Nitric oxide synthase expression and enzymatic activity in human brain tumors.

Nitric oxide (NO) is synthesized by NO synthases (NOS), existing in 3 isoforms. NO influences a great variety of vital functions including vascular tone and neurotransmission. Under conditions of excessive formation, NO emerges as an important mediator of neurotoxicity in a variety of disorders of the central nervous system (CNS). Inhibitors of NOS are available that may modify the activity of all isoforms, which may be of clinical relevance. The expression of the 3 NOS isoforms nNOS, iNOS and eNOS and NOS enzymatic activity was examined in 40 patients with primary CNS tumors (gliomas WHO grades I - IV and meningeomas WHO grades I - III) and in 13 patients with metastases from adenocarcinomas or malignant melanomas. A polyclonal antibody directed against nNOS and monoclonal antibodies directed against iNOS and eNOS were used for immunohistochemical staining. NOS enzymatic activity, measured by labeled arginine to citrulline conversion, was assessed in tissue specimens obtained from the same tumors. NOS data were compared with clinical variables and the degree of edema as judged from MR scanning. nNOS expression was increased in tumor cells of glial neoplasms and most pronounced in high-grade tumors, WHO grades III and IV, and in the carcinoma and melanoma metastases. Low-grade gliomas, WHO grades I and II and meningeomas expressed no or only little nNOS. iNOS was only expressed in a few tumors. eNOS was expressed sporadically in the tumor cells while the expression was increased in vascular endothelial cells in both the tumor itself and the peritumoral area of glial neoplasms, and in metastases. eNOS expression was sporadic in endothelial cells of meningeomas. NOS enzymatic activities were heterogeneous among tumor types (0 - 13.8 pmol/min/mg of protein) without correlation to the NOS expression found by immunohistochemical techniques. Likewise, NOS activity and expression was not correlated to the clinical scores or brain edema. In conclusion, nNOS expression may be a putative useful indicator of brain tumor differentiation and malignancy. The enhanced expression of eNOS in vascular endothelial cells of glial neoplasms and metastases raises the possibility that NO production in tumor endothelial cells may contribute to tumor blood flow regulation and possibly brain edema.

US-guided fine-needle aspiration biopsy of thyroid nodules.

PURPOSE: To determine which nodular areas provide most adequate and accurate material for cytology during US-guided fine-needle aspiration biopsy (FNAB) of thyroid nodules. MATERIAL AND METHODS: In a prospective multicentre study, US-guided FNAB (0.8 mm) was performed in 342 thyroid nodules (338 patients, 285 women, 18-83 years, 285 palpable). Histology was obtained in 169 nodules, revealing 75 neoplasms (44.4%) including 18 cancers (10.7%). In 206 solid nodules, 2 passes (central and peripheral) were compared. In 82 complex nodules, 2 passes (solid area and cystic area) were compared. RESULTS: The inadequacy rate was 16.4% and the false-negative rates were 2.2% and 28.1% for the diagnosis of cancer and neoplasm, respectively. In solid nodules, material was more adequate with peripheral passes compared to central passes, but the difference was not significant. In complex nodules, the inadequacy rate was much higher for passes in cystic areas (80.5%) compared to passes in solid areas (46.3%). CONCLUSION: Cystic areas due to degeneration seldom provided adequate material when aspirated. Aspiration should always be obtained from solid areas, at best using US-guidance. Adenomas and non-neoplastic nodules were difficult to distinguish.

Nitric oxide synthase expression of oligodendrogliomas.

In the central nervous system, nitric oxide (NO) has a variety of biological functions including vasorelaxation and neurotransmission. The synthesis of NO is catalyzed by NO synthases (NOS) existing in 3 isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). NO synthase has implications in the pathophysiology of primary glial brain tumors with enhanced expression of nNOS and eNOS in high-grade astrocytic tumors, WHO grades III and IV. Only minor groups of pure oligodendrogliomas have been investigated. The aim of the investigation was to study the expression of the 3 NOS isoforms in this genetically divergent group of primary gliomas and to correlate the findings with tumor grade and expression pattern for the major group of gliomas--the astrocytomas. We examined the NOS expression in 35 oligodendrogliomas, WHO grade II, and 7 anaplastic oligodendrogliomas, WHO grade III, by immunohistochemical methods using formalin-fixed paraffin-embedded material. We observed only a minor expression of nNOS and sparse expression of eNOS in the tumor cells, but a vivid expression of eNOS in the vascular endothelial cells in both the tumor and the surrounding tissue. The rich expression of eNOS in oligodendroglioma vessels independent of tumor grade may suggest that blood flow and angiogenesis in these richly vascularized tumors are modified by NO. Interestingly, enhanced expression of inducible NOS was observed in the oligodendroglial tumor cells in 19 of 35 oligodendrogliomas (54%) and in 2 of 7 anaplastic oligodendrogliomas (29%). This is diverging for iNOS expression in astroglial tumors and the data could be indicative of iNOS exerting anti-tumor activity which may protract the progression from low-grade oligodendrogliomas to more anaplastic types.

Immunopathology of in situ seminoma.

In this study of the seminomatous human testis the composition, activity and apoptosis of lymphocytes infiltrating the immune-privileged seminiferous tubules with in situ seminoma were studied by immunohistochemistry and DNA fragmentation detection. Likewise the lymphocytes infiltrating the invasive seminomas were studied. The study showed equal numbers of CD4+ and CD8+ T cells and B cells, about 30% of the cells. Very few T gamma/delta and NK cells were present. The activity in terms of IL-2-R, FasL and perforin expression was low. Apoptosis of the lymphocytic cells was limited. No differences were observed between the lymphocytes in seminiferous tubules with in situ seminoma and the lymphocytes in invasive tumours. The study suggests that either specifically committed lymphocytes are not present or, if present, immune-suppressing mechanisms in addition to FasL may be working.

Immunohistochemical investigation of p53 and EGFR expression of oligodendrogliomas.

The biological behavior of oligodendrogliomas is somewhat unpredictable. A supplementing prognostic factor is, therefore, desirable. Thirty-two supratentorial pure oligodendrogliomas were studied immunohistochemically by exposing the tumors to a monoclonal antibody towards the p53 protein, and a polyclonal antibody against the epidermal growth factor receptor (EGFR). A mean p53 labeling index (% of tumor cells stained) of 8.6% and a weak EGFR expression in 18 of the oligodendrogliomas were found. Univariate analysis showed no correlation between p53, EGFR expression and prognosis. Multivariate analysis showed that age was a prognostic factor for survival.

Fas and Fas-ligand expression in seminomatous testes.

Sixteen seminomas with surrounding tissue containing normal and precancerous (cis) seminiferous tubules were examined for the expression of Fas (CD95, APO-1) and Fas ligand (FasL) (CD95L). This was done by analyzing frozen specimens using immunohistochemistry with antibodies directed against Fas and FasL. The study showed that varying numbers (mean approx. 20%) of Fas-positive lymphocytes were present among tumor-infiltrating lymphocytes, but very few FasL-positive lymphocytes. Fas was not expressed by normal seminiferous tubules and only occasional Fas-positive epithelial cells were seen in cis tubules. FasL was expressed in 9 out of 10 cases in virtually all normal seminiferous tubules, mainly as a thin layer at the base of the seminiferous epithelium. In precancerous tubules, this layer was discontinuous and less pronounced. Rete testis expressed FasL in 2 out of 2 cases with rete present and Fas in 1 out of 1 case. Invasive tumor cells did not express Fas or FasL. The data are discussed in relation to immune reactions to seminomas and to the concept of the testis being an immunologically privileged area.

No prediction of recurrence of meningiomas by PCNA and Ki-67 immunohistochemistry.

Immunohistochemistry for the expression of the proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 was studied in 16 non recurring meningiomas, 11 meningiomas recurring as benign tumors, 6 recurring as atypical meningiomas and in 9 recurring as malignant meningiomas. Non recurring meningiomas were defined in this study as tumors without recurrence at least 8 years after surgery. In addition 16 benign recurrences, 14 atypical- and 12 malignant meningiomas were studied. In each group great variation of labeling indices (LI) = per cent of tumor cells labeled was observed, especially of PCNA LIs. The non recurring meningiomas displayed lower mean LI for PCNA and Ki-67 than did the recurring meningiomas of all groups but the differences were not statistically significant. The same pattern was seen when totally resected tumors were studied alone. Benign-, atypical-, and malignant meningiomas had labeling indices that were related to the grade of malignancy. Only PCNA LIs of atypical- and malignant meningiomas were statistically significantly higher than PCNA LIs of non recurring meningiomas. The study indicates that PCNA and Ki-67 are of minor value as predictors of recurrence of benign meningiomas.

Sertoli cells, but not tumor cells, of seminoma in situ express ICAM-1.

Tumor and surrounding testicular tissue from six seminomas and one combined seminoma/embryonal carcinoma were examined for the expression of ICAM-1, VCAM-1 and ELAM-1. This was done by immunohistochemical staining of frozen samples using monoclonal antibodies and the avidin-biotin/ peroxidase or alkaline phosphatase staining method. ICAM-1 was expressed by Sertoli cells of intratubular germ cell neoplasia, but not by any of the cells in normal seminiferous epithelium, or by neoplastic germ cells whether invading or not. In addition inflammatory cells and endothelium expressed ICAM-1. VCAM-1, and also occasionally ELAM-1, was expressed only on endothelial cells in and outside the tumors. These results are discussed in relation to lymphocytic infiltration and immune surveillance of seminomas and T-cell tolerance to the antigens of the immunologically privileged seminiferous epithelium.

HLA class I antigens are expressed by Sertoli cells of intratubular germ cell neoplasia.

Five frozen samples from seminomas and one from a combined seminoma/embryonal carcinoma and surrounding tissue were examined for the presence of HLA class I molecules using an antibody directed against a monomorphic epitope of the 45 kDa long chain of HLA-A, -B and -C molecules. Only tubules with intratubular germ cell neoplasia stained positively, while normal seminiferous tubules and invasive tumor cells stained negatively. The immunohistology with anti-HLA class I antibody was compared to that with anti-beta 2-microglobulin antibody which reacts with the light nonpolymorphic chain of all HLA class I molecules. Immunohistology with these two antibodies was indistinguishable. This result combined with immunohistology using anti-PLAP indicates that the stained cells are Sertoli cells. The results are discussed in relation to lymphocytic infiltration and immune surveillance of seminomas.

Sertoli cells of intratubular germ cell neoplasia express beta 2 microglobulin.

The cells in intratubular germ cell neoplasia in the vicinity of 38 germ cell tumors of the testis, including 20 pure seminomas, were studied for the expression of beta 2-microglobulin (beta 2m), the constant component of all HLA class I molecules. Immunohistochemistry using antibodies towards beta 2m, vimentin, placental alkaline phosphatase, and ferritin was employed. Whereas the intratubular cells in normal testis are beta 2m negative, beta 2m positive cells were identified in intratubular germ cell neoplasia tubules in 55 per cent of all tumors and in 60 per cent of the seminomas. The tubules with beta 2m positive cells were located in areas with invasive tumor or in the vicinity of such areas. The beta 2m positive cells were identified as Sertoli cells by morphology and by their staining with anti-vimentin. Neoplastic germ cells, identified by morphology and staining with anti-placental alkaline phosphatase and anti-ferritin were beta 2-microglobulin negative. The most intensely beta 2m-stained Sertoli cells were found in tubules with high concentrations of neoplastic germ cells. Intensely stained Sertoli cells were also found in 'Sertoli cell only' tubules inside invasive tumors and in areas without lymphocytic infiltration. The cells in adjacent normal tubules were beta 2m negative.

Beta 2-microglobulin expression of AIDS-related and classical Kaposi's sarcoma.

The expression of beta 2-microglobulin, the invariable light chain of HLA class I molecules, of Kaposi's sarcoma from 11 AIDS patients and from 11 patients without known immunodeficiency was studied by immunohistochemistry using a polyclonal antibody to beta 2-microglobulin. The staining intensity of spindle cells in these lesions was scored in a semiquantitative system. We found that the spindle cells of Kaposi's sarcomas from AIDS patients showed significantly increased staining intensity for beta 2-microglobulin compared to those of Kaposi's sarcomas from non-AIDS patients. The results may indicate that Kaposi's sarcomas developing in immunocompromised individuals, such as AIDS patients, are not subject to immune selection by T cells eliminating HLA class I high-expressing tumor cells, while this may be the case in non-AIDS patients. Alternatively, the results may be caused by differences in the activity of cytokines, which upregulate the expression of HLA class I molecules on the cell surface.

Observer variability in the assessment of type and dysplasia of colorectal adenomas, analyzed using kappa statistics.

PURPOSE: The aim of this study was to establish the intraobserver and interobserver variability in the assessment of histologic type (tubular, villous, and tubulovillous) and grade of cytologic dysplasia (mild, moderate, and severe) in colorectal adenomas. METHODS: One hundred eighty-seven slides of adenomas were assessed twice by three experienced pathologists, with an interval of two months. Results were analyzed using kappa statistics. RESULTS: For agreement between first and second assessment (both type and grade of dysplasia), kappa values for the three specialists were 0.5345, 0.9022, and 0.4100, respectively. Agreement was better for type than for dysplasia. The strength of agreement was moderate for Observers A and C and almost perfect for Observer B. Agreement between all three observers was seen in 35.2 percent for both type and dysplasia in 61 percent for type and in 47.8 percent for dysplasia. The kappa values for Observer A vs. B and Observer C vs. B were 0.3480 and 0.3770, respectively (both type and dysplasia). Values for type were better than for dysplasia, but agreement was only fair to moderate. CONCLUSION: The interobserver agreement was moderate to almost perfect, but the intraobserver agreement was only fair to moderate. A simpler classification system or a centralization of assessments would probably increase kappa values.

Are pathologists biased by clinical information?: A blinded cross-over study of the histopathological diagnosis of mesothelial tumours versus pulmonary adenocarcinoma.

In a blinded cross-over design, we studied whether three pathologists were biased by clinical information when making histopathological diagnoses of adenocarcinoma of the lung and benign and malignant mesothelial tumours. Furthermore, the interobserver variation of these diagnoses was assessed. Forty-one cases of adenocarcinoma of the lung and mesothelial tumours were assessed by three pathologists in four rounds. In the first two rounds, slides stained by H&E and clinical information were available. Slides and information were matched so that a specific slide in one round was given clinical information suggesting adenocarcinoma and in the other round, the clinical information suggested mesothelial tumour. In the third and fourth rounds, a panel of immunohistochemical stains was added. The clinical information was matched in the same way as in the first and second rounds. Bias by clinical information was observed when the diagnoses were made on slides stained by H&E, while no bias could be demonstrated when immunohistochemical reactions were included. The reproducibility also improved significantly when these slides were available.

Expression of beta 2-microglobulin by human benign and malignant mesenchymal and neurogenic tumours.

Human myosarcomas, liposarcomas, meningosarcomas, glioblastomas and malignant schwannomas, their benign counterparts and normal cells from which these tumours derive, were examined for the expression of beta 2-microglobulin (beta 2m). Formalin fixed specimens from these tumours were studied by light microscopy employing the immunoperoxidase method with the use of antibodies directed towards beta 2m. The malignant tumours showed a broad spectrum from unstained to strongly stained tumours, most pronounced among myosarcomas. In addition, most stained tumours displayed a mosaic staining pattern in that unstained areas alternated with stained. There was a tendency towards increased staining for beta 2m in malignant compared to normal cells; this was also observed in the benign tumours although to a lesser degree. The results differ from most earlier studies, mainly of carcinomas which have shown a tendency towards down-regulation of MHC I molecules on the tumour cells. The results are discussed in relation to concepts of immune surveillance of tumours.

Expression of beta 2-microglobulin by premalignant epithelium.

Many human tumors express low amounts of HLA class I molecules relative to the normal cells from which they are derived. From experimental work it is clear that the malignant behavior of a tumor cell may depend on its MHC class I expression. Therefore, it is of obvious interest to study the HLA class I expression of human tumors in their various stages. We have studied the HLA class I expression by the cells in premalignant epithelial lesions and invasive carcinoma of the bladder and uterine cervix using immunoperoxidase staining for beta 2-microglobulin of paraffin-embedded tissue. We here assume that beta 2-microglobulin expression by malignant and premalignant cells equals HLA class I expression. Thirty-two of the 36 invasive tumors expressed less overall beta 2-microglobulin than cells from the normal epithelium. In contrast, approximately two-thirds of 34 premalignant bladder epithelia and 47 premalignant cervix epithelia displayed higher overall beta 2-microglobulin expression than the normal epithelium. Thus, a systematic large-scale elimination of HLA class I high-expressing tumor cell variants may take place only after the tumor penetrates the basement membrane.

Melanotic neuroectodermal tumour as a predominant component of an immature testicular teratoma. Case report with immunohistochemical investigations.

A case of melanotic neuroectodermal tumour (MNT), or so-called retinal anlage tumour, as a predominant component of an immature testicular teratoma is presented. The patient was a 17-year-old man who furthermore had a mature mediastinal teratoma. The MNT was composed mainly of two cell types: small immature neuroblast-like cells and large columnar or cuboidal epithelial-like cells with or without melanin granules. The tumour cells were arranged in solid formations, nests, cords, alveolar and pseudoglandular structures with cleft-like or glomeruloid-like spaces. Myogenic differentiation was found in minor foci. Immunohistochemistry showed both neuroepithelial and mesenchymal features with positive staining reaction for neuron-specific enolase (NSE), S-100 protein (S-100), melanoma antigen (HMB45), cytokeratin and vimentin. Vimentin, desmin and actin were present in the myoid cells. To the best of our knowledge this is the first reported case of MNT originating in the testis. As this tumour component occurred in an immature teratoma, neuroectodermal differentiation of germ cell origin is considered most likely.

The muscularis mucosae of the human urinary bladder. Implications for tumor staging on biopsies.

The human urinary bladder wall is traditionally described as being without a muscularis mucosae. A consecutive one year material of 772 bladder biopsies from 171 patients were examined for the proposed presence of lamina muscularis mucosae (MM). MM was observed in 15% of the biopsies and in 35% of the patients and graded into three patterns according to its continuity. Biopsies with transitional cell carcinomas were reviewed in order to find out whether MM-positive biopsies had been staged correctly. The data are discussed in relation to earlier studies on the subject.

Bilateral mixed germ cell sex-cord stroma tumour in a young adult woman. Case report.

The first Scandinavian case of mixed germ cell-sex cord stroma tumour is presented. The tumour occurred in an anatomically and genetically normal young adult woman. The tumour exhibited mainly two histopathological patterns. Larger parts consisted of abundant connective tissue containing cellular nests with numerous germ cells and a few sex cord derivatives. Minor parts showed a tubular pattern composed of tubules of sex cord elements containing scattered germ cells. Immunohistochemical stains for cytokeratin, vimentin, placental alkaline phosphatase and various other substances were all negative. This case is in addition the first reported case where the tumour was bilateral. The second tumour occurred in the contralateral ovary three years after excision of the first one and showed the same histopathological pattern as in the predominant part of the first.

Influence of drainage conditions on mucosal bladder damage by indwelling catheters. II. Histological study.

A previous study demonstrated a strong but short-lasting suction through the catheter eyes by a hydro-dynamically generated negative pressure fluctuation terminating bladder evacuation in some frequently occurring circumstances of indwelling catheter drainage. This report regards the biological effect. Fifteen successive evacuations on such drainage conditions in each of 4 anesthetized pigs were followed by cystectomy and histological examination. All bladders presented small swollen areas, histologically showing localized mucosal elevations dominated by edema of lamina propria and submucosa, occasionally with urothelial thinning or defects. Drainage with suction prevented in 3 animals caused normal bladders. The changes were similar to those following hydro-statical suction and much like those of the "polypoid cystitis" so commonly occurring with indwelling catheters. This suggests both types of suction by ordinary drainage as a major pathogenetic factor in the latter condition. The clinical significance and the occurrence during regimes of straight drainage or intermittent clamping are discussed.