Detection of early squamous metaplasia in bladder biopsies of spinal cord injury patients by immunostaining for cytokeratin 14.

STUDY DESIGN: A prospective, immunohistochemical study of bladder biopsies taken from spinal cord injury (SCI) patients. OBJECTIVES: To investigate whether cytokeratin 14 immunostaining may be useful to detect early squamous metaplasia in bladder biopsies from patients with SCI. SETTING: Southport, United Kingdom. METHODS: Biopsy of bladder mucosa was taken from adults with SCI, while they underwent an elective therapeutic procedure in the urinary tract. A total of, 54 biopsies, which showed transitional epithelium only with no evidence of squamous metaplasia on routine H&E staining, formed the study group. In all, 22 biopsies, which showed squamous metaplasia on routine H&E staining, acted as controls. All biopsies were benign with no evidence of dysplasia or malignancy. Immunohistochemical staining for cytokeratin 14 was performed on all biopsies in a single batch, using a standard avidin-biotin complex method. RESULTS: All control biopsies showed positive immunostaining for cytokeratin 14 in basal and parabasal cells in areas of squamous metaplasia. Of the 54 biopsies, which showed only transitional epithelium on H&E staining, immunohistochemistry for cytokeratin 14 showed no staining in 47 biopsies. The remaining seven biopsies showed positive immunostaining for cytokeratin 14 in the epithelium, in individual cells or clusters of basal cells, revealing unexpected early squamous metaplasia in these biopsies. CONCLUSION: Immunostaining for cytokeratin 14 identifies an early phenotypic switch from transitional to squamous epithelium in bladder mucosa. Cytokeratin 14 staining is sufficiently sensitive to identify early squamous metaplasia, which is not yet evident on examination of routine H&E stained sections. This early identification may be of use in alerting physicians to change bladder management regimens to prevent predisposition to recurrent urinary infection and progression of squamous metaplasia. A cost/benefit analysis should be performed to assess the feasibility of routine cytokeratin 14 immunostaining of bladder biopsies from SCI patients.

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.

Parathyroid hormone-related protein (1-34) and urothelial redifferentiation in the neuropathic urinary bladder.

STUDY DESIGN: A comparative study of immunostaining for parathyroid hormone-related protein (1-34) (PTHrP (1-34)) in the vesical epithelium of biopsies obtained from patients with non-neuropathic bladder and those with neuropathic bladder. OBJECTIVES: To investigate the immunostaining for PTHrP (1-34) in the control cases and in neuropathic bladders showing (1) normal transitional epithelium, (2) hyperplastic transitional epithelium, and (3) squamous metaplasia. SETTING: Regional Spinal Injuries Centre, and Department of Cellular Pathology, Southport & Ormskirk Hospitals NHS Trust, Southport, Department of Pathology, Royal Liverpool University Hospital and the Departments of Clinical Chemistry and Cell Biology, The University of Liverpool, Liverpool, England. METHODS: Cold cup biopsies of bladder mucosa were taken from patients suffering from neuropathic urinary bladder when they were undergoing a therapeutic procedure in the urinary tract. Immunohistochemistry was performed on these biopsy specimens using a rabbit polyclonal antibody raised to a synthetic peptide corresponding to human PTHrP (1-34). Control group (n=10) consisted of archival biopsies taken from non-neuropathic bladders. RESULTS: In the control group, the transitional epithelium showed no immunostaining, or at the most, very faint positive staining was seen in the transitional epithelium of non-neuropathic bladder. Positive immunostaining to PTHrP (1-34) was seen in the normal transitional epithelium of neuropathic bladder in nine of 13 cases. Hyperplastic transitional epithelium showed positive immunostaining for PTHrP (1-34) in 11 of 13 biopsies from patients with neuropathic bladder. Immunostaining for PTHrP (1-34) was observed in the metaplastic squamous epithelium in 14 of 17 cases with neuropathic bladder. CONCLUSION: The transitional epithelium of non-neuropathic bladder showed no immunostaining, or at the most, very faint positive staining for PTHrP (1-34). In contrast to this, positive immunostaining for PTHrP (1-34) was observed more frequently in the vesical epithelium of neuropathic bladder. This observation opens up avenues for innovative therapy with PTHrP or its analogues for possible modulation of urothelial differentiation in the neuropathic bladder.

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.

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.

Immunohistochemical study of parathyroid hormone-related protein in vesical transitional epithelium of patients with spinal cord injury.

INTRODUCTION: Parathyroid hormone-related protein (PTHrP), in addition to the well-established role in endochrondral bone development, is believed to be an important mediator of cellular growth and differentiation in a number of non-bony tissues. OBJECTIVES: To compare the immunohistochemical staining of vesical transitional epithelium to antibodies raised to synthetic peptides of PTHrP composed of amino acid sequences 43 - 52 and 127 - 138 in patients with spinal cord injury (SCI) and neuropathic bladder (n=14), and control patients with intact neuraxis and no history of bladder cancer (n=10). SETTING: Male SCI patients registered with Regional Spinal Injuries Centre, Southport, England. INTERVENTION: Endoscopic cold cup biopsy from the trigone of the urinary bladder was taken from patients with SCI while they were undergoing a therapeutic procedure in the urinary bladder. The control samples of bladder biopsies were taken from the archives of the Department of Histopathology, District General Hospital, Southport. Immunohistochemistry was performed using rabbit antibodies raised against synthetic peptides of human PTHrP (43 - 52) and PTHrP (127 - 138). The biopsies were examined for immunostaining of transitional epithelium. RESULTS: Of the 14 biopsies of SCI patients, positive immunostaining using antibodies to both the PTHrP peptides was found in four cases; five biopsies showed positive immunostaining only to anti-PTHrP (43 - 52); and five biopsies showed no immunostaining with either of the PTHrP peptides. In contrast, transitional epithelium in the biopsy specimens of ten control subjects with no history of bladder cancer showed no immunostaining with either of the PTHrP peptides. CONCLUSION: This study revealed that the transitional epithelium of neuropathic urinary bladder exhibits increased predilection for positive immunohistochemical staining for PTHrP (43 - 52), and to a lesser extent, to PTHrP (127 - 138), as compared to the vesical transitional epithelium of able bodied individuals with no history of vesical malignancy. The possible role of PTHrP in the cellular differentiation of urothelium of neuropathic bladder, and thereby, in the pathogenesis of cystitis in SCI patients, needs to be explored.

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.

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.

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.

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.

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.

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.

Light chain nephropathy. Case report.

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

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.

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.

Radionuclide imaging of epithelial ovarian tumours with 123I-labelled monoclonal antibody (H317) specific for placental-type alkaline phosphatase.

A murine monoclonal antibody, H317, specific for placental-type alkaline phosphatase was labelled with 123I and assessed as an imaging agent using a gamma camera computer system in 18 patients suspected of possible recurrent or metastatic ovarian cancer 1-4 years after removal of the primary tumour. Four patterns of distribution were visible: (1) normal uptake; (2) focal accumulation; (3) diffuse uptake; and (4) 'cold' areas. Six patients, five of whom were clinically negative for ovarian cancer, had normal scans; 11 patients, eight of whom were clinically positive, had increased uptake. One patient had visibly 'cold' areas. Findings were confirmed, where possible, at surgery.

Detection of placental-type alkaline phosphatase in ovarian cancer.

A monoclonal antibody, H317, has been used for the sensitive and specific detection of placental-type alkaline phosphatase (PLAP) in sera, solubilized tissue extracts and fixed tumour tissue sections from patients representing a variety of ovarian tumours. PLAP was detected in over 30% of these sera and in most solubilized tumour tissue extracts. There was no association between circulating PLAP levels and either tissue extract levels or immunohistological staining of ovarian tumour tissue sections with H317. Nevertheless, immunohistology demonstrated the heterogeneity of cellular localization of PLAP within different tumours, and can often be of value in localizing tumour tissue.

Serum progesterone concentration during the luteal phase in women with benign breast disease.

Earlier studies of the serum concentration of progesterone during the luteal phase in women with benign breast disease have produced conflicting results. Serum progesterone profiles were therefore measured in relation to cyclical breast pain and in biopsied benign disease. No evidence of progesterone deficiency was found.

Serum oestradiol-17 beta and prolactin concentrations during the luteal phase in women with benign breast disease.

Serum profiles of both oestradiol and prolactin were measured during the luteal phase in normal women, in women with cyclical breast pain and in women with recently biopsied benign breast disease. The results suggest that, in women with benign breast disease, the concentration of both hormones may be increased, when compared to the normal controls, during the evening in the latter part of the luteal phase.