Reliability of proliferation assessment by Ki-67 expression in neuroendocrine neoplasms: eyeballing or image analysis?

BACKGROUND: The latest WHO classification for neuroendocrine neoplasms (NEN) of the gastrointestinal tract defines grade according to Ki-67 and mitotic indices. Some have questioned the reproducibility and thus the reliability of Ki-67 assessment. We therefore investigated the accuracy of this proliferation marker in NEN. METHODS: The Ki-67 index of tumor specimens of NEN (n = 73) was assessed by two pathologists as in routine practice with eyeballing and twice by image analysis using ImageJ freeware at different magnifications. RESULTS were correlated with overall survival. RESULTS: The intraclass correlation coefficient (ICC) between pathologists was 0.88. The ICC for the measurements using image analysis was 0.85. The ICC between all four measurements (pathologists and ImageJ) was 0.80. If the Ki-67 index was translated to grade as prescribed by the current WHO classification (<3% = grade 1, 3-20% = grade 2, >20% = grade 3), kappa was between 0.61 and 0.75. Grades based on pathologist scoring were often (16-29%) higher than grades assigned by image analysis (p < 0.001). Grade was significantly correlated with survival (p < 0.0001) irrespective of the way Ki-67 was assessed. CONCLUSION: Assessment of the Ki-67 index by eyeballing correlates remarkably well with the Ki-67 index as calculated by image analysis and is therefore an accurate parameter. Moreover, it is significantly related to survival irrespective of the method used. Yet if the Ki-67 index is translated to grade, the grade should be interpreted with caution due to values around threshold levels.

Distinct defects in collagen microarchitecture underlie vessel-wall failure in advanced abdominal aneurysms and aneurysms in Marfan syndrome.

An aneurysm of the aorta is a common pathology characterized by segmental weakening of the artery. Although it is generally accepted that the vessel-wall weakening is caused by an impaired collagen metabolism, a clear association has been demonstrated only for rare syndromes such as the vascular type Ehlers-Danlos syndrome. Here we show that vessel-wall failure in growing aneurysms of patients who have aortic abdominal aneurysm (AAA) or Marfan syndrome is not related to a collagen defect at the molecular level. On the contrary our findings indicate similar (Marfan) or even higher collagen concentrations (AAA) and increased collagen cross-linking in the aneurysms. Using 3D confocal imaging we show that the two conditions are associated with profound defects in collagen microarchitecture. Reconstructions of normal vessel wall show that adventitial collagen fibers are organized in a loose braiding of collagen ribbons. These ribbons encage the vessel, allowing the vessel to dilate easily but preventing overstretching. AAA and aneurysms in Marfan syndrome show dramatically altered collagen architectures with loss of the collagen knitting. Evaluations of the functional characteristics by atomic force microscopy showed that the wall has lost its ability to stretch easily and revealed a second defect: although vascular collagen in normal aortic wall behaves as a coherent network, in AAA and Marfan tissues it does not. As result, mechanical forces loaded on individual fibers are not distributed over the tissue. These studies demonstrate that the mechanical properties of tissue are strongly influenced by collagen microarchitecture and that perturbations in the collagen networks may lead to mechanical failure.

Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.

Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (ß3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no gradient formation. Most chondrocytes from human osteochondromas showed normal PEI staining. However, approximately 10% of tumour chondrocytes were similar to those found in the dak mutant (e.g., lack of PEI gradients). The cells in the reduced PEI-stained areas are likely associated with loss-of-function mutations in the EXT genes, and they might contribute to tumour initiation by disrupting the gradients.

Cartilage ultrastructure in proteoglycan-deficient zebrafish mutants brings to light new candidate genes for human skeletal disorders.

Proteoglycans are molecules consisting of protein cores onto which sugar chains, i.e., glycosaminoglycans (GAGs) such as heparan or chondroitin sulphates, are attached. Proteoglycans are produced by nearly all cells, and once secreted they become a major component of the extracellular matrix. Cartilage is particularly rich in proteoglycans, and changes in the structure and composition of GAGs have been found in osteochondromas and osteoarthritis. The zebrafish (Danio rerio) exhibits fast development, a growth plate-like organization of its craniofacial skeleton and an availability of various mutants, making it a powerful model for the study of human skeletal disorders with unknown aetiology. We analysed skeletons from five zebrafish lines with known mutations in genes involved in proteoglycan synthesis: dackel (dak/ext2), lacking heparan sulphate; hi307 (ß3gat3), deficient for most GAGs; pinscher (pic/slc35b2), presenting defective sulphation of GAGs and other molecules; hi954 (uxs1), lacking Notch and most GAGs due to impaired protein xylosylation; and knypek (kny/gpc4), missing the protein core of the Glypican-4 proteoglycan. Here we show that each mutant displays different phenotypes related to: (a) cartilage morphology; (b) composition of the extracellular matrix; (c) ultrastructure of the extracellular matrix; and (d) the intracellular ultrastructure of chondrocytes, proving that sulphated GAGs orchestrate the cartilage intra- and extracellular ultrastructures. The mild phenotype of the hi307 mutant suggests that proteoglycans consisting of a protein core and a short sugar linker might suffice for proper chondrocyte stacking. Finally, knypek supports the involvement of Glypican-4 in the craniofacial phenotype of Simpson-Golabi-Behmel syndrome and suggests GPC4 as a modulator of the overgrowth phenotype that is associated with this syndrome and is primarily caused by a mutation in GPC3. Moreover, we speculate on the potential involvement of SLC35B2, ß3GAT3 and UXS1 in skeletal dysplasias. This work promotes the use of zebrafish as a model of human skeletal development and associated pathologies.

IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides.

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

Symptoms in patients with ulcerative colitis in remission are associated with visceral hypersensitivity and mast cell activity.

OBJECTIVE: Patients with ulcerative colitis in remission (UCR) frequently report irritable bowel syndrome (IBS)-like symptoms. Recent studies have pointed to the role of mast cells in mediating visceral hypersensitivity in IBS. We hypothesized that visceral hypersensitivity is frequently present in patients with UCR and is related to the quantity and activity of mast cells in the sigmoid mucosa. MATERIAL AND METHODS: A group of 17 controls and 19 patients with UCR were studied. Rectal compliance and perception were measured by electronic barostat. Sigmoid biopsies were taken to quantify the amount of mast cells, degranulating mast cells and mast cells in close proximity to mucosal nerve endings. RESULTS: Visceroperception significantly increased in UCR (p < 0.05) versus controls. Rectal perception correlated positively with IBS-like symptoms in UCR (r = 0.969; p < 0.05). The amount of mucosal mast cells (per 100 crypts) was significantly increased in UCR versus controls: 228 ± 20 versus 163 ± 18 (p < 0.05). In the UCR patients a higher percentage of mucosal mast cells was in close proximity to nerve endings (58 ± 4 vs. 38 ± 3% in controls; p < 0.05) or was degranulating (40 ± 7 vs. 16 ± 4% in controls; p < 0.05). There was a significant but weak correlation between quantity of mucosal mast cells and pain perception (r = 0.32; p < 0.05). CONCLUSION: Rectal hypersensitivity is associated with mucosal presence and activation of mast cells and with IBS-like symptoms in patients with UCR.

Aberrant chemokine receptor expression and chemokine production by Langerhans cells underlies the pathogenesis of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by a clonal proliferation and retention of cells with a Langerhans cell (LC)-like phenotype at various sites within the body. The present study set out to elucidate whether aberrant expression of chemokine receptors or dysregulation of chemokine production in LCH lesions could explain abnormal retention of these cells. Immunohistochemical analysis on 13 LCH biopsies of bone, skin, and lymph node all expressed the immature dendritic cell (DC) marker CCR6 on the lesional LCs and absence of the mature DC marker CCR7. Furthermore, regardless of the tissue site, LCH lesions markedly overexpressed CCL20/MIP-3alpha, the ligand for CCR6. The lesional LCs appeared to be the source of this CCL20/MIP-3alpha production as well as other inflammatory chemokines such as CCL5/RANTES and CXCL11/I-TAC. These may explain the recruitment of eosinophils and CD4+CD45RO+ T cells commonly found in LCH lesions. The findings of this study emphasize that, despite abundant TNF-alpha, lesional LCs remain in an immature state and are induced to produce chemokines, which via autocrine and paracrine mechanisms cause not only the retention of the lesional LCs but also the recruitment and retention of other lesional cells. We postulate that the lesional LCs themselves control the persistence and progression of LCH.

Anti-C1q autoantibodies deposit in glomeruli but are only pathogenic in combination with glomerular C1q-containing immune complexes.

Anti-C1q autoantibodies are present in sera of patients with several autoimmune diseases, including systemic lupus erythematosus (SLE). Strikingly, in SLE the presence of anti-C1q is associated with the occurrence of nephritis. We have generated mouse anti-mouse C1q mAb's and used murine models to investigate whether anti-C1q autoantibodies actually contribute to renal pathology in glomerular immune complex disease. Administration of anti-C1q mAb JL-1, which recognizes the collagen-like region of C1q, resulted in glomerular deposition of C1q and anti-C1q autoantibodies and mild granulocyte influx, but no overt renal damage. However, combination of JL-1 with a subnephritogenic dose of C1q-fixing anti-glomerular basement membrane (anti-GBM) antibodies enhanced renal damage characterized by persistently increased levels of infiltrating granulocytes, major histological changes, and increased albuminuria. This was not observed when a non-C1q-fixing anti-GBM preparation was used. Experiments with different knockout mice showed that renal damage was dependent not only on glomerular C1q and complement activation but also on Fcgamma receptors. In conclusion, anti-C1q autoantibodies deposit in glomeruli together with C1q but induce overt renal disease only in the context of glomerular immune complex disease. This provides an explanation why anti-C1q antibodies are especially pathogenic in patients with SLE.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, ß-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB­EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB­EGF had the highest mRNA expression and HB­EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB­EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB­EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB­EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB­EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB­EGF. Therefore, we propose an autocrine EGFR stimulation model in cervical carcinomas.

Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections.

Immune-cell infiltration is frequently seen within human solid tumors. A detailed phenotypic analysis of these cells may aid in the understanding of an antitumor immune response. Standard hematoxylin/eosin and conventional immunohistochemical stainings are helpful, but have major limitations in the number of markers that can be identified and localized per tissue section. Therefore, we developed a combined fluorescence and brightfield microscopic technique by using both immunofluorescence and immunogold-silver methods, thereby discriminating three different leukocyte markers plus one tumor marker simultaneously in a single section. This enabled us to study both phenotype and location of infiltrating immune cells in colorectal tumors. We used a two-step staining in which primary and secondary antibodies were selected for minimal cross-reactivity. Furthermore, the secondary fluorescent antibody conjugates were selected for minimal spectral overlap. For computer-assisted analysis the brightfield microscopy image was combined with the fluorescence microscopy images. This combination of techniques provides a powerful tool for detailed multiparameter microscopic analysis of formalin-fixed, paraffin-embedded tissue sections in general and for tumor-immune cell infiltration in particular.

Association of Lung Inflammatory Cells with Small Airways Function and Exhaled Breath Markers in Smokers - Is There a Specific Role for Mast Cells?

BACKGROUND: Smoking is associated with a mixed inflammatory infiltrate in the airways. We evaluated whether airway inflammation in smokers is related to lung function parameters and inflammatory markers in exhaled breath. METHODS: Thirty-seven smokers undergoing lung resection for primary lung cancer were assessed pre-operatively by lung function testing including single-breath-nitrogen washout test (sb-N2-test), measurement of fractional exhaled nitric oxide (FeNO) and pH/8-isoprostane in exhaled breath condensate (EBC). Lung tissue sections containing cancer-free large (LA) and small airways (SA) were stained for inflammatory cells. Mucosal (MCT) respectively connective tissue mast cells (MCTC) and interleukin-17A (IL-17A) expression by mast cells was analysed using a double-staining protocol. RESULTS: The median number of neutrophils, macrophages and mast cells infiltrating the lamina propria and adventitia of SA was higher than in LA. Both MCTC and MCT were higher in the lamina propria of SA compared to LA (MCTC: 49 vs. 27.4 cells/mm2; MCT: 162.5 vs. 35.4 cells/mm2; P<0.005 for both instances). IL-17A expression was predominantly detected in MCTC of LA. Significant correlations were found for the slope of phase III % pred. of the sb-N2-test (rs= -0.39), for the FEV1% pred. (rs= 0.37) and for FEV1/FVC ratio (rs=0.38) with MCT in SA (P<0.05 for all instances). 8-isoprostane concentration correlated with the mast cells in the SA (rs=0.44), there was no correlation for pH or FeNO with cellular distribution in SA. CONCLUSIONS: Neutrophils, macrophages and mast cells are more prominent in the SA indicating that these cells are involved in the development of small airway dysfunction in smokers. Among these cell types, the best correlation was found for mast cells with lung function parameters and inflammatory markers in exhaled breath. Furthermore, the observed predominant expression of IL-17A in mast cells warrants further investigation to elucidate their role in smoking-induced lung injury, despite the lack of correlation with lung function and exhaled breath parameters.

Sdhd and SDHD/H19 knockout mice do not develop paraganglioma or pheochromocytoma.

BACKGROUND: Mitochondrial succinate dehydrogenase (SDH) is a component of both the tricarboxylic acid cycle and the electron transport chain. Mutations of SDHD, the first protein of intermediary metabolism shown to be involved in tumorigenesis, lead to the human tumors paraganglioma (PGL) and pheochromocytoma (PC). SDHD is remarkable in showing an 'imprinted' tumor suppressor phenotype. Mutations of SDHD show a very high penetrance in man and we postulated that knockout of Sdhd would lead to the development of PGL/PC, probably in aged mice. METHODOLOGY/PRINCIPAL FINDINGS: We generated a conventional knockout of Sdhd in the mouse, removing the entire third exon. We also crossed this mouse with a knockout of H19, a postulated imprinted modifier gene of Sdhd tumorigenesis, to evaluate if loss of these genes together would lead to the initiation or enhancement of tumor development. Homozygous knockout of Sdhd results in embryonic lethality. No paraganglioma or other tumor development was seen in Sdhd KO mice followed for their entire lifespan, in sharp contrast to the highly penetrant phenotype in humans. Heterozygous Sdhd KO mice did not show hyperplasia of paraganglioma-related tissues such as the carotid body or of the adrenal medulla, or any genotype-related pathology, with similar body and organ weights to wildtype mice. A cohort of Sdhd/H19 KO mice developed several cases of profound cardiac hypertrophy, but showed no evidence of PGL/PC. CONCLUSIONS: Knockout of Sdhd in the mouse does not result in a disease phenotype. H19 may not be an initiator of PGL/PC tumorigenesis.

TGF-beta1 drives partial myofibroblastic differentiation in chondromyxoid fibroma of bone.

Chondromyxoid fibroma (CMF) is a rare benign cartilaginous bone tumour with a lobular architecture containing stellate and myofibroblast-like spindle cells. The aim of this study was to investigate the presence, spatial distribution, and extent of myoid differentiation in CMF and to evaluate a possible causative role for TGF-beta1 signalling, which is known to promote smooth muscle actin (SMA) expression. Twenty cases were studied for immunoreactivity for muscle-specific actin (MSA), SMA, desmin, h-caldesmon, calponin, TGF-beta1, and plasminogen activator inhibitor type 1 (PAI-1). The extent of myofibroblastic differentiation was further investigated ultrastructurally, including immuno-electron microscopy using antibodies against MSA and SMA, focusing upon the different cell types in CMF. The expression of potential genes driving this process was quantified by Q-RT-PCR (TGF-beta1, fibronectin, its EDA splice variant, and PAI-1). Tumour cells, especially those with a spindled morphology, showed diffuse immunoreactivity for MSA, SMA, TGF-beta1, and PAI-1, while desmin, h-caldesmon, and calponin were absent. Ultrastructurally, neoplastic cells showed the presence of myofilaments and rare dense bodies, which were more prominent in spindle cells and less so in chondroblast-like cells. Immuno-electron microscopy confirmed the actin nature of these myofilaments. No fibronexus was identified. The functional activity of TGF-beta1 was demonstrated by the identification of PAI-1, a related downstream molecule both immunohistochemically as well as by Q-RT-PCR. There was a linear correlation between TGF-beta1 and PAI-1 expression. Fibronectin-EDA levels were low. We have therefore substantiated the presence of morphological, immunohistochemical, and immuno-electron microscopic partial myofibroblastic differentiation in CMF, driven by TGF-beta1 signalling.

EMMPRIN-induced MMP-2 activation cascade in human cervical squamous cell carcinoma.

Tumor progression and recurrence of cervical cancer is associated with upregulation of matrix metalloproteinase 2 (MMP-2). We evaluated the location, origin and activity of MMP-2 in cervical squamous cell carcinomas in comparison with MT1-MMP (MMP-14), TIMP-2 and extracellular matrix metalloproteinase inducer (EMMPRIN). Positive immunostaining for MMP-2 in malignant cells was detected in 83% of the patients. Two patterns of tumor cell MMP-2 staining were observed: either homogenous in all tumor cells or confined to the cells neighboring the stroma (tumor-border staining pattern, TBS). Fluorescence in situ zymography showed active MMP-2 mainly around tumor nodules displaying TBS. The MMP-2 staining of TBS tumors correlated significantly with the presence of TIMP-2 and MT1-MMP, proteins involved in docking MMP-2 to the cell surface and essential for MMP-2 activation. In situ mRNA hybridization in TBS tumors demonstrated more abundant presence of MMP-2 mRNA in neighboring myofibroblasts than in the adjacent tumor cells. Moreover, the TBS MMP-2 pattern correlated with the presence of EMMPRIN (p = 0.023), suggesting that tumor cells induce MMP-2 production in nearby stromal cells. This pro-MMP-2 could subsequently be activated on tumor cells via the presence of MT1-MMP and TIMP-2. The biological relevance of this locally activated MMP-2 was underscored by the observation that only the TBS pattern of MMP-2 significantly correlated with decreased survival. In conclusion, the colocalization of EMMPRIN, MT1-MMP and TIMP-2 in human cervical carcinomas seems to be involved in a specific distribution pattern of tumor cell bound MMP-2, which is related with local proteolytic activity and therefore might be associated with worse prognosis of the patients.

High-resolution multi-parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin-embedded tissues.

The accuracy of DNA ploidy measurements of paraffin-embedded tissues is limited by the lack of resolution and the inability to identify the DNA diploid population unequivocally in bimodal DNA histograms. A multi-parameter DNA flow cytometric method has been developed that enables the simultaneous detection of neoplastic and stromal cells in samples from dewaxed 50 microm sections or 2 mm diameter punches of archival tissue blocks. The method combines heat pretreatment in sodium citrate buffer and subsequent enzymatic dissociation with a collagenase/dispase mixture. Cells were simultaneously stained for keratin (FITC), vimentin (R-PE), and DNA (PI) before flow cytometric analysis. The method was applied to 12 paraffin-embedded cervical carcinomas and four colorectal carcinomas. In all cervical cancers, distinct keratin-positive and vimentin-positive cell populations were observed. While the exclusive vimentin-positive cell fractions always yielded unimodal DNA content distributions, bimodal distributions were observed for the keratin-positive cell fractions in nine cervical carcinomas, whereas one cervical carcinoma showed three distinct G0G1 populations. Coefficients of variation of the G0G1 peaks ranged from 1.70% to 4.79%. Average background, aggregate, and debris values were 14.7% (vimentin-positive fraction) and 33.8% (keratin-positive fraction). Flow sorting confirmed that the exclusively vimentin-positive cell fractions represent different normal stromal and infiltrate cells that can serve as an internal ploidy reference enabling discrimination between DNA hypo-diploid and DNA hyper-diploid tumour cell subpopulations. The neoplastic origin of the keratin-vimentin co-expressing cells from two cervical carcinomas was confirmed by genotyping of flow-sorted samples revealing loss of heterozygosity (LOH) of 6p. This improved method obviates the need for fresh/frozen tumour tissue for high-resolution DNA ploidy measurements and enables the isolation of highly purified tumour subpopulations for subsequent genotyping.

High numbers of tumour-infiltrating activated cytotoxic T lymphocytes, and frequent loss of HLA class I and II expression, are features of aggressive B cell lymphomas of the brain and testis.

Loss of both HLA class I and class II expression in B cell lymphomas is a mechanism of escape from a cytotoxic T lymphocyte (CTL) immune response and will therefore give a strong selective survival advantage in tumours expressing strong immunogenic antigens. We investigated loss of HLA expression using specific antibodies on tissue sections from 254 B cell lymphomas originating from nodal and different extranodal sites in relation to numbers of tumour-infiltrating T cells. Complete loss of HLA class I and II was observed in a minority of the nodal, stomach, and skin lymphomas but in the majority of the lymphomas originating from the testis and the CNS. Interestingly, relatively high percentages of activated CTLs were detected in both primary testicular and CNS lymphomas compared to lymphomas at other sites, with highest percentages in the testis (p < 0.0001). We conclude that loss of both HLA class I and II expression occurs very frequently in lymphomas originating from the testis and the CNS as compared to nodal and some other extranodal sites. The presence of high percentages of activated CTLs in the testicular and CNS lymphomas suggests that loss of HLA expression provides a strong growth advantage for lymphoma cells in these immune-privileged sites.

Beta-glucan enhanced killing of renal cell carcinoma micrometastases by monoclonal antibody G250 directed complement activation.

Metastases from renal cell carcinomas (RCC) are resistant to radiation and chemotherapy but are relatively immunogenic. We have investigated the possibility to eliminate human RCC micrometastases using MAb G250. G250 penetrates human micrometastases completely in a spheroid model and induces complement deposition rapidly on the outmost cell layers. However, complement dependent cytotoxicity (CDC) was barely detected using either (51)chromium release assays or confocal microscopy, due to relatively low expression of the G250 antigen and the effect of membrane bound complement regulatory proteins. Addition of blocking anti-CD59 MAbs enhanced formation of C5b-9 and consequently complement mediated lysis (13%). Complement assisted cellular cytotoxicity (CACC) was not detectable, although the iC3b ligand and CR3 receptor were present on respectively target and effector cells. Addition of soluble beta-glucan induced the killing of MAb and iC3b opsonized spheroids by effector cells (6-21%). Despite a lower affinity for G250 antigen, a bispecific anti-G250*anti-CD55 MAb enhanced cell killing in spheroids comparable to the parental G250 MAb. Our results suggest that complement-activating G250 in combination with anti-mCRP MAbs is able to kill human RCC cells in micrometastasis in vitro. For CACC the presence of CR3-priming beta-glucan seems to be obligatory. In vivo, bi-MAb may be more effective as therapeutic agent due to its increased C5a generating properties.