. Between each incubation dialysis, 2) protein-A sepharose affinity chromatography or 3) step, the sections were extensively washed with 0.1 M TBS. protein-A chromatography followed by ammonium sulphate Immunostaining of vascular structures in lymph node tissue precipitation and then dialysis. Control MAb’s (anti-Brucella section was used as a positive control and a negative control was abortus and Heartworm) were of the same isotype. obtained by omission of the primary antibody in one section. With AEC as the chromogen, sections were washed with distilled water Growth hormone receptor immunohistochemistry. The presence of and mounted in aqueous mounting medium. GHR was demonstrated immunohistochemically by the streptavidin-biotin horseradish peroxidase complex (ABC) Proliferative indices. Immunohistochemical analysis of nuclear technique. Endogenous peroxidase activity was eliminated by antigen-associated cell proliferation was used to demonstrate levels incubation with 0.5% hydrogen peroxide in absolute methanol for of Ki-67 and of proliferating cell nuclear antigen (PCNA). 15 minutes at room temperature (RT). Non-specific protein Polyclonal anti-proliferating cell nuclear antigen (PCNA) antibody, binding was blocked by incubation with 20% normal goat serum in clone 19A2 and of IgM isotype, and the polyclonal Ki-67 primary PBS containing 1% bovine serum albumin (BSA) for 1 hour at RT. antibody in the form of ascites fluid were obtained from Dakopatts Deparaffinized tissue sections of 4 Ìm thickness were incubated 1) (Denmark). Prior to immunostaining, the Ki-67 specimen sections overnight at 4ÆC with primary antibody (MAb 263 at 12.5 Ìg/ml in were subjected to microwave oven treatment. Tissue sections in PBS-1% BSA); 2) with biotinylated goat anti-mouse IgG for 1 hour citrate buffer (pH 6.0) were placed in the microwave oven for two at RT (1:200 in PBS-1% BSA); 3) with streptavidin-biotin cycles of 15 minutes each at 600 Watts. Sections were allowed to horseradish peroxidase complex (1:250 in PBS-1% BSA) for 1 hour cool to RT and washed well. The presence of Ki-67 and PCNA at RT. Between each step, sections were washed several times in antigen was demonstrated immunohistochemically by the PBS-1% BSA. Visualisation was with 1 mM 3-amino-9- peroxidase, anti-peroxidase (PAP) method and all incubation ethylcarbazole, containing 0.015% H2O2 in 0.1 M acetate buffer, reactions were carried out at RT unless otherwise stated. Phosphate pH 5.2 for 5 minutes. Controls were performed by a) replacing the buffered formalin-fixed and paraffin wax embedded tissues sections anti-GHR MAbs with unrelated heartworm MAb 50.6 and/or of 4 Ìm in thickness were deparaffinised through xylene and Brucella abortus MAb at the same or greater concentration; b) hydrated by treatment in graded series of alcohols for 5 minutes substituting the anti-GHR MAbs with normal mouse serum at each, followed by the removal of endogenous peroxidase activity different concentrations; c) omission of the primary antibody; d) with 0.3% hydrogen peroxide in methanol for 30 min. Following pre-incubation for 2 hours at 20ÆC with serial dilutions of extensive washes with phosphate-buffered saline (PBS), pH 7.2, a recombinant GH (Genentech, USA) at 1, 5, 10, 20 and 30 Ìg/ml in 2% normal goat serum was applied for 30 min and the sections were PBS-10 mM MgCl2-1% BSA prior to incubation with primary incubated overnight with primary anti Ki-67 and/or anti-PCNA monoclonal antibodies. All incubations were carried out in antibody, diluted 1 in 50 in PBS at 4ÆC in humidified chambers. The humidified chambers to prevent evaporation.

4204 Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours

Quantitative analysis. The tumour sections were scanned in the endothelial cells ranged from bland to focally more plump, entire area with a counting grid and for each field the number of somewhat larger and atypical forms that were polygonal to points in coincidence with the area to be measured was recorded spindle shaped, with individual cells showing mitotic figures (Point count method). The quantitative analysis of PCNA, Ki-67, and intracellular cytoplasmic vacuoles, producing a slight CD-31 and GHR on paraffin sections was done by calculating the percentage of stained cells in 10 low power magnification fields or pleomorphism to the cell pattern. Varying degrees of 16 high power fields using a Zeiss microscope connected to a intervening collagenous stromal fibrous tissue and focal area computer using image analysis software. To ensure reproducible of myxoid stromal change was present. The immunohisto- and objective assessment of staining, 10 representative areas, each chemical profile confirmed the endothelial and vascular containing 1000 tumour cells, were observed under high-power nature of the lesion with both the intervening as well as field (objective lens, X 40) in a vertical section taken from the vascular space lining cells were positive for the endothelial centre of the lesion. The proportion of positive cells was expressed cell marker CD-31. The cases of haemangiopericytomas as percentage of total cells counted. Estimation of PCNA, Ki-67 and GHR was as follows: + = labelling of great majority; +(–) = investigated displayed variation of cytological and labelling of great majority, some negative; +/– = positive labelling histological features of branching networks of capillaries is between 50-70%; –/+ = more than 50% are negative; –(+) = surrounded by clumps, trabeculae, or sheets of polygonal more than 90% are negative; – = no labelling. Intra-tumoural and spindle cells, thought to be derived from the vascular microvessel density was subjectively assessed by light microscopic pericyte. The tumour obtained from the abdomen of a analysis. Counting of microvessels was performed without female patient (Table I) was composed of a variety of cells, knowledge of the stage, grade or clinical information. Each section some of which contained cytoplasmic fibrillar bundles similar was scanned at low magnification (x100) to identify the areas with the greatest density of microvessels ("hot spots"). A Chalkley point to those found in smooth muscle and glomus tumour cells, graticule was used for counting individual microvessels, as pinocytic vesicles similar to those in endothelial and smooth delineated by the stained endothelial cells. Microvessel count was muscle cells and long cytoplasmic projections as in the then performed at 100x, 200x and 400x magnification (10x, 20x and pericytes surrounding capillaries. Silver reticular fibres stain 40x objective and 10x ocular) on 10 different fields (1.0 mm2). To revealed that the tumour cells were surrounded by varying 2 facilitate the counting, a 1 mm gradicle divided into 100 squares amounts of basement membrane material. All three axillary and fitted into the eye piece, was used. The result was expressed as lymph node cases of Castleman’s disease investigated the highest number of microvessels identified within a single field. The mean of the Chalkley counts for each tumour was calculated presented with marked follicular dendritic cell hyperplasia and used in the statistical analysis. In accordance with published and prominent vascular proliferation. There was no clear procedures, the maximum number of microvessels staining positive difference in histological appearance in the case of the at x200 was graded using the following scoring system: up to 25 female patient to distinguish between solitary and vessels = 1+, 26-50 vessels = 2+, 51-75 vessels = 3+, 76-100 multicentric types. The two axillary lymph node cases from vessels = 4+, while >100 vessels was graded 5+. a 70 year old male patient were of multicentric, plasma cell type, displaying characteristic ‘Kaposi-like lesions’ with Results mononuclear cells scattered in inter-follicular areas and follicular dendritic cell dysplasia. In addition, the adhesion Histopathology of vascular tumours. Histologically, the molecule interleukin 6 and the Kaposi’s sarcoma herpes haemangiomas investigated were well defined and not virus (KSHV) were also present. Patients who presented encapsulated. In patients who presented with lesions of the with angiosarcoma had localizations in a variety of organs skin, the tumours were made up of closely packed (Table I). The skin (45%) was the site most frequently aggregations of thin-walled capillaries (capillary involved. Bulky disease was found in 27% of the patients. haemangioma), at times partly filled with red blood cells and Histologically the tumours show a wide spectrum of separated by scant connective tissue stroma. The lumina had differentiation, ranging from Grade 1 to Grade 3. The well signs of intra-vascular thrombosis or vessel rupture showing differentiated tumours showed irregular anastomosing, scarring and occasional haemosiderin pigment. The tumours blood-filled vascular channels, lined by variably atypical obtained from the liver and oral cavity (Table I) consisted of endothelial cells with display of papillary intra-luminal spongy tissue mass, made up of large, cavernous, vascular tufting. The less differentiated tumours had solid strands and spaces (cavernous haemangiomas) that were partly or sheets, resembling carcinoma or lymphoma. Immunohisto- completely filled with fluid and blood and separated by a chemically, the cells strongly expressed the endothelial scant connective tissue stroma. All haemangioendotheliomas marker CD-31. Of the 24 Kaposi’s sarcoma cases obtained in this investigated were from skin tissue. The investigated, 29% were HIV +ve with focal infiltration of lesions consisted of vascular tumours composed largely of an the skin lesions by lymphoma (Table I). Histologically, the irregular aggregate of vascular channels of varying size with early plaque stage lesions consisted of thin-walled, dilated an endothelial cell lining, in addition to the presence of vascular spaces in the epidermis, with interstitial dominant masses and sheets of spindle-shaped cells. The inflammatory cells. The older nodular lesions consisted of

4205 ANTICANCER RESEARCH 27: 4201-4218 (2007)

Table I. Expression of PCNA, Ki-67, GHR and MVD in vascular tumours.

Tumour Tissue Gender Age PCNA* Ki-67* GHR* MVD**

Haemangioma 03-617B Skin F 24 –/+ –(+) –/+ 3+ 02-393C#3 Skin F 30 –/+ –(+) –/+ 2+ 02-465A#1 Skin F 18 –(+) – –(+) 2+ 98-1069A Skin F 37 –/+ –(+) –/+ 2+ 97-848#1 Skin M 55 –(+) – –(+) 2+ 97-858C#1 Oral cavity F 29 +/– –(+) +/– 2+ 96-1047 Liver M 44 +(–) –/+ +/– 3+ 94-612A#1 Skin M 39 –(+) – –/+ 3+ 93-435A#2 Skin F 16 –(+) –(+) –(+) 2+ 93-860 Skin M 28 –/+ –(+) –/+ 3+ 92-23A Skin M 25 –(+) – –/+ 3+ 92-125B#1 Skin M 19 –(+) –(+) –/+ 2+ Haemangioendothelioma 02-2991 Skin M 45 –/+ –(+) –/+ 2+ 01-2564 Skin M 51 –/+ –(+) –/+ 2+ 99-887A Skin M 22 +/– –/+ +/– 3+ 98-2111 Skin M 65 –/+ –(+) –/+ 3+ 97-2412B Skin F 41 +/– –/+ –/+ 2+ 96-4025A Skin M 39 +/– –/+ +(–) 4+ 96-4025B#1 Skin M 39 +/– –(+) +/– 3+ 93-2398B Skin F 30 –(+) – –/+ 2+ 92-600E#2 Skin F 48 +/– –/+ +/– 3+ 92-600E#4 Skin F 48 +/– –/+ +/– 3+ Kaposi’s sarcoma 02-8577#1 Skin nodule F 41 –/+ –(+) –/+ 2+ 02-8870#1 Skin M 40 +/– –/+ + 4+ 02-8870#2 Skin M 40 –/+ –(+) –/+ 2+ 01-7363 Skin nodule F 46 –(+) –(+) –/+ 2+ 01-8193 Skin M 38 +/– –/+ +(–) 2+ 97-2033 Skin M 60 +/– –(+) + 3+ 97-2657A Skin M 63 +/– –/+ + 3+ 97-2657B Skin M 63 +/– –/+ + 3+ 95-1797 Skin M 52 +/– –/+ + 4+ 95-2691B Skin M 70 –(+) –(+) –(+) 1+ 94-209 Skin F 45 –(+) –(+) –/+ 2+ 94-368 Duodenum F 45 +/– –/+ + 5+ 93-245 Skin plaque M 65 –(+) –(+) –/+ 2+ 92-1937 Skin M 43 –/+ –(+) +/– 3+ 92-1960#1 Stomach M 56 +(–) –/+ +/– 4+ 92-1960#2 Stomach M 56 –(+) –(+) –/+ 2+ 92-2072 Colon M 37 +/– –/+ + 5+ Kaposi’s sarcoma with lymphoma 99-6549 Skin, HIV+ve M 27 +(–) +/– +(–) 4+ 97-7200 Skin, HIV+ve M 19 +/– –(+) + 3+ 96-229#2 Skin, HIV+ve M 25 +/– –(+) + 5+ 95-393 Skin, HIV+ve M 37 –/+ –(+) +(–) 2+ 94-131 Skin, HIV+ve M 29 –/+ –/+ +(–) 2+ 92-235A Skin, HIV+ve M 44 –/+ –/+ +(–) 4+ 92-235B Skin, HIV+ve M 44 –/+ –(+) +/– 3+ Castleman’s disease 96-2790A#3 Lymph node F 55 –/+ –(+) +/– 3+ 95-2691A#1 Lyymph node M 70 +/– –/+ +/– 3+ 95-2691A#2 Lymph node M 70 +/– –/+ +/– 3+

Table I. continued

4206 Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours

Table I. continued

Tumour Tissue Gender Age PCNA* Ki-67* GHR* MVD**

Angiosarcoma 01-5548A7 Flank, Grade I-II M 50 –/+ –(+) +/– 3+ 98-4700#1 Liver M 67 +/– –/+ + 4+ 97-539 Breast F 44 +/– –/+ +(–) 4+ 97-6023 Skin M 38 –/+ –/+ +/– 3+ 97-2412A#1 Bladder F 42 –/+ –/+ +/– 5+ 97-7736#1 Anorectal M 64 +/– –/+ +/– 5+ 93-120E#1–3 Skin, Grade III M 63 –/+ –/+ +/– 3+ 93-120E#1-5 Chest, GradeIII M63 +(–) –/+ + 4+ 93-120 E#10 Skin, Grade III M 63 +/– –(+) +/– 4+ 93-337A#3-2 Axillary mass F 59 +/– –/+ +(–) 3+ Haemangiopericytoma 98-7684A#1 Neck mass M 56 –/+ –(+) –/+ 5+ 98-7684B Neck mass M 56 +/– –/(+) +(–) 5+ 97-814A#1 Abdomen F 29 + –(+) + 5+ 96-868 Trunk mass M 63 +/– –/+ +/– 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 magnification using the following scoring system: up to 25 neoplastic, cytologically atypical endothelial and spindle- vessels = 1+, 26-50 vessels = 2+, 51-75 vessels = 3+, 76-100 shaped cells. The immunoperoxidase staining demonstrated vessels = 4+, while >100 vessels was graded 5+. The median strong expression of the endothelial cell marker CD-31 grade score of the vascular tumours was as follows: 2.4 for antigen in both the endothelial and spindle-shaped cells. The haemangiomas; 2.7 for haemangioendotheliomas; 2.9 for neoplastic channels had infiltrated the surrounding Kaposi’s sarcomas; 3.3 for Kaposi’s sarcomas with structures, mostly following perivascular and perineural lymphomas; 3.0 for Castleman’s disease; 3.8 for spaces. In the skin lesions, the neoplastic vascular channel angiosarcomas and 4.7 for haemangiopericytomas. MVD was infiltrate was around skin adnexa and between individual significantly higher in HIV +ve patients with skin nodules of collagen fibres. Kaposi’s sarcomas displaying focal infiltration by lymphoma.

Microvascular density. Table I shows the clinicopathological Expression of cycling nuclear protein (Ki-67). Table I shows characteristics and results from the CD-31 immunostaining, the clinicopathological characteristics and cycling nuclear providing a measure of microvascular density (MVD) and protein (Ki-67) index obtained from counting a total of 1000 yielding an ‘angiogenic index’ of the vascular tumours. CD- cells in each of 10 representative fields from each vascular 31 immunoreactivity was present in the vascular endothelial neoplasm specimen investigated. Immunoreactivity of Ki-67 cells of the larger blood vessels (arteries and veins) and in the as a measure of cycling nuclear protein was present to endothelium of the capillaries. All other components of the varying degree in all tumour specimens. Staining was mostly vascular tumour tissue, including connective tissue stromal confined to the nuclei of the tumour cells. Cells in mitosis at cells, lymphocytes, neutrophils, plasma cells, adipocytes, times also displayed weak staining in their cytoplasm. There smooth muscle cells and nerve cells were CD-31 negative. was no Ki-67 immunoreactivity present in the nuclei of MVD was not affected by age, gender or by previous medical stromal, vascular and nerve cells in the normal tissue. conditions and treatment, including radiotherapy and did not Depending on the type of vascular tumour, the number of show a uniform increase with worsening pathological stage or cells with Ki-67-positive nuclei varied greatly between grade, but varied significantly in different categories of labelling more than 90% negative and labelling of great benign and malignant vascular tumours. Grading of majority of nuclei with some being negative. Among the microvascular density was done according to the maximum benign haemangiomas, in 58% of cases the tumour cell number of microvessels staining CD-31 positive at X200 nuclei were more than 90% Ki-67 negative, 33% displayed

4207 ANTICANCER RESEARCH 27: 4201-4218 (2007)

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.

4208 Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours

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.

4209 ANTICANCER RESEARCH 27: 4201-4218 (2007)

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.

4210 Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours no labelling and in one case of haemangioma of the liver, investigated, positive nuclear labelling of between 50-70% the nuclei were more than 50% immune-positive. Of the ten of tumour cells was observed in 6 cases, while in four cases benign haemangioendothelioma tumours investigated, in less than 50% of nuclei were PCNA positive and in one case half of the cases more than 50% of nuclei were Ki-67 nuclear PCNA staining was more than 90% negative. Of the negative, while in 4 cases more than 90% of the nuclei were 24 malignant Kaposis sarcoma investigated, PCNA labelling negative and one case displayed no labelling. Of the of the great majority of nuclei with some negative was in Castleman’s disease investigated, in two cases more than two cases (stomach and HIV +ve skin). Positive nuclear 50% of the tumour cell nuclei were Ki-67 negative and in labelling of between 50-70% was observed in ten tumours one case less than 10% were immune-positive. Of the 24 (41.7% cases) and in seven cases (29.2%) less than 50% malignant Kaposis sarcoma investigated, the tumour cell tumour cell nuclei were immune-positive, while in the nuclei were more than 90% Ki-67 immune-negative in 13 remaining tumours (20.6% cases) less than 10% of nuclei cases (54%, mostly skin lesions) and in ten cases (42%) expressed PCNA. Of the Castleman’s disease investigated more than 50% of nuclei were negative, while one of the (lymph nodes), in two tumours (66.7% cases) positive seven HIV +ve cases had Ki-67 positive labelling between PCNA labelling of tumour cell nuclei was between 50-70% 50-70%. Of the haemangiopericytomas investigated, in the and staining in the remaining case was less than 50% of the majority of cases the tumour cell nuclei were more than nuclei. In two cases of the haemangiopericytomas 90% negative and in one case less than 50% of the nuclei investigated, labelling was between 50-70% of tumour cell were stained positive. In the great majority (82%) of nuclei, while in one case (abdomen) labelling of great malignant angiosarcomas cases, positive Ki-67 nuclear majority of nuclei was observed; in the remaining case less staining was less than 50%, while more than 90% tumour than 50% nuclei expressed PCNA immunoreactivity. Of cell nuclei were Ki-67 negative in 18% of the tumours. the eleven cases of angiosarcomas investigated, positive nuclear PCNA labelling of between 50-70% was present Expression of proliferating cell nuclear antigen (PCNA). Table in approximately half (54.5%) of the malignant tumours; I shows the clinicopathological characteristics and in four cases (36.4%) less than 50% of the tumour cell proliferating cell nuclear antigen (PCNA) index obtained nuclei were stained, while in one case (skin, grade III) from counting a total of 1000 cells in each of ten labelling, was of the great majority of tumour cell nuclei, representative fields from each vascular neoplasm specimen with some negative. investigated. Results from this investigation demonstrate that overnight application of primary anti-proliferating cell Expression of growth hormone receptors (GHR). MAb 263 nuclear antigen (PCNA) at a dilution of 1:50 dilution at 4ÆC consistently showed immunoreactivity in paraffin wax overnight in humidified chambers revealed distinct nuclear embedded vascular tumour tissues. Table I shows the labelling and low background with diaminobenzedine clinicopathological characteristics and GHR expression, (DAB) as the visualizing agent. Somewhat superior staining obtained from counting a total of 1000 cells in each of 10 results were achieved with 3-amino-9-ethyl carbazole (AEC) representative fields from each vascular neoplasm specimen as the visualising chromogen. Proliferating cell nuclear investigated. Immunoreactivity of MAb 263 as a measure of antigen immunoreactivity was present in all vascular tumour GHR expression was present to varying degree in all vascular cases investigated and the expression of PCNA tumour specimens and consistently showed prominent immunopositive tumour cell nuclei varied from being more immunoreactivity in malignant Kaposi’s sarcoma and than 90% negative to a positive labelling of the great angiosarcoma tumour cells and to a lesser extend in the cells majority, with a variable proportion of stained cells in of the benign vascular neoplasms. Expression of the GHR was different vascular tumour types. A distinct to strongly well marked in cell membranes, was predominantly positive staining intensity was present in the nuclei of these cytoplasmic, but strong nuclear immunoreactivity was also cells. The nuclei of normal tissue cells, including fibroblasts, apparent in many instances (Figures 1A-3D). Nuclear staining vascular endothelial cells, adipocytes, smooth muscle cells, was heterogeneous, sometimes with an open chromatin lymphocytes, neutrophills, Mast cells and plasma cells were pattern being more dominant (Figures 1D, 2D, 3B, 3C). Not mostly negative. Of the twelve benign haemangioma all cells, displaying localization of the chromagen, possessed tumours investigated, in one case of the liver (8.3%) PCNA nuclear GHR immunoreactivity. The presence of intracellular tumour cell nuclei displayed labelling of great majority with GHR is the result of endoplasmic reticulum and Golgi some negative. In haemangioma of the oral cavity, positive localisation. Nuclear localisation is due to identical nuclear labelling was between 50-70%, while nuclear labelling in GHR/binding protein. Localized immunoreactivity was also lesions of the skin were less than 50% positive in four cases present in the vascular endothelial cells of tumours (Figures and more than 90% negative in half of all cases investigated. 2A, 2C, 3D) obtained from the different organ sites and this Of the ten benign haemangioendothelioma tumours supports the concept of a direct role for GH in endothelial

4211 ANTICANCER RESEARCH 27: 4201-4218 (2007) cell proliferation and angiogenesis during carcinogenesis. Of primary antibodies of identical IgGIK isotype (Brucella the twelve benign haemangioma tumours investigated, GHR abortus, Heartworm MAb 50.6) at the same concentration as expression in tumour cells was more than 90% negative in 3 MAb 263, did not display detectable immunoreactivity. Pre- cases (25%). In the majority of lesions (58.3%), treatment of tissue sections with recombinant human growth immunoreactivity was more than 50% negative, while GHR hormone (hGH) before application of the primary antibody expression was positive between 50-70% of tumour cells in the (MAb 263) abolished staining both in the cell haemangiomas of the liver and oral cavity (16.7%). Of the ten membrane/cytoplasm and the nucleus. On serial dilutions of benign haemangioendothelioma tumours investigated, positive GH at 1, 5, 10, 20 and 30 Ìg/ml, graded levels of GHR labelling of between 50-70% of tumour cells was immunoreactivity were observed. Immunoreactivity was observed in 4 cases, while in one case the great majority of reduced after pre-absorption at 5 Ìg/ml followed by tumour cells were immunopositive, with some cells negative. incubation with MAb 263 and was absent with pre-incubation In half of all haemangioendothelioma investigated, GHR at 20 to 30 Ìg/ml. Immunoreactivity was more prominent in expression was less than 50% of the tumour cells. Of the 24 sections when pre-incubated with 5 Ìg/ml recombinant hGH malignant Kaposis sarcoma investigated, GHR labelling of the followed by MAb 263, instead of the control monoclonal great majority of tumour cells was in nine tumours (37.5%), antibodies Brucella abortus or Heartworm 50.6. while in 5 cases (20.5%) receptor labelling was of the great majority of tumour cells, with some negative. Positive GHR Discussion labelling of between 50-70% was observed in three of the malignant tumours (12.5% cases) and in 6 cases (25%) less Vascular tumours, composed of endothelial cells of blood than 50% tumour cell nuclei were immune-positive, while in and lymph vessels, are very common lesions of the skin and only one tumour less than 10% of the tumour cells were subcutaneous tissue, but they also occur in many other tissues positive. Of the Castleman’s disease investigated, GHR and in internal organs. Although most vascular tumours are expression of vascular tumour cells was between 50-70% in all benign, low-grade malignant tumours such as cases. In the haemangiopericytomas specimen from the haemangioendothelioma and highly malignant angiosarcoma abdomen, GHR expression was in the great majority of and Kaposi’s sarcoma are common. Most benign vascular vascular tumour cells; receptor labelling of tumour cells in the neoplasms (haemangiomas) arise in the skin and trunk mass specimen was between 50-70%, while in the other subcutaneous tissues, but no organ or tissue is exempt. two haemangiopericytomas specimen of neck mass tumours Haemangiomas are common benign vascular neoplasm that investigated, GHR labelling was in the great majority, with do not metastasize, they occur in all age groups and may some tumour cells negative in one specimen and was more present in many clinical and pathological forms. than 50% negative in the second trunk mass specimen. Of the Haemangioendothelioma is a true neoplasm of vascular eleven cases of angiosarcomas investigated, GHR expression origin, composed predominantly of masses of endothelial of between 50-70% of the tumour cells was in approximately cells growing in and about vascular lumen (30) and represents half (54.5%) of the malignant tumours; while receptor an intergrade between the well-differentiated haemangiomas labelling of the great majority of tumour cells with some and the frankly anaplastic, totally cellular haemangiosarcoma negative (27.3%) was in breast and axillary mass tissue (angiosarcoma). It follows the pattern of distribution of the specimen. In angiosarcomas of the liver and abdomen, GHR haemangiomas and is most frequently encountered in the expression was present in the great majority of the tumour skin, but may affect the liver and the spleen. cells. The strong immunoreactivity exhibited with MAb 263 is Haemangiopericytomas are primarily tumours of adults. In concordant with the specificity of this antibody. The intensity this investigation, the youngest patient was a 29 year old of GHR immunoreactivity, but not the location, was found to female and the oldest a male of 63 years. Large tumours (6.5 be dependent on the antibody preparation technique. The cm), tumours with foci of necrosis and haemorrhage, and most intense immunoreactivity was observed with ascitic fluid those with increased mitotic rate, cellularity and anaplasia are precipitated with ammonium sulphate and dialysed. Protein-A more likely to exhibit malignant biological behavior The purification resulted in a decrease in immunoreactivity, a cytological variation of haemangiopericytomas is a reflection degree of which was restored by ammonium sulphate of the property of the pericyte to act as a precursor cell for precipitation and dialysis subsequent upon protein-A the formation of fibroblasts, endothelial cells and histiocytes. purification. Use of protein-A purified and protein- Castleman’s disease represents an atypical lympho- A/ammonium sulphate precipitated antibody at a four-fold proliferative disorder, highly associated with infection by concentration (5 mg/ml; 1:5 dilution) resulted in comparable human herpes virus 8 (HHV8), and patients have an staining to ascitic fluid/ammonium sulphate precipitated increased risk for the development of other HHV8-associated antibody. Tissue sections incubated with 20% normal serum, neoplasms, or subsequent development of malignancy such without the primary monoclonal antibody or with unrelated as Kaposi’s sarcoma, malignant extra-nodal B-cell lymphoma

4212 Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours and plasmacytoma. Its clinicopathological features depend on tumour size, histological grade, vascular invasion and axillary various etiological factors such as Kaposi’s sarcoma herpes lymph node status (45-47). In univariate analysis of survival virus (KSHV), over-secretion of the cytokine interleukin 6 data, Haersley et al. (48) showed that Ki-67 was a parameter (IL-6) adhesion molecule (31) and follicular dendritic cell of a poor overall survival in lymph node-positive and - dysplasia (32, 33). Although the histogenesis of Kaposi’s negative patients. In a multivariate analysis using a Cox sarcoma is a matter of some debate, the balance of evidence model stratified by nodal status, Ki-67 failed to be of is that the cells are of endothelial origin. The pathogenesis of prognostic significance, whereas classical histopathological Kaposi’s sarcoma is unknown. It often occurs in association parameters such as tumour size and histological grade, are with other immune-deficient state of the patient. In 30 to of independent prognostic significance in both lymph node- 40% of AIDS patients, Kaposi’s sarcoma develops and this positive and -negative patients. figure is even higher in patients who die of AIDS. The viral Compared to benign haemangiomas and haemangio- etiology is suggested by the epidemiologic features. Human endotheliomas, the results from this study show that immunodeficiency virus (HIV) itself is a cofactor in patients proliferative activity is greatly increased in the highly with AIDS. Growth factors released by retrovirus-infected malignant angiosarcoma and Kaposi’s sarcoma vascular CD4+ T-lymphocytes and from Kaposi’s sarcoma cells tumours (Table I). PCNA may help to predict prognosis, themselves seem to play a role in the vascular proliferation since the measurement of PCNA immunolabelling has been and growth of stromal cells that characterize histological shown to provide important prognostic information in T1-2 lesions (Figures 2C, 3A). Another unfortunate aspect of NOMO tumours (49). Although the proliferative activity Kaposi’s sarcoma is that about one third of all patients can be variable in some vascular tumours, it nevertheless subsequently develop a second malignancy, usually correlates well with the expression of GHR. Indeed, PCNA lymphoma, leukemia or myeloma. In this investigation, seven has a significantly higher expression in those malignant patients out of a total of 24 Kaposi’s sarcoma cases were HIV vascular tumours in which GHR immonostaining is positve with infiltrating lymphoma. Angiosarcoma is a highly pronounced (Table I). Since both the Ki-67 and/or PCNA malignant tumour and encompasses several clinical entities proliferative activity are also significantly higher in vascular such as cutaneous tumours that occur without pre-existing tumours with high GHR expression, the simultaneous lymphedema or that arise in lymphedematous extremities; validation of these markers may serve as a useful tool in the angiosarcoma of the breast and of deep soft tissues. On cross- process of therapeutic strategy planning. Proliferating cell examination, tumours are often haemorrhagic and poorly nuclear antigen is a nuclear protein associated with DNA demarcated from normal tissue. In this study, the polymerase, which is present throughout the cell cycle in immunohistochemical expression of growth hormone proliferating cells. In immunohistochemical staining, PCNA receptors (GHR), proliferative indices (Ki-67, PCNA) and is commonly found in G1- and G2-phases of the cell cycle. It vascular density was determined in a total of 61 vascular is a 36 kD eukaryotic nuclear protein, primarily associated tumours, ranging from benign haemangioma to malignant with the synthesis phase of the cell cycle and is involved in Kaposi’s sarcomas. Many of the malignant tumours showed DNA repair, replication, post-replication modifications and a high Ki-67 and PCNA proliferative activity, which is chromatin assembly. Active PCNA is a trimeric protein indicative of poor prognosis (34, 35). A positive correlation forming a sliding clamp around DNA and interacts with has been demonstrated between Ki-67 staining and both eukaryotic DNA polymerase to form a replisome. The pathological and nuclear grade (35-37) and with mitotic index interaction of cell cycle regulatory proteins may be and over-expression of c-erbB-2 (38). fundamental for cell survival, since it represents the link The differentiation and proliferative activity of tumour between the DNA damage response and the regulation of cells are important predictors of the aggressiveness of a DNA replication and repair. When this mechanism is tumour (39, 40). Immunohistochemical analysis of nuclear disrupted, mutations accumulate, genetic integrity is lost, antigen-associated cell proliferation has successfully been and the cell cycle is deregulated (50). PCNA is highly used to demonstrate levels of Ki-67 and proliferating cell representative of proliferating activity and is associated with nuclear antigen (PCNA) in fresh and fixed tissues (41). poor prognostic factors such as high histological grade, Estimation of Ki-67, which labels a nuclear protein expressed numerous mitosis and presence of metastases (51, 52) The in cycling cells, correlates well with other proliferative immunohistochemical evaluation of PCNA is considered to markers such as thymine labelling (22), mitotic activity and s- be a very useful index of tumour behaviour. In particular, it phase fraction, and when present in high levels is an has been postulated that the PCNA index can be used as an independent prognostic indicator (42). Ki-67 monoclonal objective and quantitative means for evaluation of the antibodies are specific for a nuclear antigen that is expressed malignancy of a tumour (53) and PCNA expression was the only in proliferating cells in late G1, S, M and G2 phases of only independent prognostic factor in lymph node negative the cell cycle (43, 44). Ki-67 staining correlates directly with cancer patients (54).

4213 ANTICANCER RESEARCH 27: 4201-4218 (2007)

As a result of the availability of a specific monoclonal simple up-regulation of angiogenic activity. Concomitant antibody to the GHR and probes for GHR mRNA, it has down-regulation of endothelial inhibitors, naturally present now been clearly established that GHR expression is far in cells before and after they become neoplastic, also seems more widespread than previously realized and that GH is to be necessary. Tumour angiogenesis is the growth of new involved in many functions not involving local IGF-1 vessel toward and within the tumour and is mediated by generation. Our results show that GHR immunoreactivity factors secreted by the tumour cells and/or tumour was up-regulated from benign to malignant vascular associated inflammatory cells. Micro-vessel density in the tumours respectively and strongly over-expressed in the area of the most intense neo-vascularization in invasive majority of angisarcomas and Kaposi’s sarcoma (Table I). tumours has been reported to correlate with the presence The results also show that vascular endothelial cells are of tumour metastases, patient survival and vascular targets for GH and thus indicate a direct role for GH in quantification was found to be an independent prognostic endothelial cell proliferation and in angiogenesis during factor (56, 57). The established method of enumeration of tumour growth. Expression of GHR in vascular tumour cells micro-vessel density is to count the vessels using an ocular was well marked in cell membranes (Figure 2B), was raster. It is critical to carefully select fields for predominantly cytoplasmic (Figures 1A, 1B, 1C, 2A, 2C, 3A, measurement. In this study, the areas showing the highest 3D), but strong nuclear immunoreactivity was also apparent estimated vascular density were chosen for the assay, using (Figure 1D, 2D, 3B, 3C). The presence of intracellular the sensitive endothelial cell marker CD-31. A total of 64 GHR is the result of endoplasmic reticulum and Golgi vascular tumours (Table I) were quantified for localisation. Nuclear localisation is due to identical nuclear microvascular density (MVD) each with ten x20 fields. In GHR/binding protein. The human GHR specific other neoplasia, vascular surface area has been reported to monoclonal antibody (MAb 263) consistently resulted in correlate with stage of the disease, tumour size, nodal status strong GHR expression in both the aggressive malignant and combined histological grade (25). The importance of angiosarcomas and Kaposi’s sarcomas but to a lesser extend endothelial cells to vascular tumour growth is emphasized in benign vascular tumours. The strong immunoreactivity by our finding of a positive correlation between vascular exhibited by MAb 263 is concordant with the specicifity of tumour cells having endothelial cell characteristics and both this monoclonal antibody. It recognizes the human GHR on tumour vascularity and tumour growth rates, using CD31 IM-9 human lymphocyte tumour cells and blocks binding of and the proliferative index markers PCNA and Ki-67 about 50% of 125I hGH to these cells (55). The expression respectively. Our findings also demonstrate that GH of GHR in the cell membrane, endoplasmic reticulum, peri- receptor are strongly expressed in vascular tumour cells and nuclear Golgi apparatus and in the nuclei of vascular raises the possibility of a direct role for GH in endothelial neoplastic cells strongly supports the concept that GH also cell proliferation during angiogenesis. acts locally to stimulate both growth and cellular This investigation on vascular tumour cells followed the differentiation directly at the gene level and supports the discovery that dissociated cell suspensions from both hypothesis that GH is directly involved in paracrine- experimental and human paediatric tumour biopsies could autocrine mechanism acting locally in regulating vascular form two types of cellular colonies after plating in semi- tumour growth. solid nutrient agar medium (22). The first type varied from An interesting finding is the marked expression of GHR tumour to tumour and generally consisted of tight colonies in the endothelium of the blood vessels (Figures 2A, 2C, of round neoplastic cells. The second type consisted of loose 3D), which is especially pronounced in the newly forming colonies of larger, inter-connecting, elongated atypical capillaries. The receptor expression is especially prominent "sprouting" or "variant" cells. The highest numbers of in the solid buds of newly forming capillaries of infiltrating vascular tissue-derived endothelial colony forming vascular tumours (22). This indicates to a role of GHR in progenitor cell (VECPC) colonies were obtained from tumour angiogenesis, which may indirectly contribute to highly vascular tumour tissue, whilst poorly vascularised, tumour growth. The contribution of neovascularisation to fibrotic and semi-necrotic tumour tissue yielded the lowest tumour growth lies not only in perfusion of the tumour, but number of colonies (22), showing that this cell is of also in the paracrine effect of vascular endothelial cells on importance in vascularization and hence tumour growth. neoplastic cells. The former can also release growth factors Both tritiated thymidine suiciding and gamma-radiation that stimulate tumour cells; thus, a bidirectional paracrine clearly showed that faster cycling cells are more vulnerable relationship emerges in which endothelial cells stimulate to therapeutic attack than non-cycling cells. tumour cells and chemical signals from tumour cells shift Growth hormone and its receptor, similar to other growth resting vascular endothelial cells into a phase of rapid factors and their cognate receptors, facilitates the growth, leading to an angiogenic response. Nevertheless, the uncontrolled proliferation of transformed cells through "switch" to the angiogenic phenotype involves more than potential autocrine and/or paracrine pathways. Changes in

4214 Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours the level of their expression are important in the Signal transducers and activators of transcription pathogenesis of vascular tumours. IGF-1, produced in (STATs) are a family of cytoplasmic proteins with roles as response to GH by most tissues in the body, is involved in signal messengers and transcription factors that participate normal growth but also increases proliferation of cancer in normal cellular responses to cytokines and growth cells. The pathogenesis of GH-related disorders, including factors. These proteins directly link growth factor receptor vascular neoplasms, is not mediated solely by IGF-1, activation to nuclear gene transcription by serving as both although GH regulation of IGF-1 gene transcription is second messengers and nuclear transcription factors. STATs rapid. The expression of the alternative 5' untranslated are found in the cytoplasm of resting cells, and become region in the rat IGF-1 gene is differentially regulated by activated by the phosphorylation of a single conserved GH in various tissues and the administration of GH induces tyrosine residue (64). This phosphorylation can be catalysed a rise of IGF-1 in GH-responsive patients. However, it is by a variety of kinases, including Jak family tyrosine kinases not necessary to invoke co-localization of IGF-1 and GHR associated with cytokine receptors, intrinsic tyrosine kinases expression because the role of GH in tumour cells may be of polypeptide growth factor receptors, and other cellular to regulate the expression of mature cell function rather tyrosine kinases such as c-src. Once tyrosine than to promote cellular proliferation through local IGF-1 phosphorylated, STATs form dimers through reciprocal synthesis. These two hormones do not always act in series, phosphotyrosine-SH2 interactions, translocate to the in some tissues IGF-1 is synthesized independently of GH, nucleus, and bind to specific DNA elements, thereby despite the fact that these tissues possess GHR, as is modulating transcription of target genes. STAT activation evidenced by potent mitogenesis independent of IGF-1 in has been identified in a variety of malignancies and in some response to GH. tumours appears to occur via autocrine activation of The GHR is a single-pass transmembrane protein . For example, STAT 3 and STAT 5 without intrinsic tyrosine kinase activity. Using MAb to activation are present in T cell lymphomas and may be the GHR extracellular domain (ECD) to act as receptor related to autocrine activation of the interleukin (IL)- agonists, Fuh et al. (58) proposed GHR activation by GH- receptor (65). There is growing evidence that STATs play dependent dimerization and this observation supported an important role in the pathogenesis of haematological, the ligand-dependent receptor dimerization model for epithelial and mesenchymal tumours (66), reflecting the tyrosine kinase receptors such as the epidermal growth importance of STATs in mediating cellular proliferation factor receptor (EGFR). GH binding to a constitutive stimulated by a variety of growth factor receptors. The receptor dimer results in relative rotation of receptor growing number of cancers that demonstrate inappropriate subunits in the homodimer, producing realignment of STAT activation suggests that this pathway may play an JAK2 kinases bound to the membrane-proximal receptor important role in the pathogenesis of malignancy. sequence below the cell membrane, which in turn are than GHR immunoreactivity in the cytoplasm of vascular able to activate each other by transphosphorylation, tumour cells is due to endoplasmic reticulum/Golgi initiating signalling cascades (59). This activation of GHR localization as a result of receptor mediated intracellular results in the initiation of a multiplicity of downstream GH transport. Once internalized, the hormone-receptor signals, including STATs 1, 3 and 5 a/b, extracellular- complex dissociates and the hormone is translocated by a signal-regulated kinase, stress-activated protein kinase receptor independent pathway, complexed to the growth pathways and increased cellular calcium. GHR signalling hormone binding protein (GHBP). Growth hormone in is essential for cellular growth and loss of STAT 5 serum circulates complexed with GHBP, which contains the signalling correlates progressively with loss of postnatal extracellular portion of the GHR. The MAb used in this growth enhancement. Furthermore, Woelfe et al. (60, 61) investigation recognizes epitopes shared by both the GHR identified a functional STAT5 element in the second and the GHBP. The identity of the extracellular domain of intron of the insulin-like growth factor (IGF)-1 promoter. the GHR and the serum GHBP has been confirmed by Specific STAT 5b gene mutation resulting in major sequencing. A consequence of the ability of GHR to retardation of postnatal growth in humans was also modulate transcription is the localization of these receptors reported by Kofoed et al. (62). Using mice possessing in the nucleus (Figures 1D, 2D, 3B, 3C), indicating that GH homozygous mutations to the GHR cytoplasmic domain, internalized to the nucleus binds to chromatin GHBP and Waters et al. (63) showed that trancation at residue 569 in modulates transcription of GH specific mRNA. Thus, the cytoplasmic domain, a loss of 70% of STAT 5 nuclear GHR expression shows that GH, internalized to the signalling results in approximately 50% loss of postnatal nucleus, binds to chromatin GHBP and modulates growth enhancement by GH, and around 75% loss of transcription of GH specific mRNA. The nuclear function circulating IGF-1., while truncation at residue 391 of GH seems to be dependent upon the presence in the removed all STAT5a/b signalling. nucleus of the receptor intracellular domain. The nuclear

4215 ANTICANCER RESEARCH 27: 4201-4218 (2007) chromatin shares two identical determinants with the 6 Hynes MA, Van Wyk J, Brooks PJ, D’Ercole AJ and Lund PK: membrane receptor and the nuclear chromatin associated Growth hormone dependence of somatomedin C-insulin like GHR may be involved in transcriptional regulation. The growth factor I and insulin like growth factor II messenger ribonucleic acids. Mol Endocrinology 1: 233-242, 1987. heterogeneity of the GHR nuclear immunoreactivity 7 Murphy LJ, Bell GI and Friesen HG: Growth hormone observed in this study suggests a relationship of the nuclear stimulates sequential induction of c-myc and insulin-like receptor to specific cell events such as cell cycle and a growth factor I expression in vivo. Endocrinol 120: 1806- functional significance of the nuclear GHR and nuclear 1812, 1987. translocation of GH is indicated. 8 Waters MJ: The growth hormone receptor. In: Handbook of In seeking new approaches for the therapy of malignant Physiology, Oxford University Press, JL Kostyo (eds.), 1996. diseases, understanding the roles played by not only the 9 Lincoln DT, Waters MJ, Breipohl W, Sinowatz F and Lobie P: neoplastic but also the vascular tissue cells in tumour Growth hormone receptor expression in proliferating rat . Acta Histochem Suppl. 40: 47-49, 1990. growth, is a prerequisite to any therapeutic approach which 10 Lincoln D, Berger H, Breipohl W, Hifnawi E, Waters M and might attempt to control tumour growth by exerting control Lobie P: Growth hormone receptor localization in primary over the vascular tissue elements. Because of the specific human melanomas. Arch Dermatol Res 283: 64, 1991. action of GH on vascular endothelial cells, the identification 11 Lincoln DT, Breipohl W and Wegener A: Immunohistochemical and localization of GHR in vascular tumours may have demonstration of growth hormone receptors (GH-R) in stromal considerable clinical implications in cancer treatment and cells from long-term human bone marrow cultures (LTBMC). GH and its receptor represent promising targets for anti- Histochem J 24: 626-627, 1992. 12 Lincoln DT, El-Hifnawi E, Sinowatz F and Waters MJ: angiogenesis agents. Since location of specific receptors at Immunohistochemical localization of growth hormone receptor the cell surface is required for hormones and growth factors binding protein in the mammalian cerebellum. Ann Anatomy to interact with a cell, antibodies against receptors which 176: 419-427, 1994. block ligand binding or interfere with signal transmission 13 Lincoln DT, Sinowatz F, El-Hifnawi E, Hughes RL and Waters after ligand binding occurs, offer a new therapeutic MJ: Evidence of a direct role for growth hormone in mammary approach to hormone control of cancer cell growth. The use gland proliferation and lactation. Anat Histol Embryol 24: 107- of anti-GHR antibodies to block tumour progression is an 115, 1995. interesting possibility, as is the use of long acting 14 Lincoln DT, Temmim L, Al-Jarallah MA, Mathew TC and Dashti H: Primary Ki-1 lymphoma of the skin: expression of analogues, and is a possible alternative to growth hormone receptors. Nutrition Suppl 11: 627-631, 1995. tumour specific antibodies; the spread of proliferating 15 Lincoln DT, Temmim L, Al-Jarallah MA, Lobie PE:Growth tumour cells might be arrested or slowed down and this may hormone receptors in human keratinocyte cells. Kuwait Medical be a more gentle way than existing therapies to shrink Journal 10: 86-93, 1995. tumours. Monoclonal antibodies conjugated to 16 Lincoln DT, Temmim L, Al-Jarallah MA, Lincoln JD and radioisotopes, immunotoxins, pro-drug-activating enzymes, Mathew TC: Tumour Stromal Cell Progenitors Adapt to and chemotherapeutic agents are likely to be more effective Tumour Growth. In: Adaptation Biology and Medicine, Volume 1, Subcellular Level. Sharma BK, Takeda N, Ganguly cytotoxic agents than antibodies alone. Antibodies may be NK, Singal PK (eds.). Narosa Publishing House, New Delhi, useful for tumours in which the GHR is highly expressed. Chapter 29, pp. 299-314, 1997. Antibody-directed therapy is also likely to play a role in the 17 Lincoln DT, Sinowatz F, Gabius H,. Gabius S, Baker H and treatment of minimal residual disease that persists after Mathew C: Subpopulations of stromal cells from long-term conventional treatments. human bone marrow cultures: ontogeny of progenitor cells and expression of growth hormone receptors. Anat Hist Embryol References 26: 11-28, 1997. 18 Lincoln DT, Sinowatz F, Temmim-Baker L, Baker HI, Kölle S 1 Longati P, Comoglio PM and Bardelli A: Receptor tyrosine and Waters MJ: Growth hormone receptor expression in the kinases as therapeutic targets: The model of the MET nucleus and cytoplasm of normal and neoplastic cells. oncogene. Current Drug Targets 2: 41-55, 2001. Histochem Cell Biol 109: 141-159, 1998. 2 Green H, Morikawa M and Nixon T: A dual effector theory of 19 Lincoln DT, Sinowatz F, Kölle S, Parsons P and Waters MJ: growth hormone action. Differentiation 29: 195-198, 1985. Up-regulation of growth hormone receptor immunoreactivity in 3 Isaksson OG, Lindahl A, Nilsson A and Isgaard J: Mechanism human melanoma. Anticancer Res 19: 19919-1932, 1999. of the stimulatory effect of growth hormone on longitudinal 20 Lincoln DT, Snyder JR, Sinowatz F and Waters MJ: Adaptation bone growth. Endocrine Rev 8: 426-438, 1987. of growth hormone receptor expression to tumour growth. In: 4 Mathews LS, Norstedt G and Palmiter RD: Regulation of Adaptation Biology and Medicine, Volume 2, Pandolf KB, insulin-like growth factor I by growth hormone. Takeda N, Singal PK (eds.). Narosa Publishing House, New Proc Natl Sci USA 83: 9343, 1986. Delhi, Chapter 19, pp. 119-138, 1999. 5 Mathews LS, Enberg B and Norstedt G: Regulation of rat 21 Lincoln DT, Kaiser HE, Raju GP and Waters MJ: Growth growth hormone receptor gene expression. J Biol Chem 264: hormone and colorectal carcinoma: Localization of receptors. 9905-9910, 1989. In Vivo 14: 41-50, 2000.

4216 Lincoln et al: Growth Hormone Receptor Expression in Vascular Tumours

22 Lincoln DT, Kaiser HE, Abul HT and Al-Jarallah MA: 38 Vanlemmens L, Hebbar M, Peyrat J and Bonneterre J: Age as Vascular stroma-derived endothelial colony forming progenitor a prognostic factor in breast cancer. Anticancer Res 18: 1891- cells adapt to tumour growth. In Vivo 16: 1-14, 2002. 1896, 1998. 23 Lincoln DT and West P: Growth hormone receptor expression 39 Sakr WA, Sarkar FH, Sreepathi P, Drozdowicz S and Crissman in the central nervous system. Journal of Anatomy 168: 55, 2002. JD: Measurement of cellular proliferation in human prostate by 24 Lincoln DT, Abul HT and Al-Khaledi M: Breast cancer and AgNOR, PCNA and SPF. Prostate 22: 147-154, 1993. growth hormone: Receptor expression in young Kuwaiti 40 Cher M,Chew K, Rosenau W and Carroll P: Cellular proliferation women. Annals of Anatomy 185(Suppl.): 94, 2003. in prostatic carcinoma as assessed by bromodeoxyuridine uptake, 25 Lincoln D T, Singal PK, Al-Khaledi M, Al-Yatama F and Ki-67, PCNA expression. Prostate 26: 87-93, 1995. Mohammed FM: Clinicopathological aspects and risk factors of 41 Leong A, Milios J and Tang S: Is immuno-localisation of breast cancer: Adaptation of biologically agressive carcinomas proliferating cell nuclear antigen (PCNA) in paraffin sections a in young Arab women. In: Adaptation Biology and Medicine, valid index of cell proliferation? Appl Immunohistchem 1: 127- Volume 5, Health Potentials, Lukyanova L, Takeda N, Singal 135, 1993. PK (eds.). Narosa Publishing House, New Delhi, Chapter 17, 42 Simpson JF and Page DL: Status of breast cancer prognostication pp. 211-244, 2007. based on histopathologic data. Am J Clin Pathol 102: S3-S8, 1994. 26 Sinowatz F, Breipohl W, Waters MJ, Lincoln DT, Lobie PE and 43 Charpin C, Andrac L and Vacheret H: Multiparametric Amselgruber W: Growth hormone receptor expression in the evaluation of growth fraction (Monoclonal Ki-67) in breast Dunning R 3327 prostatic carcinoma of the rat. Prostate 19: carcinoma tissue sections. Cancer Res 48: 4368, 1988. 273-278, 1991. 44 Iatropoulos M and Williams G: Proliferation markers. Exp 27 Sinowatz, F, Schams D, Kölle S, Plath A, Lincoln D and Waters Toxicol Pathol 48: 175-181, 1996. MJ: Cellular localization of growth hormone receptor in the 45 Bouzzubar N, Walker RJ and Griffiths K: Ki-67 immunostaining bovine mammary gland during mammogenesis, lactation and in primary breast cancer. Pathological and clinical associations. involution. J Endocrinology 166: 503-510, 2000. Br J Cancer 59: 943, 1989. 28 Barnard R, Mulcahy J, Garcia-Aragon J and Wyse B: Serum 46 Brown RW, Allered DC and Clark G: Prognostic significance growth hormone binding protein and hepatic GH binding sites and clinical pathological correlations of cell cycle measured by in the Lewis dwarf rat: effects of IGF-1 and GH. Growth Ki-67 immunocytochemistry in axillary node negative carcinoma Regulation 4: 147-154, 1994. of the breast. Breast Cancer Res Treat 16: 191, 1990. 29 Werther GA, Haynes KM, Barnard R and Waters M: Visual 47 Brown RS and Wahl R: Overexpression of glut-1 glucose demonstration of GH receptors on human growth plate transporter in human cancer. Cancer 72: 2979-2985, 1993. chondrocytes. J Endo Metab 70: 1725-1731, 1990. 48 Haarrslev T, Jacobson G and Zedeler K: Correlation of 30 Brennan JW, Midha R, Ang LC and Perez-Ordonez B: Epithelioid growth fraction by Ki-67 and proliferating nuclear antigen hemangioendothelioma of the spine presenting as cervical immuno-histochemistry with histopathological parameters and myelopathy: case report. Neurosurgery 48: 1166-1169, 2001. prognosis in primary breast cancer. Breast Cancer Res Treat 31 Nishimoto H and Norihiro T: Humanized anti-interleukin-6 37: 101-113, 1996. receptor antibody treatment of multicentric Castleman’s 49 Vesalainen T, Lipponen PK, Talja MT, Alhava EM and Syrjanen disease. Blood 106: 2627-2632, 2005. KJ: Proliferating cell nuclear antigen and p53 expression as 32 Kim JE, Kim CJ, Park IA and Kim WH: Clinicopathologic prognostic factors in T1-2MO prostatic adenocarcinoma. Int J study of Castleman’s disease in Korea. J Korean Med Sci 15: Cancer 58: 303-303, 1994. 393-398, 2000. 50 Jonsson ZO and Hubscher U: Proliferating cell nuclear antigen: 33 Du, M Q, Liu H, Diss TC, Ye H and Hamoudi RA: Kaposi’s more than a clamp for DNA polymerases. Bioessays 19: 967- sarcoma associated herpes virus infects monotypic (IgM 975, 1997. lambda) but polyclonal naive B cells in Castleman’s disease and 51 Visakorpi T: Proliferative activity determined by DNA flow associated lymphoproliferative disorders. Blood 97: 2130-2136, cytometry and proliferating cell nuclear antigen (PCNA) 2001. immunohistochemistry as a prognostic factor in prostatic 34 Sirvent JJ, Santage M, Salvado M, Alvaro T, Raventos A and carcinoma. J Pathol 168: 7-13, 1992. Palacios J: Hormonal receptors, cell proliferation fraction (Ki- 52 McNeal JE, Haillot O and Yemoto C: Cell proliferation in 67) and c-erbB-2 amplification in breast cancer, relationship dysplasia of the prostate: analysis by PCNA immunostaining. between differentiation, degree and axillary lymph node Prostate 27: 258-268, 1995. metastases. Histol Histopathol 9: 563-570, 1994. 53 Cappello F, Palma A, Martorana A, Rappa F, Cabibi D and 35 Bacus S, Goldschmidt R, Chin D, Moran G and Weinberg D: Barresi E: Biological aggressiveness evaluation in prostate Biological grading of breast cancer using antibodies to proliferating carcinoma: immunohistochemical analysis of PCNA and p53 cells and other markers. Am J Pathol 135: 783-792, 1998. in a series of adenocarcinomas. Eur J Histochem 47: 129-132, 36 Bacus S, Zelnick CR, Plowman G and Yaeden Y: Expression 2003. of the erbB-2 family of growth factor receptors and their ligands 54 Schonborn I, Minguillon C, Lichtenegger W, Zschiesche W and in breast cancer. Am J Clin Pathol 102: S13-S24, 1994. Spitzer E: The prognostic significance of recent tumour markers 37 Barbareschi M, Leonardi F and Mauri F: p53 and c-erbB-2 protein for lymph node negative breast cancer. Geburtshilfe expression in breast carcinomas. An immunohistochemical study Frauenheilkunde 55: 566-571, 1995. including correlation with receptor status, proliferation markers 55 Asakava K, Heda JA, McElduff A, Rouiller DG, Waters, MJ and clinical stage in human breast cancer. Am J Clin Pathol 98: and Gordon P: The human growth hormone receptor of 408-418, 1992. cultured human lymphocytes. Biochem J 238: 379-386, 1986.

4217 ANTICANCER RESEARCH 27: 4201-4218 (2007)

56 Weidner N, Semple JP, Welch WR and Folkman J: Tumor 62 Kofoed EM, Hwa V, Little B, Woods KA, Buckway CK, angiogenecies and metastasis correlation in invasive breast Tsubaki J, Pratt KL, Bezrodnik L, Jasper H and Tepper A: carcinoma. New Engl J Med 324: 1-8, 1991. Growth hormone insensitivity associated with a STAT5b 57 Weidner N, Folkman J and Pozza F: Tumor angiogenecies: A mutation. New Engl J Med 349: 1139-1147, 2003. new significant and independent prognostic indicator in early- 63 Waters MJ, Hoang HN, Fairlie DP, Pelekanos RA and Brown stage breast carcinoma. JNCI 84: 1875-1887, 1992. RJ: New insights into growth hormone action. J Mol 58 Fuh G, Cunningham BC, Fukunaga S, Nagata S, Goeddel DV Endocrinology 36: 1-7, 2006. and Wells JA: Rational design of potent antagonists to the 64 Bowman T, Garcia R, Turkson J and Jove R: STATs in human growth hormone receptor. Science 256: 1677-1680. oncogenesis. Oncogene 19: 2474-2488, 2000. 59 Brown RJ, Adams JJ, Pelekanos RA, Wan Y, McKinstry WJ, 65 Somerville L and Cory JG: Enhanced roscovitine-induced Palethorpe K, Seeber RM, Monks TA, Eidne KA, Parker MW apoptosis is mediated by a caspase-3-like activity in and Waters MJ: A model for GH receptor activation based on deoxyadenosine-resistant mouse leukemia L1210 cells. Anticancer subunit rotation within a receptor dimer. Nat Struct Mol Biol Res 20: 3347-3355, 2000. 12: 814-821, 2005. 66 Turkson J and Jove R: STAT proteins: Novel molecular targets 60 Woelfe J, Chia DJ and Rotwein P: Mechanisms of growth for cancer drug discovery. Oncology 19: 6613-6626, 2000. hormone action. Identification of conserved Stat5 binding sites that mediate GH-induced insulin-like growth factor 1 gene activation. J Biol Chem 278: 1261-1266, 2003. 61 Woelfe J, Billiard J and Rotwein P: Acute control of IGF-1 gene transcription by growth hormone through Stat5b. J Biol Received June 26, 2007 Chem 278: 22696-22702, 2003. Accepted October 8, 2007

4218