Detection of interleukin-8 mRNA and protein in human colorectal carcinoma cells.

Interleukin-8 (IL-8) is a member of the chemokine family of pro-inflammatory chemotactic cytokines and is secreted by some human colorectal carcinoma cell lines. We have used in situ hybridisation and immunohistochemistry to determine whether IL-8 mRNA and protein, respectively, are produced by human colorectal carcinoma cells in vivo. IL-8 mRNA was detected within the cytoplasm of tumour cells in all nine samples tested, including that of a tumour which had metastasised to a lymph node. Non-involved colonic mucosa within the same tissue blocks showed much weaker labelling. IL-8 protein was detected in 74% (23/31) of tumour samples and was mainly localised to the tumour cell cytoplasm. In 30% of cases, staining was heterogeneous, with between 1 and 30% of cells being positive. In some tumour cells, IL-8 showed a perinuclear distribution resembling that found by in situ hybridisation. Some infiltrating leucocytes, endothelial cells and fibroblast-like cells within the tumour sections were also positive for IL-8 mRNA and protein. The possibilities that colorectal tumours produce IL-8 to aid invasion and/or metastasis or as a tumour growth factor are discussed.

Loss of cytokeratin 14 expression is related to human papillomavirus type and lesion grade in squamous intraepithelial lesions of the cervix.

In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells. In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle--associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin 14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.

Implications on laboratory workload of breast cancer screening.

AIMS: To determine whether the recommended methods for the handling of breast excision biopsy specimens from screening patients cause more laboratory work than those used for non-screening patients. METHODS: All breast excision biopsy specimens from 1990 were identified. Ninety one came from patients identified during the prevalent round of breast screening. These were compared with 127 from non-screening patients operated on by the same surgeon. The workload in terms of initial blocks taken, cases which needed extra blocks or other additional work, and the time taken for delivery of slides to the pathologist were assessed. RESULTS: The screening cases required significantly more initial blocks than the non-screening cases (8.03 v 4.95; p = 0.000001). When looking at the malignant diagnoses this difference was maintained (7.74 v 6.02; p = 0.00014). CONCLUSIONS: Excision biopsy specimens from screening patients require more laboratory work per case than those from non-screening patients. The reasons for these differences lie in the nature of the specimens and their subsequent diagnoses, and in the initial indications for biopsy.

Human papillomavirus in squamous cell carcinoma of the oesophagus associated with tylosis.

Human papillomavirus (HPV) may have a pathogenic role in squamous cell carcinoma of the oesophagus. Tylosis, an inherited thickening of the skin of the palms and soles, was associated with a high risk of developing squamous cell carcinoma of the oesophagus among members of a large family in Liverpool. The resected carcinomas of the oesophagus was examined from four such patients with DNA probes to HPV types 6,11,16,18,31,33 and 35 using in situ hybridisation under conditions of high stringency. No reaction was detected. The oesophageal biopsy specimens from 10 tylotic subjects without carcinoma were also examined. No HPV DNA was detected. It is concluded that there is no evidence that HPV infection has a role in the development of squamous cell carcinoma of the oesophagus in tylosis.

Localization of C-X-C and C-C chemokines to renal tubular epithelial cells in human kidney transplants is not confined to acute cellular rejection.

Chemokines are important mediators of leucocyte chemoattraction to inflammatory sites. Previous work has shown that the expression of some chemokines is upregulated during renal transplant rejection. The objectives of the present study were to determine whether chemokine expression is increased during renal transplant rejection. Immunohistochemistry was used to localize the C-X-C (alpha) chemokine interleukin-8 (IL-8) and the C-C (beta) chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1beta (MIP-1beta) in 30 needle biopsies of human kidney transplants taken for diagnosis of renal dysfunction. Urine samples from transplant patients taken immediately prior to biopsy were assayed for chemokine content using enzyme-linked immunosorbent assays (ELISAs). Results from groups of patients having different clinicopathological diagnoses were then compared. All three chemokines were detected in most renal transplant biopsies showing acute cellular rejection but, although infiltrating leucocytes were often positive, staining was predominantly localized to renal tubular epithelium. Staining for MCP-1 was generally weaker than for the other chemokines, and collecting tubules were usually stained more strongly than proximal convoluted tubules. Tubular epithelial staining was also found in biopsies from patients without signs of acute cellular rejection. There were significantly higher amounts of IL-8 in the urine of patients with acute cellular rejection, even when patients with urinary tract infections were excluded, but mean titres of urinary MIP-1beta did not differ between patient groups. This was also found when titres were normalized for urine volume and creatinine levels. Production of IL-8, MCP-1 and MIP-1beta is not confined to kidney transplants showing acute cellular rejection, and may be a relatively nonspecific response of tubular epithelial cells to renal damage.

Demonstration of placental and placental-like alkaline phosphatase in non-malignant human tissue extracts using monoclonal antibodies in an enzyme immunoassay.

The occurrence and nature of heat-stable placental-type alkaline phosphatase (Pl-ALP) in extracts from a variety of non-malignant human tissues has been investigated using monoclonal antibodies in a sensitive solid-phase enzyme immunoassay. The presence of Pl-ALP was confirmed in testicular, cervical and lung tissue extracts, and trace amounts were also detected in extracts from mammary and ovarian tissue. Evidence is presented that normal testis contains at least two forms of Pl-ALP, the major component being an L-leucine-inhibitable placental-like enzyme which is not the D-variant of Pl-ALP. These results have a bearing on the occurrence of Pl-ALP and placental-like ALP activity in malignancy.

The immunohistological demonstration of carcinoembryonic antigen in intra-epithelial and invasive squamous carcinoma of the cervix.

Using the sensitive peroxidase, anti-peroxidase immunohistological technique, carcinoembryonic antigen (CEA) was studied in formalin-fixed, paraffin-embedded cervical specimens from 225 patients. CEA was demonstrated in 33% of moderate dysplasia, 78.5% of severe dysplasia, 79% of in-situ carcinoma and 78% of invasive carcinoma. CEA was not demonstrated in normal squamous epithelium, squamous metaplasia, reserve cell hyperplasia nor in mild dysplasia.

The presence and distribution of carcinoembryonic antigen in tumors of human minor salivary glands.

Using an immunoperoxidase method we have demonstrated carcinoembryonic antigen (CEA) in more than half of a series of 62 tumors of minor salivary glands. CEA was present in both benign and malignant tumors. Its presence was related mainly to the formation of well differentiated ductal tissue in the neoplasms rather than to the histologic type of tumor. Although of only limited diagnostic value, the histologic presence of CEA in a particular tumor may indicate the possibility of postoperative monitoring of saliva and plasma, particularly where residual tumor, metastases or recurrences are considered likely.

Carcinoembryonic antigen levels in cervico-vaginal fluid from patients with intra-epithelial and invasive carcinoma of the cervix.

Carcinoembryonic antigen (CEA) levels have been measured in cervico-vaginal washout fluid using the direct CEA Roche radioimmunoassay. Raised CEA levels were found associated with cervical intra-epithelial neoplasia and invasive carcinoma (85% above 300 ng/ml) compared with benign cervical lesions (29% above 300 ng/ml), suggesting that estimation of local CEA levels might be a useful adjunct to cytology and biopsy in the investigation of cervical neoplasia.

Evidence for keratinocyte immortalization in high-grade squamous intraepithelial lesions of the cervix infected with high-risk human papillomaviruses.

In this study, we demonstrate that expression of cyclin B protein is up-regulated and persists into the upper epithelial layers in parallel with cyclin A expression in high-grade squamous intraepithelial lesions (SIL) infected with human papillomaviruses 16, 31, 33, 51, 58, 66, and 67 (n = 33). In contrast, low-grade SIL infected with human papillomaviruses 16, 18, 31, 33, 39, 51, 52, 56, 58, and 66 (n = 27) show weaker cyclin B expression confined to basal and parabasal cells despite extension of cyclin A and Ki67 expression into superficial cells. Moreover, aneusomy is present in 20% of the high-grade lesions but in none of the low-grade lesions. The persistent expression of cyclin B in high-grade SIL, and the restriction of aneusomy to high-grade SIL suggest that there is cell cycle progression. In combination with in vitro studies, this provides evidence that high-grade SIL lesions have undergone immortalization.

Numerical abnormalities of chromosomes 1, 11, 17, and X are associated with stromal invasion in serous and mucinous epithelial ovarian tumours.

The clinical behaviour of ovarian tumours of low malignant potential (LMP) is unpredictable and it has been suggested that the majority of these lesions have no invasive potential. This study has analysed 92 epithelial ovarian tumours [11 mucinous cystadenomas, 18 mucinous LMP tumours, 15 mucinous carcinomas (9 FIGO stage I), 16 serous cystadenomas, 15 serous LMP tumours, and 17 serous carcinomas (11 FIGO stage I)] for numerical abnormalities of chromosomes 1, 11, 17, and X by interphase cytogenetics. Overall, numerical aberrations were identified in none of the cystadenomas, 15 per cent of serous LMP tumours, 17 per cent of mucinous LMP tumours, 67 per cent of mucinous carcinomas, and 82 per cent of invasive serous carcinomas. In mucinous LMP tumours, chromosome gains were associated with spindled nuclear morphology. Chromosome abnormalities were significantly more frequent in invasive mucinous (overall p< 0.01; stage I p< 0.05) and serous (overall p< 0.001; stage I p< 0.01) carcinomas than in the corresponding LMP tumours. No significant relationship between either stromal invasion or tumour type and the pattern of chromosome loss or gain was identified, although monosomy X was identified almost exclusively in invasive serous carcinomas. These observations are consistent with the concept that LMP tumours are unlikely to be precursors of ovarian carcinoma, but suggest that chromosome instability is important in the development of the invasive phenotype.

An analysis of referral patterns from a breast screening unit.

The reasons for referral from a breast screening unit are reviewed, together with the subsequent outcome and biopsy findings. 4.94% of persons attending were referred for assessment in a breast clinic, biopsy being performed on 2.09% with a detection rate of 4.1 cancers per 1000. 24.4% of the cancers were palpable and 36.6% were non-invasive. A rounded ill-defined mass was the commonest mammographic reason for biopsy. Cancer was found in 15.2% of cases with microcalcification. In no case did clinical examination in the screening unit reveal cancer which was not detected on mammography.

Light chain nephropathy. Case report.

Two patients with light chain nephropathy are described. The diagnosis and management of this rare condition is discussed.

Expression of human placental-type alkaline phosphatase in primary breast cancer.

A monoclonal antibody (H317) with specificity for the heat-stable placental-type alkaline phosphatase (PI-ALP) isoenzyme has been used to investigate the occurrence of PI-ALP in patients with primary breast carcinoma. All pre-operative plasma samples were negative for PI-ALP in a sensitive solid-phase enzyme immunoassay with a lower limit of detection of 0.1 U/l. In contrast, using a peroxidase-anti-peroxidase staining technique on fixed tissue sections, as well as enzyme immunoassay on fresh tissue extracts, PI-ALP could be demonstrated in the carcinomatous tissue of all seven patients investigated.

Interphase cytogenetic evidence for distinct genetic pathways in the development of squamous neoplasia of the uterine cervix.

Human papillomavirus (HPV) infection has been implicated as an etiologic factor in most cervical cancers. However, additional genetic alterations are thought to be required for the development of a carcinogenic genotype. In the present study, interphase cytogenetics utilizing pericentromeric probes specific for chromosomes 1, 3, 11, 17, 18, and X was performed on paraffin-embedded tissue sections from 25 high-grade squamous intraepithelial lesions (SILs) and 25 invasive squamous cell carcinomas (ISCCs) of the cervix. HPV infection was determined by both in situ hybridization and broad-spectrum GP5+/GP6+ PCR. HPV was identified in all high-grade SILs (HPV 16, n = 16; 18, n = 2; 26, n = 1; 31, n = 4; 45, n = 1; 66, n = 1) and 23 (92%) ISCCs (HPV 16, n = 19; 18, n = 2; 31, n = 1; 39, n = 1). Aneusomy was identified in 11 (44%) high-grade SILs and 18 (72%) ISCCs. In 18 (62%) of these, relative under-representation of chromosomes 3, 11, 17, and/or 18 was identified (8 high-grade SILs and 10 ISCCs). Tetrasomy of all six chromosomes was present in two high-grade SILs but no ISCCs. Twelve (48%) high-grade SILs and seven (28%) ISCCs were disomic with all six chromosome probes, and there was no relationship between HPV presence or type and chromosome pattern. The presence of distinct patterns of numerical chromosome abnormality in these lesions suggests that progression to high-grade SIL or invasive carcinoma can occur by more than one genetic pathway. The lack of correlation between chromosome pattern and HPV type indicates that these pathways are not HPV type-specific. Whether these patterns reflect differences in early gene expression, possibly related to viral integration, or differences in the biologic properties of HPV type variants remains to be established.

Investigation of expression of 5T4 antigen in cervical cancer.

A monoclonal antibody detecting amniotrophoblastic antigen 5T4 has shown reactivity against various neoplastic cell lines and tumour specimens but with a relatively restricted normal tissue expression. This antibody has been investigated as a potential indicator of premalignant changes identified as cervical intra-epithelial neoplasia and malignant cervical lesions using immunohistochemistry on frozen tissue biopsies. The basal cells of normal cervical stratified epithelium exhibited faint staining, but a general increase in intensity and extent of specific labeling of this tissue was seen from the first premalignant stage through to carcinoma. In most cases, this was in accordance with the distribution of dysplastic cells, and was accompanied by increased specific staining of the stromal tissue. All invasive squamous carcinomas of the cervix were 5T4 antigen positive. Common inflammatory non-malignant diseases did show a certain degree of epithelial and stromal reactivity. These results, showing 5T4 reactivity with neoplastic and pre-neoplastic lesions, may provide a quantitative basis for its potential use as a tumour marker in the immunochemical detection on immunoassay of cervical cancer.

Secretory immunoglobulin A in the vesical urothelium of patients with neuropathic bladder--an immunohistochemical study.

STUDY DESIGN: A pilot study was carried out on archival material of bladder biopsies taken during 1994 and 1995 from patients with neuropathic bladder. OBJECTIVES: To compare the pattern of immunostaining for sIgA in the urothelium of biopsies taken from neuropathic bladder with the biopsies obtained from patients with non-neuropathic bladders. SETTING: Regional Spinal Injuries Centre, Southport and Department of Pathology, Royal Liverpool University Hospital, Liverpool. METHODS: Formalin-fixed, paraffin-embedded biopsies of bladder mucosa taken from patients with neuropathic urinary bladder (n=43) during 1994 and 1995 were processed for immunostaining with rabbit polyclonal antibody for secretory component of IgA. Archival specimens of bladder biopsies from non-neuropathic bladder were stained as controls. All sections were stained contemporaneously. RESULTS: In all the control biopsies, strong immunostaining for sIgA was observed in the superficial cells of transitional epithelium. In the biopsies taken from patients with neuropathic bladder, immunostaining in the transitional epithelium was variable: strong in 14 cases; moderate in four; faint in 16; and absent in three. Immunostaining for sIgA was absent in all the five biopsies in which the urothelium had undergone squamous metaplasia. One biopsy showed intestinal metaplasia; immunostaining for sIgA was seen in the basal cells. CONCLUSION: Strong immunostaining for sIgA was observed in the urothelium of all biopsies taken from non-neuropathic bladder. In contrast to this, only 18 of 37 biopsies obtained from neuropathic bladder showed strong or moderate immunostaining for sIgA in the transitional epithelium. Spinal Cord (2000) 38, 378 - 381.