Angiogenesis and lymphangiogenesis in thyroid proliferative lesions: relationship to type and tumour behaviour.

The role of angiogenesis and lymphangiogenesis in thyroid cancer pathogenesis has not been elucidated. Patterns for tumour behaviour and metastasic spread vary according to tumour type and whether differences in the angiogenic or lymphangiogenic phenotype influence the route for tumour metastases or determine a more aggressive behaviour has not been fully explored. The angiogenic and lymphangiogenic phenotypes of a large cohort of thyroid proliferative lesions (n=191) were studied. Using immunohistochemistry for CD34, lymphatic vessel endothelial receptor-1 (LYVE-1) (specific markers for vascular and lymphatic endothelium respectively), vascular endothelial growth factor (VEGF-A), VEGF-C and fibroblast growth factor-2 (FGF-2), this study analyses microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic factors in normal thyroid (NT; n=19), multinodular goitre (n=25), toxic multinodular goitre (n=8), Graves' hyperplasia (n=22), follicular adenoma (n=54), papillary carcinoma (PC; n=27), incidental papillary microcarcinoma (PMC; n=8), follicular carcinoma (FC; n=20) and medullary carcinoma (MC; n=8). MVD was decreased in proliferative lesions, benign and malignant, compared with NT (P<0.0001). In contrast, VEGF-A expression was increased in thyroid carcinomas (PC, FC and MC) when compared with PMC, benign lesions and NT (P<0.0001). LVD was higher in PC and PMC (P=0.001), and VEGF-C expression was increased in PC (P<0.0001). Despite higher LVD and increased expression of VEGF-A and VEGF-C in thyroid cancers, these markers were not related to poor prognosis in terms of tumour size, multifocality and/or presence of lymphatic or distant metastases. In conclusion, angiogenesis is reduced in thyroid proliferative lesions compared with NT tissue. However, VEGF-A expression is upregulated in thyroid cancers. Lymphangiogenesis and VEGF-C expression are increased in thyroid tumours prone to lymphatic metastases. This may be an important mechanism underlying the differences in metastatic behaviour between papillary and follicular thyroid cancer.

The NHS breast screening programme (pathology) EQA: experience in recent years relating to issues involved in individual performance appraisal.

BACKGROUND: The original role of the National Health Service breast screening programme (pathology) external quality assessment (EQA) scheme was educational; it aimed to raise standards, reinforce use of common terminology, and assess the consistency of pathology reporting of breast disease in the UK. AIMS/METHODS: To examine the performance (scores) of pathologists participating in the scheme in recent years. The scheme has evolved to help identify poor performers, reliant upon setting an acceptable cutpoint. Therefore, the effects of different cutpoint strategies were evaluated and implications discussed. RESULTS/CONCLUSIONS: Pathologists who joined the scheme improved over time, particularly those who did less well initially. There was no obvious association between performance and the number of breast cancer cases reported each year. This is not unexpected because the EQA does not measure expertise, but was established to demonstrate a common level of performance (conformity to consensus) for routine cases, rather than the ability to diagnose unusual/difficult cases. A new method of establishing cutpoints using interquartile ranges is proposed. The findings also suggest that EQA can alter a pathologist's practice: those who leave the scheme (for whatever reason) have, on average, marginally lower scores. Consequently, with the cutpoint methodology currently used (which is common to several EQA schemes) there is the potential for the cutpoint to drift upwards. In future, individuals previously deemed competent could subsequently be erroneously labelled as poor performers. Due consideration should be given to this issue with future development of schemes.

Impact of a national external quality assessment scheme for breast pathology in the UK.

BACKGROUND: This article presents the results and observed effects of the UK National Health Service Breast Screening Programme (NHSBSP) external quality assurance scheme in breast histopathology. AIMS/METHODS: The major objectives were to monitor and improve the consistency of diagnoses made by pathologists and the quality of prognostic information in pathology reports. The scheme is based on a twice yearly circulation of 12 cases to over 600 registered participants. The level of agreement was generally measured using kappa statistics. RESULTS: Four main situations were encountered with respect to diagnostic consistency, namely: (1) where consistency is naturally very high-this included diagnosing in situ and invasive carcinomas (and certain distinctive subtypes) and uncomplicated benign lesions; (2) where the level of consistency was low but could be improved by making guidelines more detailed and explicit-this included histological grading; (3) where consistency could be improved but only by changing the system of classification-this included classification of ductal carcinoma in situ; and (4) where no improvement in consistency could be achieved-this included diagnosing atypical hyperplasia and reporting vascular invasion. Size measurements were more consistent for invasive than in situ carcinomas. Even in cases where there is a high level of agreement on tumour size, a few widely outlying measurements were encountered, for which no explanation is readily forthcoming. CONCLUSIONS: These results broadly confirm the robustness of the systems of breast disease diagnosis and classification adopted by the NHSBSP, and also identify areas where improvement or new approaches are required.

Telomerase activity in lesions of the thyroid: application to diagnosis of clinical samples including fine-needle aspirates.

The telomerase enzyme is capable of replacing telomeric DNA sequences that are lost at each cell division. It has been suggested that the function of this enzyme is necessary for cells to become immortal, and in concordance with this hypothesis, telomerase activity has been detected in malignant tumor cells, whereas the enzyme is inactive in normal somatic cells. The measurement of this activity in human tissue samples may have diagnostic value, and in this study, we examined whether such a measurement may be useful for the detection of malignant cells within the thyroid. Telomerase activity was assayed using the telomeric repeat amplification protocol and related to the histological diagnosis of thyroid biopsy tissue samples and of cells obtained from the thyroid by fine-needle aspiration (FNA). Extracts from 9 of 11 (82%) carcinoma biopsy tissue samples contained telomerase activity, whereas enzyme activity was detected in only 2 of 14 (14%) benign tissue sample extracts. These two positive cases were subsequently diagnosed as Graves' disease with severe lymphocytic infiltration. Five of six (83.3%) histologically confirmed carcinoma FNA samples were identified by using the telomeric repeat amplification protocol assay, and two samples considered to be suspicious by FNA cytology were also positive. Conversely, only 4 of 48 (8.3%) benign FNA samples had telomerase. These promising data indicate that this sensitive assay could become a useful adjunct to microscopic cytopathology in the detection of cancer cells in small tissue biopsies and in fine-needle aspirates of the thyroid.

Angiogenesis and lymphangiogenesis in parathyroid proliferative lesions.

Angiogenesis and lymphangiogenesis are involved in tumoral growth and metastatic spread. There is little information on angiogenesis and no available data on lymphangiogenesis in parathyroid glands (PTG). Using immunohistochemistry for CD34, LYVE-1 (specific markers for vascular and lymphatic endothelium, respectively), vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, this study analyzes microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic growth factors in 13 normal PTG, 77 parathyroid adenomas (PTA), and 17 primary parathyroid hyperplasia (PPH). MVD was higher in PPH and PTA, compared with PTG (P < 0.001). There was no difference in VEGF-A expression among groups. In contrast, FGF-2 expression was higher in PPH, compared with PTA and PTG (P < 0.0001). FGF-2 scores and MVD were significantly correlated (r = 0.43). LVD did not differ among groups, and VEGF-C expression was unrelated to LVD. There was no relationship between MVD and tumor behavior (adenoma size, PTH, or calcium). In conclusion, this study shows increased angiogenesis in parathyroid proliferative lesions compared with normal glands and suggests that FGF-2 is proangiogenic in parathyroid tissue. In PTA, tumor behavior is not related to angiogenic phenotype. This is the first demonstration of lymphatic vessels in PTG, but the lack of correlation with VEGF-C expression suggests that VEGF-C is not the primary lymphangiogenic factor.

Expression of intermediate filament proteins in normal and diseased thyroid glands.

A total of 67 samples from normal and pathological thyroid glands were stained (as formalin fixed paraffin sections) with a panel of monoclonal antibodies directed against intermediate filament proteins. The study confirmed previous reports of cytokeratin and vimentin coexpression in primary thyroid carcinomas, but coexpression was also detected in normal thyroid and in a range of benign conditions including follicular adenomas, Hashimoto's thyroiditis, and diffuse hyperplasia (thyrotoxicosis). Prekeratin expression was found (using antibodies recognising higher molecular weight cytokeratins) predominantly in areas of squamous change, independent of the underlying thyroid pathology. This study does not therefore support previous findings that prekeratin expression provides a reliable means of distinguishing follicular pattern papillary carcinoma from follicular carcinoma with its poorer prognosis or that it helps distinguish benign from malignant papillary lesions. No evidence of desmin or neurofilament expression was seen, and in particular, neurofilaments could not be detected in any of the cases of medullary carcinoma studied.

Histopathological assessment of metastasis.

In spite of advances in the fields of immunohistochemistry and molecular biology, in clinical practice much of the assessment of metastases still relies on light microscopy using conventional histological stains. This is not so much a reflection of a reluctance by histopathologists to adopt new techniques, but more an indication that for most malignancies an enormous amount of useful prognostic data can be gained from relatively unsophisticated assessment of tissues, and that many of the strongest studies of prognostic factors in malignancy predate the era of molecular diagnostics. Although it is undoubtedly true that newer techniques have added prognostic information in the assessment of many tumors, and many, such as the measurement of estrogen receptor status in breast cancer, could be considered routine, a skilled assessment of the morphology of the tissues still provides the fundamental basis of assessing prognosis in the vast majority of cases.

Experts and performance in histopathology--a study in breast pathology.

This study was undertaken to determine if it was possible to identify expertise within Histopathologists (trainees, district general pathologists and pathologists with a special interest in breast disease) using an objective measure of performance. The method of assessment of performance is based on the CWS (Cochran-Weiss-Shanteau) ratio formed by the individual's ability to discriminate between a spectrum of disease categories and their level of inconsistency when assessed at intervals. The slides circulated represented the spectrum of breast disease seen in routine practice. The results demonstrated the average CWS ratio to be lowest in trainees and highest in pathologists with a special interest in breast pathology although there was no statistical difference in the CWS scores obtained between the district general pathologists and pathologists with a special interest. Differences in inconsistency rather than discriminatory ability mainly accounted for the difference in the CWS ratio observed between the groups studied. The study shows that the CWS ratio is potentially a very useful tool in the assessment of pathologists with regard to assessing their progress through training.

Uterine tumours are a phenotypic manifestation of the hyperparathyroidism-jaw tumour syndrome.

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by parathyroid tumours, which are frequently carcinomas, and ossifying jaw fibromas. In addition, some patients may develop renal tumours and cysts. The gene causing HPT-JT, which is referred to as HRPT2 and is located on chromosome 1q31.2, encodes a 531 amino acid protein called PARAFIBROMIN. To date 42 mutations, of which 22 are germline, have been reported and 97% of these are inactivating and consistent with a tumour suppressor role for HRPT2. We have investigated another four HPT-JT families for germline mutations, searched for additional clinical phenotypes, and examined for a genotype-phenotype correlation. Mutations were found in two families. One family had a novel deletional-insertion at codon 669, and the other had a 2 bp insertion at codon 679, which has been reported in four other unrelated patients. These five unrelated patients and their families with the same mutation were not found to develop the same tumours, thereby indicating an absence of a genotype-phenotype correlation. An analysis of 33 HPT-JT kindreds revealed that affected women in 13 HPT-JT families suffered from menorrhagia in their second to fourth decades. This often required hysterectomy, which revealed the presence of uterine tumours. This resulted in a significantly reduced maternal transmission of the disease. Thus, the results of our analysis expand the spectrum of HPT-JT-associated tumours to include uterine tumours, and these may account for the decreased reproductive fitness in females from HPT-JT families.

GalNAc glycoprotein expression by breast cell lines, primary breast cancer and normal breast epithelial membrane.

Over-expression of N-acetylgalactosamine glycoproteins as detected by binding of the lectin from Helix pomatia (HPA), is associated with metastatic competence and poor patient prognosis in a range of human adenocarcinomas. These glycoproteins remain poorly characterised, and their functional role has yet to be elucidated. This study describes characterisation of a range of human breast/breast cancer cell lines for the expression of the N-acetylgalactosaminylated glycoproteins of interest, and their comparison with normal breast epithelium and a range of clinical breast carcinoma samples. Confocal and light microscopy studies revealed cytochemical HPA-binding patterns consistent with a fundamental disruption in normal glycobiosynthetic pathways attending increasing metastatic potential. We report the most complete comparative analysis of HPA-binding ligands from cultured breast cells, clinical breast carcinoma samples and normal breast epithelium to date. Lectin blotting identified 11 major HPA-binding glycoprotein bands common to both clinical tumour samples and breast cell lines and 6 of these bands were also expressed by samples of normal breast epithelium, albeit at much lower levels. Moreover, very marked quantitative but not qualitative differences in levels of expression consistent with metastatic capability were noted.

Measuring proliferation in routine fine needle aspirates. Immunocytochemical detection of bromodeoxyuridine incorporation and Ki-67 expression in breast aspirates.

Two simple quantitative means of measuring tumour proliferation which can be applied to cytological material are described. One method involves immunocytochemical staining of cytological smears prepared from breast aspirates with the monoclonal antibody Ki-67. The other method involves incubation of aspirated material with 5-Bromo-2-deoxyuridine (BrdU). Direct measurement of the S phase of the cell cycle is feasible in breast fine needle aspirates by Bromodeoxyuridine incorporation and subsequent immunocytochemical detection. The proliferation indices obtained correlate with those derived from Ki-67 staining. This technique is suitable for routine use in the assessment of tumour proliferation.

Immunocytochemical staining of serous effusions with the monoclonal antibody Ber-EP4.

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One effusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.

Granular cell tumours revisited. An immunohistological and ultrastructural study.

Twenty-five granular cell tumours were stained with a panel of antibodies to histiocytic, muscle, neural, neural crest, epithelial and endothelial markers. Electron microscopy was also performed in six cases. Twenty-four of the cases were similar morphologically and immunocytochemically. One case with features of an endothelial origin is described. The present study strongly supports the viewpoint that granular cell tumours are a distinct entity rather than being the common appearance of a group of lesions of differing histogenesis. Origin from a neural crest-derived peripheral nerve-related cell is favoured.

Medullary carcinoma of the thyroid: management of persistent hypercalcitonaemia utilizing [99mTc] (v) dimercaptosuccinic acid scintigraphy.

Whole body scintigraphy with [99mTc] (v)dimercaptosuccinic acid (pentavalent DMSA) was performed in seven patients with histologically confirmed medullary carcinoma of the thyroid (MCT). Six of these patients had undergone previous thyroid resections for MCT and, although asymptomatic at the time of pentavalent DMSA scintigraphy, had persistent and serial elevations in their plasma calcitonin levels. One additional patient was scanned before and after total thyroidectomy for MCT. The pentavalent DMSA scintigram demonstrated either local neck recurrence (three patients) or distant metastases (two patients) in five of the six asymptomatic patients. In one asymptomatic patient only equivocal neck uptake was demonstrated. Since he had only minimal calcitonin elevations, repeat neck exploration was not performed. The one patient studied before thyroid resection for MCT demonstrated neck uptake before, but not after, total thyroidectomy. The results of the scintigrams had significant impact on patient care and resulted in neck re-exploration (three patients), neck biopsy (one patient), and lumbar spine biopsy and subsequent radiotherapy (one patient). These data demonstrate pentavalent DMSA to be a sensitive localizing agent in the evaluation of asymptomatic MCT patients with hypercalcitonaemia. Accurate targeting of treatment may be shown in due course to have a beneficial impact on survival.

Fine needle aspiration in plastic surgery outpatients: a retrospective study.

A survey suggested that fine needle aspiration cytology of masses in plastic surgery outpatient clinics was suboptimal. A cytopathologist gave training in the technique and the effectiveness of this intervention was audited. A total of 236 aspirates were taken from 147 patients in the earlier time period and 215 from 149 in the later period. The overall inadequate aspirate rate remained constant at 43%. The most common reasons for poor aspirates were excess blood, unrepresentative adipose tissue and insufficient cellular material. When the specimen was adequate after training, the sensitivity and specificity of the investigation were 96% and 100%, respectively. We present methods for sample optimization. Alternative strategies may be to limit aspiration to one clinician or to refer the patient to a cytopathologist experienced in the technique.

An immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration.

The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P. falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.

Autoimmunity to insulin receptor and hypoglycaemia in patient with Hodgkin's disease.

A 76-year-old man with fasting hypoglycaemia had impaired in-vitro binding of insulin to erythrocyte receptors. The immunoglobulin fraction of his plasma inhibited binding of insulin to normal donor erythrocytes in vitro. Autoantibodies may have stimulated the insulin receptor and produced hypoglycaemia. Hodgkin's disease developed and may have induced the autoimmunity. The hypoglycaemia did not respond to plasmapheresis or azathioprine alone, but it remitted after the addition of prednisolone, and the erythrocyte receptor binding of insulin became normal.