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In cultured clear-cell renal carcinoma (CCRCC) 786-0 cells transfected with HIF1alpha (HIF-1+), HIF-2alpha (HIF-2+), or empty vector (EV), no significant differences were observed in the growth rates in vitro, but when grown in vivo as xenografts HIF-2alpha significantly increased, and HIF-1alpha significantly decreased growth rates, compared to EV tumors. Factors associated with proliferation were increased and factors associated with cell death were decreased in HIF-2+ tumors. Metabolite profiles showed higher glucose and lower lactate and alanine levels in the HIF-2+ tumors whilst immunostaining demonstrated higher pyruvate dehydrogenase and lower pyruvate dehydrogenase kinase 1, compared to control tumors. Taken together, these results suggest that overexpression of HIF-2alpha in CCRCC 786-0 tumors regulated growth both by maintaining a low level of glycolysis and by allowing more mitochondrial metabolism and tolerance to ROS induced DNA damage. The growth profiles observed may be mediated by adaptive changes to a more oxidative phenotype.

The pathogenesis of rheumatoid arthritis (RA) and osteoarthritis (OA) remains obscure, although angiogenesis appears to play an important role. We recently confirmed an overexpression of two angiogenic factors, namely vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF), by the lining and stromal cells of the synovium in both conditions. Because hypoxia inducible factor (HIF)-1alpha and HIF-2alpha are essential in regulating transcription of the VEGF gene, active participation of HIF-alpha molecules in the pathogenesis of these arthritides is anticipated. We investigated the immunohistochemical expression of HIF-1alpha and HIF-2alpha in the synovium of 22 patients with RA, 34 patients with OA and 22 'normal' nonarthritic individuals, in relation to VEGF, VEGF/KDR (kinase insert domain protein receptor) vascular activation, PD-ECGF and bcl-2. A significant cytoplasmic and nuclear overexpression of HIF-1alpha and HIF-2alpha was noted in the synovial lining and stromal cells of both diseases relative to normal. Overexpression of HIF-alphas was related to high microvessel density, high PD-ECGF expression and high VEGF/KDR receptor activation, suggesting HIF-alpha-dependent synovial angiogenesis in OA. By contrast, the activation of the angiogenic VEGF/KDR pathway was persistently increased in RA, as indeed was microvessel density and the expression of PD-ECGF, irrespective of the extent of HIF-alpha expression, indicating a cytokine-dependent angiogenesis. In all cases, the VEGF/KDR vascular activation was significantly lower in OA than in RA, suggesting a relative failure of the HIF-alpha pathway to effectively produce a viable vasculature for OA, which is consistent with the degenerative nature of the disease. The activation of the HIF-alpha pathway occurs in both RA and OA, although for unrelated reasons.

AIMS: The study was undertaken to demonstrate wild-type p53 in normal paraffin-embedded tissues using two widely used antibodies, DO7 and 1801 and two different antigen retrieval buffer systems. METHODS AND RESULTS: Formalin-fixed paraffin-embedded normal tissue samples were obtained from the archives of the John Radcliffe Hospital, Oxford. Antigen retrieval was performed by microwaving using two different buffer systems: (i) the commercially available Dako target retrieval solution (Cat. no. 1699) (pH 9.8-9.9), (ii) freshly prepared buffer consisting of 0.1 m EDTA with 0.1% Tween pH 6.0, and (iii) freshly prepared buffer consisting of 0.1 m EDTA with 0.1% Tween pH 8.0. Staining was performed with DO7 and 1801 antibodies using the Dako Envision kit (peroxidase/DAB). DO7 antibody elicited strong nuclear staining in the mucosal cells of the small and large intestine, lymphoid cells, decidua, neurones such as Purkinje cells of the cerebellum, glandular epithelial cells and stromal cells of the prostate, cardiac myocytes and bronchial epithelial cells. Cytoplasmic staining was noted in Purkinje cells, glandular epithelium of prostate, exocrine pancreas and renal tubular epithelium. The 1801 antibody did not produce staining in any of these tissues. CONCLUSIONS: Our study demonstrates the presence of p53 in normal paraffin-embedded tissue with nuclear and/or cytoplasmic localization in some instances. In our view, DO7 appears to be better suited for such detection.

AIMS: Angiogenesis in solid tumour pathology is well established but less is known about its role in haematological malignancies. Our study investigated the immunohistochemical expression of a variety of angiogenic and hypoxic factors and microvessel densities on 110 cases of high- and low-grade non-Hodgkin's lymphomas and reactive lymphoid tissues. methods and results: Expression of vascular endothelial growth factor (VEGF) was present in 82 (96%) of the non-Hodgkin's cases and 35 (100%) of the reactive lymphoid tissue cases. Both hypoxia inducible factors 1 alpha and 2 alpha (HIF 1 alpha, 2 alpha) were weakly expressed in the majority of high- and low-grade lymphomas. Carbonic anhydrase IX (CA IX), a HIF-inducible membrane-bound enzyme, expression was not abundant with membranous staining being present in seven (8%) of the lymphoma cases and none of the reactive cases. Thymidine phosphorylase (TP) was distributed amongst macrophages and follicular dendritic cells but was not present in the neoplastic population. The vasculature was stained using CD34 which gave rise to a distinct vascular, predominantly paracortical network present in low-grade lymphomas and reactive lymphoid tissue but which was lost in high-grade lymphomas. CONCLUSION: Our results suggest that non-Hodgkin's lymphomas may be less angiogenic and hypoxically driven than most solid tumours, which has implications for possible future therapies.

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n=18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with 'malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n=26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n=17) designated LCT; and cytologically malignant (n=9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n=13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n=4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.

In this multicentre study, we examined 60 cases of Type II enteropathy-associated T-cell lymphoma (EATL) from the Asia-Pacific region by histological review, immunohistochemistry and molecular techniques. Patients were mostly adult males (median age: 58 years, male:female 2.6:1), presenting with abdominal pain (60%), intestinal perforation (40%) and weight loss (28%). None had a history of coeliac disease and the median survival was only 7 months. Histologically, these tumours could be divided into (i) central tumour zone comprising a monotonous population of neoplastic lymphocytes, (ii) peripheral zone featuring stunted villi and morphologically atypical lymphocytes showing epitheliotropism, and (iii) distant mucosa with normal villous architecture and cytologically normal intra-epithelial lymphocytes (IELs). Characterized by extensive nuclear expression of Megakaryocyte-associated tyrosine kinase (MATK) (87%) and usually a CD8(+)CD56(+) (88%) cytotoxic phenotype, there was frequent aberrant expression of CD20 (24%). T-cell receptor (TCR) expression was silent or not evaluable in 40% but of the remainder, there was predominant expression of TCRαβ over TCRγδ (1.6:1). In keeping with the normal ratio of IEL subsets, CD8(+) cases showed predominant CD8αα homodimer expression (77%), regardless of TCR lineage. These tumours constitute a distinct entity from classical EATL, and the pathology may reflect tumour progression from IEL precursors, remnants of which are often seen in the distant mucosa.

This highly illustrated, diagnostic guidebook provides a single comprehensive source of essential information to enable non-specialists to diagnose lymph node and related diseases with confidence. The text is didactic and practical, covering reactive as well as malignant conditions. Diagnosis of Lymphoproliferative Diseases approaches the disorders based on the WHO classification and is edited by members of the WHO panel for the classification of lymphomas. This second edition contains over 1000 high quality, digitised colour images and tables of essential criteria for each diagnosis. New features include: Full discussion of immunostaining, the core of modern diagnosis Variant forms and technical issues This authoritative guide is an essential reference for haematologists, haematopathologists, general pathologists, diagnostic histopathologists and oncologists. © 2012 Kevin C. Gatter, Georges Delsol, Roger A. Warnke and Francesco Pezzella.

Nuclear expression of MATK is a novel marker of type II enteropathy-associated T-cell lymphoma

BACKGROUND/AIM: Intensified angiogenic pathways are associated with poor prognosis and resistance of multiple myeloma (MM) cells to therapy. The links of the VEGF pathway with the hypoxia inducible factor (HIF) expression in MM are herein investigated. MATERIALS AND METHODS: The vascular density (VD) and the HIF/VEGF/VEGF-receptor expression in the bone marrows of 106 MM cases were studied using immunohistochemistry. RESULTS: HIF1alpha and HIF2alpha were expressed strongly in 33% and 13.2% of the cases, respectively. VEGFR and the phosphorylated (active) form of VEGFR2/KDR receptors were up-regulated in 42.5% and 36.8% of cases, respectively. Both HIF1alpha and HIF2alpha were significantly linked with high VD and VEGF expression. Moreover, the expression of the phosphorylated (active) form of VEGFR2/KDR was significantly linked with VEGF and HIF1alpha expression. The HIF/VEGF/VEGF-receptor pathway is up-regulated in approximately 40% of MM cases and linked with increased angiogenesis. Survival analysis in 37 evaluable patients showed a significantly worse prognosis in cases with high VD. CONCLUSION: HIFs and VEGF are up-regulated in a significant percentage of MM and are strongly related to each other. Targeting HIFs and the VEGF/receptor autocrine loop may prove of importance in the treatment of the disease. Anticancer Res

In this study, 32 cases of T-cell lymphoma of angioimmunoblastic lymphadenopathy type (AILD-TCL) were investigated for their association with Epstein-Barr virus (EBV). For this purpose, three different approaches were applied: polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small nuclear RNAs (EBER), and immunohistology for EBV-encoded latent membrane protein (LMP). PCR and EBER-ISH produced almost identical results, showing that all but one case of AILD-TCL contained EBV genomes. Three distinctive patterns of EBV infection were observed after immunophenotypical characterization of EBER-positive cells: (1) in 26% of the cases, B and T cells were infected, the majority of which were B cells of immunoblastic morphology located in the remnants of lymphoid follicles; (2) in 42% of the cases, the vast majority of infected cells were neoplastic T cells diffusely distributed in the lymph nodes, but infected B cells were also present; and (3) in 32% of the cases, there were only a few infected small lymphoid cells. Detectable LMP was frequent in cases exhibiting patterns 1 and 2. These findings suggest that in AILD-TCL patients, B cells and especially T cells are highly susceptible to a persistent EBV infection, which often leads to a growth advantage of the infected cells. Thus EBV, in conjunction with genetic abnormalities and selective defects of the immune system, might be involved in the pathogenesis of AILD-TCL.

AIMS: Angiogenesis is an important factor in tumour growth and metastasis. Vascular endothelial growth factor receptor 2 (VEGFR-2) or KDR plays a crucial role in angiogenesis. The aim of this study was to raise and characterize antibodies against phosphorylated KDR which could be used for studies on human tissues to assess KDR activation and novel inhibitors of KDR activation in clinical trials. METHODS AND RESULTS: Three monoclonal antibodies and one rabbit polyclonal antiserum were produced. The specificity of the antibodies was confirmed by ELISA. One of the mouse antibodies and the rabbit polyclonal antiserum reacted with a 200-kDa band on a Western blot of human umbilical vein endothelial cell (HUVEC) lysates, the molecular weight of KDR. Immunohistochemical staining showed that phosphorylated KDR is present in a wide variety of normal tissues including liver, colon and placenta, and is not restricted to endothelium. It was also present in a number of human tumours including breast carcinomas, colonic carcinomas and non-Hodgkin's lymphomas. The pattern of staining was membranous, cytoplasmic and nuclear. CONCLUSIONS: This study has shown that phosphorylated KDR is present in a wide variety of tumour and tissue types and is not confined to endothelium.

AIM: To describe an unusual human immunodeficiency virus (HIV)-associated lymphoma in uncommon sites. Plasmablastic lymphoma is a distinctive HIV-associated tumour that was first described in the jaws and oral cavity. Only two cases (stomach and lung) have been documented in extra-oral sites. MATERIALS AND METHODS: Four cases were encountered in HIV+ patients: three in the anorectal region and one which was nasal and paranasal. The cases were routinely processed and immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue. RESULTS: The cases showed the typical morphological appearances of a high-grade, blastic non-Hodgkin's lymphoma (brisk mitotic activity and tingible body macrophages). In addition, some cells had a plasmacytoid appearance and paranuclear clearing. Immunophenotypically, the tumour cells were negative for LCA, CD20 and CD45RA. However, a small proportion of cells (5%) were immunoreactive for CD79a and the majority were positive with VS38c, indicative of plasma cell differentiation. Kappa light chain and IgG heavy chain restriction was also detected. CONCLUSIONS: Plasmablastic lymphoma may occur in extra-oral sites and has a characteristic immunophenotype including focal expression of CD31 by the neoplastic cells. Awareness of the absence of expression of conventional B-cell markers and its presence in unusual sites should facilitate the diagnosis of plasmablastic lymphoma in HIV+ patients.

AIMS: Hypoxia inducible factors 1alpha and 2alpha (HIF1alpha and HIF2alpha) are hypoxia regulated transcriptional factors, which control the expression of a variety of genes responsible for angiogenesis, glycolysis, and the inhibition of apoptosis. Because angiogenesis and tissue regeneration are integral components of the inflammatory process, this study was designed to investigate the role of HIFalpha molecules in inflammatory bowel disease. METHODS: Surgical specimens from patients with active ulcerative colitis (UC) and Crohn's disease (CD) were assessed immunohistochemically for HIF1alpha and HIF2alpha reactivity, and the expression of these molecules was compared with the expression of the angiogenic factors thymidine phosphorylase (TP), vascular endothelial growth factor (VEGF), and VEGF-KDR activated vasculature. The vascular density of the lesions was also assessed using anti-CD31 immunostaining. RESULTS: HIF1alpha was expressed focally (epithelial cells, stromal fibroblasts, and myocytes) in both UC and CD, whereas HIF2alpha was expressed focally in UC and diffusely in CD. TP expression was uniformly positive in both diseases. VEGF expression was absent in CD, and weakly positive in UC. The VEGF-KDR reactivity of the submucosal vasculature was only slightly increased in UC and CD compared with normal tissue. The inflammatory cells stained with HIF2alpha and TP in all cases, but the reactivity was generalised in CD and focal in UC. In both diseases, vascular density was significantly higher than that seen in normal tissue. CONCLUSIONS: The discordant expression of HIF2alpha and VEGF in CD suggests an inherent deficiency of the intestine to respond to various stresses by the induction of VEGF. This finding should be investigated further.

We review, with particular reference to our clinical experience, the main types of pathological conditions in bone marrow other than haematological malignancy.

The JC12 monoclonal antibody recognizes a previously unknown nuclear protein that showed a restricted distribution in normal tonsil and was also overexpressed in a subset of diffuse large B-cell lymphomas. Using this reagent, we expression cloned cDNAs encoding its antigenic target and identified this protein as a novel putative transcription factor, FOXP1. The FOXP1 protein sequence contains predicted domains characteristic of transcription factors, including a winged helix DNA-binding motif, a second potential DNA-binding motif, a C(2)H(2) zinc finger, nuclear localization signals, coiled-coil regions, PEST sequences, and potential transactivation domains. The FOXP1 gene has been mapped to chromosome 3p14.1, a region that commonly shows loss of heterozygosity in a wide range of tumors and which is reported to contain a tumor suppressor gene(s). Using tissue arrays and immunohistochemistry, we demonstrate that both the FOXP1 mRNA and protein are widely expressed in normal tissues. The levels of FOXP1 mRNA were compared in paired normal and tumor tissues (from the same patient) using a tissue array containing cDNAs extracted from 68 samples taken from kidney, breast, prostate, uterus, ovary, cervix, colon, lung, stomach, rectum, small intestine, and from nine cancer cell lines. Differences in FOXP1 mRNA expression between normal and tumor samples were observed in 51% of cases. Most striking was the comparative loss of expression in 73% of colon tumors and comparative overexpression of FOXP1 mRNA in 75% of stomach tumors. Analysis of the FOXP1 mRNA expression in normal tissues (not taken from cancer patients) indicated that loss of FOXP1 expression may occur in some histologically normal tissues adjacent to tumors. Immunohistochemical analysis of FOXP1 protein expression was performed on 128 solid tumors, including 16 renal, 9 breast, 12 lung, 20 colon, 21 stomach, 10 head and neck, 35 prostate, and 5 pancreatic cases. Complete loss of expression, increased expression, and cytoplasmic mislocalization of the predominantly nuclear FOXP1 protein were frequently observed in neoplastic cells. Our study identifies FOXP1 as a new candidate tumor suppressor gene localized to the chromosome 3p14.1 region.

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

We have reported that angiogenic primary breast carcinoma can relapse not only as angiogenic, but also as non-angiogenic, lung metastases. This non-angiogenic pathway seems to be a novel pathway of cancer progression, and such tumours are likely to be resistant to antiangogenic treatment.

Vascular endothelial growth factor (VEGF) is a cytokine that is involved in tumor angiogenesis. Wild-type p53 (wt-p53) protein has been shown in cell lines to suppress angiogenesis through thrombospondin regulation. In this study, we immunohistochemically examined the expression of VEGF, nuclear and wild-type cytoplasmic p53, bcl-2, epidermal growth factor receptor, and c-erbB-2 oncoprotein; vascular grade; proliferation index; and extent of necrosis in non-small cell lung cancer (NSCLC). We analyzed 120 cases of early-stage NSCLCs (81 squamous cell carcinomas and 39 adenocarcinomas) treated with surgery alone (median follow-up, 63 months; range, 45-74 months). VEGF expression showed a positive association with high vascular grade (microvessel score of >75 per x250 field; P = 0.008), although about half of the LVG cases also expressed VEGF. None of the p53 antibodies examined correlated with angiogenesis. However, wt-p53 expression was inversely associated with VEGF expression, suggesting that wt-p53 is involved in the suppression of the VEGF gene. Combined analysis of VEGF, wt-p53, and microvessel counting showed that, although wt-p53 loss associates with VEGF switch-on, p53 protein may not be involved in the regulation of the angiogenic events downstream of VEGF expression. Moreover, no significant association of bcl-2 and c-erbB-2 oncoprotein expression with VEGF expression was observed. T/N stage, grade, Ki67 proliferation index, and extent of necrosis were not correlated with VEGF expression. Survival analysis showed that VEGF correlated with poor survival (P = 0.04) and was significant in node-negative cases (P = 0.03). We conclude that VEGF is an important angiogenic factor in NSCLC, its expression being dependent on wt-p53 loss.

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1alpha and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1alpha transactivation) of the VEGF promoter than the LDH-A promoter.

AIMS: Human AP endonuclease 1 (HAP1) plays a major role in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA by catalysing hydrolytic cleavage of the phosphodiester backbone 5' to the site. HAP1 is also known to be a potent reduction-oxidation (redox) factor, regulating the binding activity of a number of transcription factors. The purpose of the present study was to examine the expression of HAP-1 in a wide range of human tissues. METHODS AND RESULTS: Using a recently developed specific rabbit polyclonal antibody, we performed immunohistochemistry on paraffin-embedded tissue material. Nuclear staining was detected in crypt cells of the small and large intestine, epithelial cells of breast ducts, basal cells of the skin, alveolar cells of the lung, lymphocytes of the marginal zone of the spleen, in the surface epithelium and stromal cells of the ovary and the transitional epithelium of the bladder. Unexpectedly for a presumed nuclear protein, the staining pattern in some cell populations was mainly cytoplasmic (e.g. superficial cells of gastrointestinal tract, Langerhans cells, Leydig cells and spermatocytes, epithelium of the prostate glands), or both cytoplasmic and nuclear (e.g. epithelial cells of thymus, follicular thyroid cells, parietal cells of the stomach, glandular epithelial cells of the cervix, epithelial cells of exocrine pancreas). CONCLUSION: This differential expression in a wide spectrum of cells is indicative of a potential multifunctional action of HAP1, not necessarily restricted to a role in the nucleus. Histopathology

Neoplastic growth is usually dependent on blood supply, and it is commonly accepted that this is provided by the formation of new vessels. However, tumors may be able to grow without neovascularization if they find a suitable vascular bed available. We have investigated the pattern of vascularization in a series of 500 primary stage I non-small-cell lung carcinomas. Immunostaining of endothelial cells has highlighted four distinct patterns of vascularization. Three patterns (which we called basal, papillary, and diffuse) have in common the destruction of normal lung and the production of newly formed vessels and stroma. The fourth pattern, which we called alveolar or putative nonangiogenic, was observed in 16% (80/500) of the cases and is characterized by lack of parenchymal destruction and absence of both tumor associated stroma and new vessels. The only vessels present were the ones in the alveolar septa, and their presence highlighted, through the whole tumor, the lung alveoli filled up by the neoplastic cells. This observation suggests that, if an appropriate vascular bed is available, a tumor can exploit it and grows without inducing neo-angiogenesis. This could have implications for strategies aimed at inhibiting tumor growth by vascular targeting or inhibition of angiogenesis.

A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin superfamily with similarity to the N-CAM transmembrane proteins and is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is known to occur, resulting in different isoforms of the protein. Using five antibodies against the DCC gene product (three monoclonal antibodies raised in our laboratory, one commercially available antibody, and a rabbit polyclonal antibody), we have demonstrated by immunostaining a DCC protein isoform in reticuloendothelial cells in human thymus, tonsil, and lymph node. This can be distinguished from another isoform described in normal colonic epithelium, because this latter is not demonstrable with the antibodies we have used. It could not be detected in normal colonic epithelium, polyps or colorectal carcinomas. This restrictive distribution suggests that not all DCC gene products are important in colonic cancer.

Presentation of endogenous antigenic peptides to cytotoxic T lymphocytes (CTLs) is mediated by the major histocompatibility complex (MHC) class I molecules. These antigenic peptides derived from the cytoplasmic protein pool are transported by the recently described MHC-encoded transporters (TAP1 and TAP2) into a pre-Golgi region where they take part in the assembly of MHC class I molecules. Using an affinity-purified polyclonal antibody (AK1.7) for TAP1, we analyzed 81 colorectal carcinomas, 32 adenomas, and the respective nonneoplastic mucosa. Loss of the transporter molecule (TAP1) was observed in 14% (11 of 81) of the carcinomas, either complete (7 of 11) or focal (4 of 11), whereas adenomas and normal mucosa were always positive. This study adds further information to the understanding of the mechanisms related to the defective presentation of the MHC class I molecules by tumor cells.