Comparative DNA methylation analysis in normal and tumour tissues and in cancer cell lines using differential methylation hybridisation

Immortalized human cancer cell lines are widely used as tools and model systems in cancer research but their authenticity with regard to primary tissues remains a matter of debate. We have used differential methylation hybridisation to obtain comparative methylation profiles from normal and tumour tissues of lung and colon, and permanent cancer cell lines originally derived from these tissues. Average methylation differences only larger than 25% between sample groups were considered for the profiles and with this criterion approximately 1000 probesets, around 2% of the sites represented on the array, indicated differential methylation between normal lung and primary lung cancer tissue, and approximately 700 probesets between normal colon and primary colon cancer tissue. Both hyper- and hypomethylation was found to differentiate normal tissue from cancer tissue. The profiles obtained from these tissue comparisons were found to correspond largely to those from the corresponding cancer cell lines, indicating that the cell lines represent the methylation pattern of the primary tissue rather well. Moreover, the cancer specific profiles were found to be very similar for the two tumour types studied. Tissue specific differential methylation between lung and colon tissues, in contrast, was found to be preserved to a larger extent only in the malignant tissue, but was not preserved well in the cancer cell lines studied. Overall, our data therefore provide further evidence that permanent cell lines are good model systems for cancer specific methylation patterns, but deviate with regard to tissue-specific methylation.     

Neoplastic response of turkey liver to MC29 leukosis virus.

Twelve to fourteen days after intravenous inoculation of MC29 virus (1 x 10(5)LD50) into 1-day-old turkeys, liver tumour developed in 100% of the infected animals and led to death of birds. The tumour proved to be hepatomas histologically and electron microscopically. Numerous C-type particles were detectable among the tumour cells, as well as budding from the cell membrane. C-type particles were also observed in the tumour-free liver tissue in the spaces between the cells, budding was not detectable. Large number of virus particles were found in spleen extracellularly and intracellular vacuoles. Kidney tumours did not develop, but a few extracellular virus particles were located in the tissue. The MC29 virus-induced primary liver tumour in the turkey seems to be a suitable model for the morphological study of the relationship between oncogenic viruses and eukaryotic cells.     

Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: Correlation with tumour type and progression

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.     

Immunocytochemical Staining For the C-Erbb-2 Gene Product In Breast Aspirates: A Preliminary Report

The results of the determination of c-erbB-2 gene expression by immunocytochemical staining of cytological aspirates, prepared by cytocentrifugation, have been compared with paraffin-embedded tissue sections from the same tumour. Our results show equivalent staining in 20/22 cases, with six cases being both scored positive and fourteen cases being both negative. Two samples gave conflicting results. One case was scored as being positive on the cytological aspirate, whereas in the tissue sections taken from the same tumour positive staining was only seen in areas of non-invasive intraduct carcinoma. This sample was scored as being negative. In another case, cytoplasmic staining with less than 50% of the cells showing any positivity was observed in the cytospin sample, with negative staining in the corresponding tissue section. We conclude that expression of c-erbB-2 immunostaining is detectable on cytological preparations prepared by cytocentrifugation but must be interpreted with caution in tumours which may have a large intraduct component or which give predominant cytoplasmic staining.     

The competent sentinel node: an association with an axillary presentation and an occult or a small primary invasive breast carcinoma

The concept of the sentinel node describes a primary or sentinel lymph node (SLN), which exists and through which tumour cells from a primary tumour in a particular location must first travel to spread to a particular regional lymph node group. In this series we present three patients presenting with a pathological axillary node associated with either an occult or very small primary breast cancer. In each case the primary tumour was found to have metastasised to the palpable node, however despite the significant enlargement of this node, no other axillary nodes were found to be affected on axillary node clearance. This has led us to postulate that the SLN in some cases contains unique characteristics that enable it to prevent further spread of the tumour up the lymphatic chain. Hence the term the competent sentinel node.     

Telomerase activity levels in the surgical margin and tumour distant tissue of the squamous cell carcinoma of the head-and-neck.

The survival of patients with a head-and-neck squamous cell carcinoma is determined by loco-regional recurrence and second primary carcinomas. As a complement to histopathology, molecular changes of tumour marginal and tumour distant tissue may confirm curative surgical tumour extirpation. We tested telomerase activity with PCR-ELISA kits.20 tumour margin biopsies were chosen by the surgeon from 20 patients. In addition, 3 tissue samples were taken from each of 20 additional patients, one from the carcinoma centre, the tumour margin and one distant from the tumour. 50% of the carcinoma centres were telomerase-positive. Thirteen of the 40 tumour margin samples showed increased telomerase levels, and in 3 of these residual carcinoma was histopathologically detected. Six of the 20 tumour distant tissues revealed increased telomerase levels. Telomerase positivity in carcinoma-free tumour margins correlated with a good prognosis. Confirmation of the results in a larger patient group is needed.     

Subcellular localization and polymorphism of peroxidase in horse-radish tumour and teratoma tissue

The localization of peroxidase in cells of horse-radish (Armoracia lapathifolia Gilib.) tumour and teratoma tissues was studied. Both tissue lines were derived from the same primary crown-gall tumour induced on the leaf fragments by a wild type of Agrobacterium tumefaciens B6S3. Enzymatic activity was measured in cell walls, high-density heterogeneous membrane fraction, microsomal and soluble (no particulate) fractions. The subcellular localization of enzymatic activity was distinct for each transformed tissue. Both tumour and teratoma showed similar isoenzyme patterns, but one soluble acidic isoperoxidase could be considered as a marker of cell differentiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometry

Appropriate methods for the analysis of microdissected solid tumour tissues by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing approximately 200 000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI-TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high-performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four-fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI-TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.     

''Ciliated'' Tumour Cells In Ascitic Fluid From Two Cases of Cystadenocarcinoma of the Ovary

We report two cases of primary carcinoma of the ovary in which 'ciliated' adenocarcinoma cells were found in the ascitic fluid. Transmission electron microscopy revealed that these were not true cilia but rather a prolific growth of abnormal microvilli. The cytological findings were compared with the histological appearances of the primary tumour. No ciliated cells were seen in the primary tumour, suggesting that the formation of the microvilli represented an independent proliferation of the cells in the fluid. Special staining reactions for mucin, alkaline phosphatase and epithelial membrane antigen were identical in the primary tumour and the cells in the ascitic fluid.     

MicroRNA‐379 suppresses osteosarcoma progression by targeting PDK1

Osteosarcoma is the most common primary bone tumour. Increasing evidence has demonstrated the pathogenic role of microRNA (miRNAs) dysregulation in tumour development. miR‐379 was previously reported to function as an oncogenic or tumour‐suppressing miRNA in a tissue‐dependent manner. However, its function in osteosarcoma remains unknown. In this study, we found that the expression of miR‐379 was downregulated in osteosarcoma tissues and cell lines. Further functional characterization revealed that miR‐379 suppressed osteosarcoma cell proliferation and invasion in vitro and retarded the growth of osteosarcoma xenografts in vivo. Mechanistically, PDK1 was identified as the direct target of miR‐379 in osteosarcoma, in which PDK1 expression was up‐regulated and showed inverse correlation with miR‐379. Enforced expression of PDK1 promoted osteosarcoma cell proliferation and rescued the anti‐proliferative effect of miR‐379. These data suggest that miR‐379 could function as a tumour‐suppressing miRNA via targeting PDK1 in osteosarcoma.     

Viral oncogenesis in tumours of the central nervous system: reality or random association? A retrospective study on archived material

Central nervous system (CNS) tumours have devastating effects and are recurrent, with dismal prognosis (gliomas) or life‐threatening by the compression effect (meningiomas). This disease''s aetiology remains debatable. Over the last decade, the hypothesis that human viruses may be implicated in these tumours has been proposed. In this study, our aim is to examine the presence of 11 viruses in the most frequent CNS primary tumours. Using polymerase chain reaction (PCR), we assessed the viral presence in archived, paraffin‐embedded tumour tissues from 114 patients with glioma and meningioma and in the brain tissue from 40 controls lacking tumour pathology. We focused on candidate neuro‐oncogenic types (herpesviridae and polyomaviruses) and on human papillomavirus (HPV). HPV presence, for which involvement in these tumours was hardly investigated, was found to be associated with both tumour categories compared with controls (glioma, p = 0.032; meningioma, p = 0.032), whereas the presence of the neuro‐oncogenic viruses was found in a negligible number of both categories, suggesting a lack of association with the tumour presence. Moreover, our study reveals a positive correlation between HPV presence and glioma malignancy, and a negative correlation with meningioma grading. Our results suggest that the presence of HPV seems to be significantly associated with primary tumours of the CNS and its meninges.     

Endocrine cells in gastric carcinoma and adjacent mucosa. An immunohistochemical and ultrastructural study

Endocrine cells are often found in human gastric carcinoma and may be recognized by the immunoreactivity of their chromogranin A, peptides and biogenic amines content. Anti-chromogranin A was used to investigate the morphology of endocrine cells using light and electron microscope immunohistochemical techniques. The hormone content of endocrine cells was examined in both tumour tissue and tumour-adjacent mucosa. It was found that the endocrine cells in tumour tissue were malignant, often had amphocrine differentiation and did not resemble a normal cell type. The hormone content of endocrine cells in tumour tissue seldom corresponded to the hormonal content of endocrine cells in tumour-adjacent mucosa. In intestinal-type carcinoma and in some parts of diffuse-type gastric carcinomas, endocrine cell hyperplasia and an alteration of the differentiation in the tumour-adjacent mucosa were discovered. The distribution of endocrine cells in the tumour tissue was different in both types of gastric carcinoma. The results reported here suggest that endocrine cell differentiation of malignant endocrine cells in human gastric carcinoma develops in a different way from that of endocrine cells in tumour-adjacent mucosa, and as a result, diverse hormonal products may appear in tumour tissue.     

The effect of heating on the respiration of MC7 and D23 tumour tissue

The sensitivity of the tissue respiration of two tumours and liver slices to heat has been studied. The tumour tissue is sensitive to in vitro preincubation at temperatures above 43°C, whereas liver slices were less temperature sensitive. The inclusion of 1 mM tetracaine during preincubation sensitizes the tumour tissue to heating. In vivo heating of the tumour tissue at 44°C for 1h was not inhibitory of respiration when subsequently measured at 37°C. Mitochondria isolated from the D23 hepatoma tissue showed coupled respiration, however mitochondria isolated from in vivo heated tumour did not show coupled respiration. It contrast to mitochondria isolated from unheated tissue, these mitochondria lacked cristae and contained electron-dense granules, indicators of damage. The lack of effect of an in vivo heat-dose, known to cause tumour regression, on respiration and the reports that ATP levels are unaffected by such heating, suggests that cell respiration is not a primary lesion in cellular heat injury. This implies that the observed impairment of mitochondrial function following in vivo heating is best explained if the heating sensitized the mitochondria to subsequent damage during isolation.     

Branchial Cleft Anomalies: A Review of 87 Cases Treated at the Toronto General Hospital

The embryology, anatomy and pathology of branchial cleft anomalies are discussed and 87 cases reviewed.The most frequent anomaly was branchial cleft cyst, of which there were 77 cases. Treatment in all cases consisted of complete excision.There were five cases of external branchial sinus and five cases of complete branchial fistula. Sinograms were helpful in demonstrating these lesions. Excision presented little difficulty.No proved case of branchiogenic carcinoma has been found in the Toronto General Hospital. Five cases are described in which the original diagnosis was branchiogenic carcinoma—in four of these a primary tumour has already been found. The authors believe that the diagnosis of branchiogenic carcinoma should never be accepted until repeated examinations over a period of at least five years have failed to reveal a primary tumour.     

The synthesis of E-17 alpha-[131I]iodo-vinyl oestradiol and evaluation of its use as a radiotracer for oestrogen receptor positive breast tumours

17 alpha-Iodo-vinyl oestradiol binds with high affinity to the oestrogen receptor, is metabolically stable and has been shown to accumulate in oestrogen sensitive tissues of rodents. We have synthesised this compound and its 3-acetate, labelled with carrier free 125I and shown the accumulation of both compounds in rat uterus. 17 alpha-Iodo-vinyl oestradiol-3-acetate was prepared labelled with carrier free 131I and administered to twelve patients with suspected breast cancer, approximately 1 h before surgical removal of the primary tumour mass. At the time of surgery a sample of the tumour and a peripheral blood sample were taken. The ratio of radioactivity in the tumour to blood was determined. All tumours accumulated radioactivity and ratios ranged from 1.5:1 to 3.7:1. There was no correlation between the degree of accumulation and either cytosolic oestrogen or progesterone receptor concentration in the tumour. Analysis of blood revealed a single circulating species, 17 alpha-iodo-vinyl oestradiol. The results show that 17 alpha-iodo-vinyl oestradiol is metabolically stable and accumulates in breast tumours though this accumulation is not sufficient to permit imaging.     

On the significance of in situ production of oestrogens in human breast cancer tissue.

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

On the significance of in situ production of oestrogens in human breast cancer tissue

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.