Detection of herpes virus DNA in post-operative thyroid tissue specimens of patients with autoimmune thyroid disease.

AIM: To demonstrate any differences in the detection of herpes simplex virus type 1 and 2, cytomegalovirus, human herpes virus type 6 and 7 DNA from thyroid tissue blocks of patients with autoimmune thyroid disease and multi-nodular goiter and to propose few mechanisms, which could explain the possible role of herpesvirus infection in the development of thyroid autoimmune responses. MATERIAL-METHODS: Thyroid tissue specimens were obtained postoperatively from 4 patients with multinodular goiter and 18 patients with autoimmune thyroid disease (Graves' disease and Hashimoto thyroiditis). Herpes virus DNA was detected using polymerase chain reaction based assays. RESULTS: No statistically significant differences were observed between autoimmune thyroid disease and multinodular goiter tissue specimens concerning herpes simplex virus type 1, 2 DNA isolation (44.4% vs 0%, P=0.094), human herpes virus type 6 DNA isolation (11.1% vs 0%, P=0.48), human herpes virus type 7 DNA isolation (33.3% vs 25%, P=0.75). No CMV DNA was isolated from any tissue specimen. At least one kind of herpes virus DNA was detected in 13 out of 18 (72.22%) AITD tissue specimens and in 1 out of 4 (25%) MNG tissue specimens (P=0.01). CONCLUSIONS: Although no data are available relating the direct effect of herpes infection on thyroid epithelial cells, a better understanding of how an aberrant immune response against the thyroid gland is initiated and propagated through herpes virus infection is required. Elucidation of the underlying mechanisms may allow the development of new etiologically based therapeutic modalities.

Rare XXY/XX mosaicism in a phenotypic male with Klinefelter syndrome: case report.

Klinefelter syndrome represents the most commonly found human sex chromosomal abnormality. It is characterized by small, firm testes with hyalinization of the seminiferous tubules, elevated gonadotropins and azoospermia. Males with Klinefelter syndrome may have a 47,XXY or a mosaic 47,XXY/46,XY constitutional karyotype and varying degrees of spermatogenic failure. Mosaicism 47,XXY/46,XX with clinical features suggestive of Klinefelter syndrome, is very rare and so far only 10 cases have been described in literature [1,2,5,8,10,15,22,23,25,44]. We report here a case of a mosaic 47,XXY/46,XX infertile male in whom detailed cytogenetic, histological and molecular studies were performed. Cytogenetic analysis revealed 80% and 50% mosaicism for the 46,XX cell line in blood lymphocytes and in skin fibroblasts, respectively, and the presence of 47,XXY cells only, in cultured testicular tissue. Testicular histopathology revealed atrophy of the testes with no spermatogenesis and absence of germ cells. Molecular analysis showed paternal inheritance of the extra X chromosome.

Molecular cytogenetics of chromosome 11 in pituitary adenomas: a comparison of fluorescence in situ hybridization and DNA ploidy study.

Chromosome 11 abnormalities were detected by fluorescence in situ hybridization (FISH) technique and compared with DNA ploidy in 24 surgically removed pituitary adenomas. The tumors were diagnosed and classified by histology, electron microscopy, and pituitary hormone immunocytochemistry. They included 2 densely granulated somatotroph (DG-SM) and 4 sparsely granulated somatotroph (SG-SM) adenomas, 3 SG lactotroph (LT), 2 mixed somatotroph-lactotroph (SM-LT), 4 functioning corticotroph (CRT), 1 silent CRT subtype 1, 1 thyrotroph, 1 mixed thyrotroph-somatotroph, 2 gonadotrophs, and 4 null cell adenomas. FISH analysis with an alpha-satellite DNA probe specific for chromosome 11 showed numerical abnormalities in 16 functioning (94%) and 5 nonfunctioning (71%) adenomas. Ten functioning tumors showed aneuploid histograms, whereas the remaining and all nonfunctioning adenomas were diploid. Aberrant chromosome 11 signals were noted mostly in aneuploid adenomas involving 17% to 100% of their cell population. The severity of chromosome 11 aberrations in adenomas containing extra copies often correlated with a higher DNA index (DI). Monosomy 11 as dominant aberration was noted in a mixed SM-LT and to a lesser degree in 3 CRT adenomas involving 21% to 97% of their cell population. Two of these CRT adenomas were associated with normal DI, whereas the remaining third showed a high DI, indicating increased copy number of chromosomes other than of chromosome 11. In conclusion, chromosome 11 abnormalities are common in all types of pituitary adenomas, occurring more frequently in functioning tumors. Specific numerical abnormalities, such as monosomy and trisomy, tend to be associated with certain adenoma types, whereas tumors with extra chromosome 11 copies often exhibit aneuploid histograms.

Expression of the 27-kDa heat shock protein (HSP27) in gastric carcinomas and adjacent normal, metaplastic, and dysplastic gastric mucosa, and its prognostic significance.

PURPOSE: The investigation of heat shock protein 27 (HSP27) expression in gastric cancer and adjacent normal, metaplastic, and dysplastic gastric mucosa and its correlation with clinicopathological parameters and survival of patients. METHODS: Immunohistochemical methodology was performed on formalin-fixed paraffin-embedded sections by using a monoclonal anti-HSP27 antibody. HSP27 expression was screened and compared in 86 cases of gastric carcinoma and adjacent normal, metaplastic, and dysplastic gastric mucosa. RESULTS: In the normal mucosa, HSP27 was detected in 68 out of 86 cases (79%) and was more intense in the surface and upper two-thirds of gastric foveolae. In dysplastic gastric mucosa, HSP27 immunoreactivity was usually higher than that of the adjacent normal epithelium and was parallel to the severity of dysplasia. HSP27 expression was found in 54 out of 86 (62.7%) gastric carcinomas and was significantly related to more than six metastatic lymph nodes ( P =0.03). HSP27 expression was also higher in tumors of advanced stage and in those of female patients. HSP27 expression was associated with shorter overall survival in univariate analysis ( P =0.04), but this relationship was not retained in multivariate analysis. CONCLUSIONS: Our findings indicate that: i) HSP27 is commonly expressed in normal gastric epithelium where it seems to exert a protective role; and ii) HSP27 is involved in gastric carcinogenesis and its expression appears to be associated with parameters of unfavorable prognosis and shorter overall survival.

Androgen receptor (AR) expression is an independent unfavorable prognostic factor in gastric cancer.

PURPOSE: To investigate the expression of sex steroid receptors in gastric cancer and to correlate their tumor expression profile with the clinicopathological parameters and overall survival of the patients. METHODS: Immunohistochemical methodology was employed in formalin-fixed paraffin-embedded sections from 86 patients with gastric carcinoma. Monoclonal antibodies against androgen (AR), estrogen (ER), and progesterone (PR) receptors were used. Survival rates were estimated by the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis was performed by the Cox proportional hazards model. RESULTS: Fifteen (17.4%) cases of gastric adenocarcinomas were positive for AR, two (2.3%) were positive for PR and three (3.5%) were positive for ER. Significantly higher AR expression was found in tumors with metastases to lymph nodes (P = 0.03). Patients with AR-positive tumors (AR+) had worse prognosis than (AR-) patients (median survival 9 months vs 24 months, P = 0.03). Patients with AR- and heat shock protein 27 (HSP27)-positive tumors (AR+/HSP27+) had a median survival of 6 months, whereas (AR-/HSP27-) patients had a median survival of 42 months (P = 0.017). Multivariate analysis revealed that AR expression and UICC stage were independent factors of unfavorable prognosis (P = 0.037 and P = 0.0055, respectively). CONCLUSIONS: Identification of AR-positive gastric carcinomas in gastric biopsies may warrant a more aggressive therapeutic approach and anti-androgen or AR-targeted agents may represent a novel strategy in tackling this devastating malignancy.

K-ras mutation in Greek patients with poorly and moderately differenciated tumours of the lower intestinal tract.

The K-ras protooncogene is activated via mutations in approximately 40% of primary colorectal adenocarcinoma. This finding suggests that these genetic alterations are important events in the genesis of colon cancer and should be correlated with other parameters in order to infer some conclusions relevant to the etiology and the pathogenesis of this type of cancer. In our study we examined whether the incidence of K-ras mutations detected in 23 samples with colorectal adenocarcinomas, was related to different anatomical sites within the lower intestinal tract (transverse colon, descending colon and rectosigmoid region) and also the correlation between K-ras mutations and depth of invasion, level of tumour cell differentiation and metastasis to the regional lymph nodes. For this study the critical regions for the activation of the K-ras protooncogene were amplified by the PCR technique and the particular sequences analysed, after their membrane transfer, by differential hybridization with selected synthetic oligonucleotides. Our results demonstrated that 39% of the adenocarcinomas examined contained point mutations, and 66.6% of these were located in the second position of K-ras codon 12, whereas the other 33.3% were located in the second position of codon 13. 77.8% of the mutations were located at the rectosigmoid region and the relevance of the mutations was higher in poorly differenciated tumours. The depth of invasion was associated with the presence of a mutation whereas no correlation was found between the presence of a mutation anb the regional lymph node metastasis.

Nm23 expression in breast ductal carcinomas: a ten year follow-up study in a uniform group of node-negative breast cancer patients.

The nm23 gene was originally identified in murine melanoma cell lines as a putative metastasis suppressor gene. 1a a limited number of studies in breast carcinomas nm23 mRNA and/or protein levels were found to correlate inversely with lymph node metastases, and positively with the survival of patients. Using a monoclonal antibody to nm23-Hl protein we have examined the immunohistochemical expression of nm-23 in breast ductal carcinomas of 44 lymph node-negative patients with similar tumor pathologic features. The mean follow-up period of the patients was 138 months. Thirty two out of 44 tumors (72%) disclosed high immunohistochemical expression of nm23 protein and 12 (28%) low or negative expression. No correlation was observed between nm23 expression and the relapse or death rate of the patients. Similarly, no association was found between nm23 protein levels and estrogen receptor status or p53 protein. Our results do not seem to agree with the proposed antimetastatic property of nm23 protein, and indicate that its immunohistochemical determination has no prognose significance in the management of node-negative breast cancer patients.

Prognostic significance of apoptosis related proteins Bcl-2 and Bax in node-negative breast cancer patients.

In this immunohistochemical study we investigated the expression of Bcl-2 and Bax apoptosis related proteins in node-negative breast carcinomas. The results were correlated with the recurrence rate of the patients. In order to avoid the influence of the most important tumor prognostic parameters we selected two groups of node negative breast ductal carcinomas that were grade II according to Bloom and Richardson classification, had a diameter of 1-3 cm but differed in clinical outcome: 44 of the patients had a 10 year disease-free survival while 46 developed metastatic disease in the same period of time. Bcl-2 and to a lesser degree Bax expression were inversely related with distant metastases (Pbcl-2 = 0.007, Pbax = 0.03). Combined analysis of Bax/Bcl-2 expression in relation to clinical outcome showed that the absence of both factors was more strongly associated with the development of distant metastases (P = 0.006, Ptrend = 0.001). Our findings indicate that Bcl-2 and Bax apoptosis-related proteins are good indicators of prognosis in node-negative breast cancer patients, and their combined absence, suggestive of serious deregulation of the apoptotic process, may contribute to the biologic aggressiveness of the tumors.

Topoisomerase II alpha expression in squamous cell carcinomas of the head and neck.

The aim of the present study was to examine topoisomerase II alpha (topo II alpha) expression in cancers of the head and neck and to establish a correlation with clinicopathological parameters and survival. Paraffin embedded tissue specimens (studied by immunohistochemistry), from 103 consecutive patients with squamous cell carcinoma of the head and neck regions, were examined on the primary tumor (96 patients) and on recurrence (7 patients). Immunostaining evaluation was quantified by examining at least 1,000 neoplastic cells and counting the percentage of positively stained nuclei. Topoisomerase II alpha expression was correlated with age, gender, stage, site of the disease, tumor differentiation, response to chemotherapy, disease-free survival and overall survival. More than half of the specimens had a high expression of topoisomerase II alpha (> or = 15% positive neoplastic cells). Topoisomerase II alpha expression was significantly higher in tumors of low and moderate differentiation versus tumors of high differentiation (P = 0.00001). There was also a significant difference in topo II alpha in specimens of responders to chemotherapy versus non-responders (P = 0.02), although the cytotoxic drugs used do not belong to topoisomerase II alpha antagonists. The correlation of high topoisomerase II alpha expression and stage of disease, age, gender, primary site, recurrence, disease-free survival and overall survival was not statistically significant. In conclusion, topoisomerase II alpha is highly expressed in histological specimens of the majority of patients with head and neck cancers; mainly, it is related to a significant degree to low and moderately differentiated tumors versus highly differentiated ones. High expression of topoisomerase II alpha is also significantly related to response to chemotherapy.

p53, p21 and p27 protein expression in head and neck cancer and their prognostic value.

Histological specimens from 62 laryngeal and 31 oral carcinomas were immunohistochemically assessed for p53, p21 and p27 proteins; cases with > 10% labelled nuclei were considered as positive. p21 showed higher expression in patients > 65-years-old (P = 0.04), in chemotherapy responders (P = 0.02), and in stage III patients with longer overall survival (P = 0.02), representing the only independent prognostic factor in the multivariate analysis. In addition, stage III patients with p53-/p21+ showed the longest survival whereas those with p53+/p21- tumors showed the shortest overall survival (P = 0.02). A significant influence on the survival of stage III patients was also found for the combinations of p21 and p27 proteins with p21+/p27- imparting the best and p21-/p27+ the worst prognosis (P = 0.04). p27 expression was significantly related to oral cancer specimens (P = 0.04) and to moderate and high tumor grade (P = 0.01). p53 expression was not significantly related to any of the examined clinicopathological characteristics. Our findings indicated that, by functionally promoting apoptosis, p21 seems to play a key role in the successful response to chemotherapy and may be considered as a predictive factor of a better prognosis in stage III patients with head and neck cancers.

Detection of human papillomavirus DNA in nongenital seborrhoeic keratoses.

The histological similarities of seborrhoeic keratoses and common warts led to the investigation of the possible occurrence of human papillomavirus DNA (HPV-DNA) in a large number of nongenital seborrhoeic keratoses using the in situ hybridization technique. All specimens derived from normal skin (n = 173) were negative for the applied HPV-DNA probe, whereas the HPV genome was detected in 34 of 173 seborrhoeic keratosis specimens (19.65%). Of 34 HPV-positive specimens, 15 contained types 6/11 and 14 types 31/33/35, and 5 showed no positive reaction to the applied types. These results suggest that a considerable percentage of nongenital seborrhoeic keratoses may be related to an HPV infection.

A study of apoptosis in brain tumors by in situ end-labeling method.

Recognition of apoptotic cells has recently been facilitated by in situ end-labeling (ISEL) techniques which identify DNA strand breaks. The ISEL assay has been applied in 26 brain tumors, meningiomas, and gliomas of different grade of malignancy in order to determine the apoptotic index (AI). Apoptotic cells were readily found in all the examined tumors; in most cases the AI values were below 1%. Any significant relationship between AI and grade of malignancy could not be determined. However, the AI mean value of the meningiomas was higher than that of gliomas WHO II and III, respectively. Furthermore, it seemed that in the examined gliomas, with the exception of the glioblastoma group, low grade malignancy was related with higher AI values. The latter decreased gradually in the WHO II and III group of tumors, respectively. Our preliminary results indicate that an apoptotic process may play a significant role in brain tumor kinetics and will probably contribute to our understanding of the biologic behavior of those tumors.

Detection of herpes simplex virus DNA in human spermatozoa by in situ hybridization technique.

The role of herpes simplex virus (HSV) in male infertility has been investigated using the localizing ability of in situ hybridization technique. Sperm samples obtained from 80 men attending a maternity center because of fertility problems were classified by spermogram and analyzed for the presence of HSV-1 and HSV-2 DNA using digoxigenin-labelled DNA probes. HSV DNA was detected in the nuclei of spermatozoa in 37 semen samples (46%), HSV-1 specifically in 21 cases (26%) and HSV-2 in 16 cases (20%). HSV infection was almost three times more common in semens with a sperm count lower than 20 million/ml in relation to semens with a sperm count higher than 20 million/ml (70% versus 25.5%, P = 0.0001). The mean sperm count and motility for HSV positive semens were 23.5% and 36% respectively, whereas those of HSV negative semens were 53.2% and 47% respectively (P(count) = 0.0005 and P(motility) = 0.01). The number of HSV positive sperm cells ranged from 2 cells per specimen to 60% of the sperm cells. The mean number of HSV-2 labelled sperm cells per sample was 3.7% and that of HSV-1 1.5%. The percentage of hybridization positive sperm cells was also inversely correlated with sperm count and motility. Acyclovir therapy of eight males with HSV positive semens resulted in three successful pregnancies. In conclusion, HSV seems to play an important role in male infertility and the detection of this virus in the semen will not only allow substantial therapeutic interventions for the restoration of fertility but it will also contribute to the control of the transmission of HSV infection.

Detection of herpes simplex virus DNA in maternal breast milk by in situ hybridization with tyramide signal amplification.

In this study we investigated the prevalence of herpes simplex virus 1 (HSV-1) and 2 (HSV-2) in maternal breast milk using the technique of in situ hybridization combined with the sensitive detection system of tyramide signal amplification. Breast milk samples were collected from both breasts of 34 puerperals 4-5 days after delivery. HSV DNA was detected in 16 out of 34 examined breast milk specimens. HSV DNA was found in the milk of both breasts in 10 cases (62.5%), in that of left breast in 4 cases (25%) and in the milk of only the right breast in 2 cases (12.5%). HSV DNA was localized in the nuclei of mononuclear cells and to a lesser degree in the nuclei of epithelial cells. The number of HSV infected cells ranged from a few cells per sample to 20% of cells (mean value 9%, median value 5%). According to HSV typing, we found both HSV-1 and HSV-2 in 11 out of 16 positive cases and HSV-2 only in 5 cases. In conclusion, our findings indicate that HSV-1 and HSV-2 are shed into breast milk in a significant proportion of puerperals and breast-feeding may be an important route for the transmission of these viruses to infants.

Lectin histochemistry of laryngeal squamous cell carcinomas.

A panel of five biotinylated lectins was applied to study the presence and distribution of membrane carbohydrate residues in the normal laryngeal epithelium and in laryngeal squamous cell carcinomas (SCCs) of 86 patients with the avidin-biotin peroxidase complex technique. The lectin-binding pattern of well-differentiated SCCs was comparable to that of the spinous cells of the normal laryngeal epithelium. In the less differentiated SCCs, staining of the keratinocyte plasma membrane with lectins was either reduced or absent, indicating a decline in the glycosylation of cell surface glycoconjugates. The lectins applied here could be used in the rapid assessment of less-differentiated areas within a laryngeal SCC, but they cannot be regarded as reliable markers of laryngeal keratinocytes undergoing malignant transformation.

Effect of fixation on interphase cytogenetic analysis by direct fluorescence in situ hybridization on cell imprints.

The direct fluorescent in situ hybridization (FISH) technique using a centromerespecific DNA probe for chromosome 11 was applied on fresh tissue imprints from melanomas and pituitary adenomas. The cell imprints were fixed in acetone and formalin, and the influence of fixation was evaluated. Acetone fixed imprints gave a sharp and intense fluorescent signal in a large number of cells from both tumors without any pretreatment. In contrast, formalin fixed imprints disclosed a weak hybridization signal in a few cells even after careful enzymatic digestion. Direct FISH on acetone fixed cell imprints represents a simple and time saving technique applicable to any laboratory for the study of numerical chromosomal abnormalities.

A quality assurance exercise to evaluate the accuracy and reproducibility of chromogenic in situ hybridisation for HER2 analysis in breast cancer.

BACKGROUND: Chromogenic in situ hybridisation (CISH) is an alternative to immunohistochemistry or FISH for the assessment of HER2 oncogene status in breast cancer. Although CISH is being used increasingly in routine diagnostics, there are no established inter-laboratory quality assurance programmes for this test. METHODS: The reproducibility of HER2 CISH analysis was assessed when performed by seven different centres that use the test routinely in diagnostic service. RESULTS: The results from 28 cases showed overall concordance of 98.5% (192/195 tests; kappa coefficient 0.91). One of the discrepancies was due to the invasive carcinoma having been cut out in the sections received by two of the centres, and the other two were in the non-amplified/equivocal/low-amplified category. CONCLUSION: This is believed to be the first report of a quality assurance study assessing laboratories that use HER2 CISH routinely in clinical diagnostics. The results show that CISH is a robust technique providing a suitable assay for the frontline testing of HER2 status in breast cancer.

Immunophenotypic analysis of the p53 gene in non-melanoma skin cancer and correlation with apoptosis and cell proliferation.

BACKGROUND: Sunlight precipitates a series of genetic events that lead to the development of skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The p53 tumour suppressor gene, which plays a pivotal role in cell division and apoptosis, is frequently found mutated in sunlight-induced skin tumours. OBJECTIVE: To investigate the immunoreactivity of the p53 gene in non-melanoma skin cancers and to correlate its expression with apoptotic and cell proliferation markers. METHODS: We analysed 35 non-melanoma tumours including 19 BCCs and 16 SCCs from sun-exposed skin areas. p53 protein expression was studied immunohistochemically using the DO7 monoclonal antibody against wild-type and mutant p53 forms. The percentage of p53-immunopositive nuclei was measured by image analysis. Cell proliferation and apoptosis were also assessed by image analysis following Ki-67 immunostaining and application of the TUNEL method on paraffin sections, respectively. RESULTS: The percentage of p53-expressing cells varied from 3.5 to 90 in BCCs (median value 54.4%) and from 3.7 to 94 in SCCs (median value 40.3%). The mean value of Ki-67-positive cells was comparable in both groups of tumours with a mean value of 40.6% in BCCs and 34.6% in SCCs. Conversely, the TUNEL assay showed sporadic staining of apoptotic cells within the tumours with a mean value of 1.12% in BCCs and 1.8% in SCCs. p53 protein expression was correlated positively with cell proliferation (r = 0.75, P = 0.000001) and negatively with apoptosis (r = -0.23, P = 0.05). CONCLUSION: p53 immunoreactivity was high in the majority of the skin carcinomas examined and correlated positively with cell proliferation and negatively with apoptosis. The p53 protein overexpression appears to be related to an inactivated protein resulting from mutations of the p53 gene or other unclear molecular mechanisms.

Loss of basement membrane components laminin and type IV collagen parallels the progression of oral epithelial neoplasia.

AIMS: To determine the immunohistochemical localization of basement membrane components laminin and type IV collagen in premalignant and malignant lesions of the oral epithelium. METHODS AND RESULTS: Formalin-fixed tissue sections of 12 epithelial hyperplasias with no dysplasia and 30 dysplasias, clinically diagnosed as leukoplakia and/or erythroplakia, as well as 50 invasive squamous cell carcinomas, were stained with mouse monoclonal antibodies to human laminin and type IV collagen. Statistical analysis showed that there was a linear trend for discontinuous distribution of laminin from epithelial hyperplasia to epithelial dysplasia and invasive squamous cell carcinoma (P < 0.001). Laminin staining showed a linear trend for discontinuity with increasing grade of dysplasia (P < 0.05) and was more frequently discontinuous in areas of deep tumour invasion than in central or superficial areas (P < 0.05). Brush-shaped thickening and reduplication of the basement membrane were also identified. CONCLUSIONS: Alterations in the distribution of laminin and type IV collagen in oral premalignant and malignant lesions indicate that the loss of continuity of the subepithelial basement membrane parallels the progression of the neoplastic transformation process in oral epithelium.