A Novel C-Terminal Mutation in Gsdma3 (C+/H-) Leads to Alopecia and Corneal Inflammatory Response in Mice.

Purpose: Mutations in the gene encoding Gasdermin A3 (Gsdma3) have been described to cause severe skin phenotypes, including loss of sebaceous glands and alopecia, in mice. We discovered a novel C-terminal mutation in Gsdma3 in a new mouse line and characterized a less frequently reported corneal phenotype, likely caused by degeneration of Meibomian glands of the inner eyelid. Methods: We used histologic methods to evaluate the effects of the C+/H- mutation on sebaceous gland and skin morphology as well as Meibomian glands of the inner eyelid and corneal tissue. Chromosomal aberrations were excluded by karyogram analyses. The mutation was identified by Sanger sequencing of candidate genes. Results: Analyses of skin samples from affected mice confirmed the frequently reported phenotypes associated with mutations in Gsdma3: Degeneration of sebaceous glands and complete loss of pelage. Immunologic staining of corneal samples suggested an inflammatory response with signs of neovascularization in half of the affected older mice. While the corneal phenotype was observed at irregular time points, mainly after 6 months, its appearance coincided with a degeneration of Meibomian glands in the eyelids of affected animals. Conclusions: The mutation described herein is associated with inflammation and neovascularization of corneal tissue. Simultaneous degeneration of Meibomian glands in affected animals suggested a change in tear-film composition as the underlying cause for the corneal phenotype. Our data further support that different pathogenic mechanisms underlie some of the reported mutations in Gsdma3.

Relation between human papillomavirus positivity and p16 expression in head and neck carcinomas--a tissue microarray study.

Overexpression of p16 has been demonstrated to be strongly related to the presence of HPV16, 18. Squamous cell carcinoma of the head and neck has been shown to be associated with human papillomavirus (HPV) infection. The main aim of this study was to investigate the relationship between HPV presence and p16 expression in a representative collection of 60 head and neck carcinomas by tissue microarrays. The presence of HPV (HPV6, 11-low risk, HPV16, 18-high risk) was detected by applying in situ hybridisation. P-16 protein expression was detected immunohistochemically. HPV6, 11 positivity was observed in 10 out of 60 carcinomas. HPV16, 18 presence was found in 30 out of 60 tumours. P-16 expression was detected in 35 out of 60 tumours. A statistically significant relationship was demonstrated between HPV16, 18 presence and increased expression of p16. Also the HPV6, 11 presence was significantly correlated with p16 immunoreactivity. Additionally, this study demonstrates that it is possible to analyse p16 expression and HPV presence by tissue microarrays.

Alterations of the retinoblastoma and p16 pathway correlate with promoter methylation in malignant fibrous histiocytomas.

Recent reports indicate that the alterations in the p16 and pRb pathways can influence tumour progression and poor prognosis in several tumours. The objective of this study was to analyse p16 and pRb expression in161 patients with malignant fibrous histiocytomas (MFH). By immunohistochemistry, p16 and pRb were demonstrated in 25% and 56% of MFH, respectively. Cox regression analysis demonstrated an independent prognostic influence of both genes. Generally, the loss of p16 and pRb expression correlated with poorer prognosis. Promoter methylation of p16 was found in 16/42 of p16 negative MFH and of pRb in 2/42 of pRb-negative MFH. It can be concluded that p16 and pRb alterations play an important role in the progression of soft tissue sarcomas.

Altered expression of DNA double-strand repair genes Ku70 and Ku80 in carcinomas of the oral cavity.

The relevance of double-strand DNA repair genes has been demonstrated in several tumours. The main aim of this study was to analyse the expression of the heterodimers Ku70 and Ku80, building regulatory subunits of the DNA-dependent protein kinase in 40 oral carcinomas. Ku70 expression was found in 87.5% of grade 1 and grade 3 tumours and in 82.9% of grade 2 carcinomas. Ku80 presence was noted in 87.5% of grade 1 tumours, 82.9% of grade 2 tumours and in all grade 3 tumours. Ku70-positive cells were present in 90.5% of tumours without and in 80% of tumours with lymphatic metastases. A similar relationship was found for Ku80 expression. Additionally, the expression of Ku70 was highly significantly related to smoking habits. Our results demonstrated that defects of DNA double-strand repair genes play an important role in the tumour progression of oral carcinomas.

Exonic deletions of mismatch repair genes MLH1 and MSH2 correlate with prognosis and protein expression levels in malignant melanomas.

The mutations of MLH1 and MSH2 have been reported to be responsible for malignant transformation and tumour progression in several sporadic tumours. Eighty-six primary malignant melanomas with known follow-up were investigated. Point mutations of DNA mismatch repair MLH1 and MSH2 in malignant melanomas were not found. Exon 12 (MSH2) was not present in 26 out of the 86 melanomas and exon 13 (MSH2) was lost in 25 of the tumours. The loss of exon 15 (MLH1) was observed in 22 out of the 86 tumours and the loss of exon 16 (MLH1) in 24 melanomas. The loss of exons correlated strongly with the loss of MLH1 and MSH2 protein expression. In multivariate analysis, including all 4 exons and expressions of MLH1 and MSH2, prognostic significance was found only for loss of exon 12 (MSH2) and loss of exon 15 (MLH1).

Loss of Ku70/Ku80 expression occurs more frequently in hereditary than in sporadic colorectal tumors. Tissue microarray study.

Ku70 and Ku80 proteins take part in the repairing of DNA double-strand breaks by their function as a regulatory subunit of the DNA-dependent protein kinase. In this study, expression of both genes was analyzed in colorectal carcinoma tissue arrays applying immunohistochemistry. Expression of both genes decreased along with pT stage. Significant differences in Ku70 and Ku80 expression were found between pT3 and pT4 as well as between pT2 and pT3 tumors, respectively. Loss of Ku70/Ku80 expression was more frequently observed in hereditary than in sporadic tumors. We conclude that expression of Ku70/Ku80 genes is down-regulated in colorectal carcinoma and that defects of these genes are more frequently observed in hereditary than in sporadic tumors.

Proposal of a new grading system for malignant fibrous histiocytomas.

The proposed grading system for malignant fibrous histiocytomas (MFH) comprises 3 grades of malignancy. Analogous to other grading systems, the system includes the factors of mitotic rate and necrosis. In addition to these two factors, the concept of cellularity was included. The prognostic relevance of the grading systems published by Costa, Coindre, van Unnik, Pezzi and Tsujimoto as well as the grading system proposed by the present study was tested on 161 MFH. The results showed that all grading systems tested produced clearly significant differences (p < 0.01) with regard to the survival estimated for patients with various grades of malignancy. These results revealed the superiority of systems that use 3 grades of malignancy over a 2-grade classification. The proposed grading system yielded a lower percentage of grade II tumours (37%) than the grading systems of Coindre (60%) and van Unnik (70%). In the multivariate analysis of all grading systems, the proposed grading system was the only one to show prognostic relevance (p < 0. 05).

Relationship of nm23 expression to proliferation and prognosis in malignant melanomas of the oral cavity.

Twenty-nine cases of oral melanomas were investigated for nm23 and Ki67 antigen expression, as well as for the fraction of tumour cells in S-phase, using immunohistochemical techniques and DNA cytophotometry. Nm23 expression was significantly reduced and Ki67 antigen expression increased in primary tumours with either lymph node or organ metastases in comparison to tumours without metastases. The percentages of Ki67 immunoreactive tumour cells and cells in S-phase correlated positively with each other and negatively with the percentage of nm23-expressing cells. These data argue against a significant growth stimulatory function of the nm23H1 gene product nucleoside diphopshate kinase in the progression of oral melanomas. The functional relevance of nm23 in relation to increased proliferation and metastatic spread is discussed.

Cytokeratin expression correlates with aneuploidy in cytological specimens of melanoma metastases.

It has been postulated that the high malignancy of melanomas could be connected with an increased cytokeratin (CK) expression. In order to define the relationship between CK expression and genetic instability of melanoma metastases, ploidy-related parameters were compared in cytological specimens of CK-positive and CK-negative melanoma metastases. CK expression was investigated immunohistochemically in 35 melanoma liver metastases and in 52 melanoma lymphatic metastases. Ploidy-related parameters were evaluated on Feulgen-stained specimens with a CAS200 image analyzer. Cytokeratin was detected in 14 out of 35 melanoma liver metastases and in 24 out of 52 melanoma lymphatic metastases investigated. Significant differences between CK-positive and CK-negative melanoma metastases were found for the percentage of diploid cells, percentage of tetraploid cells, percentage of aneuploid cells between 4c and 8c, as well as for 5c exceeding rate. Our results confirmed that CK is present in more advanced (unstable), clearly aneuploid forms of melanoma metastases.

Application of in situ hybridization probes for MLH-1 and MSH-2 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemistry and tumor stage.

Defects in DNA mismatch-repair genes MLH1 and MSH2 reported primarily in hereditary nonpolyposis colorectal carcinoma are present in many sporadic tumors, including malignant melanomas. The main aim of this study was to investigate the expression of these genes in malignant melanomas in relation to tumor stage. An experiment was performed on paraffin-embedded tissue microarrays of malignant melanomas applying in situ hybridization with probes produced by our research group and immunohistochemical techniques. In situ hybridization demonstrated MLH1 expression in 45 of 59 melanomas and MSH2 expression in 51 of 59 melanomas. Immunohistochemistry detected MLH1 expression in 46 of 59 melanomas and MSH2 expression in 50 of 59 melanomas. Down-regulation of expression of both DNA mismatch repair genes in malignant melanomas was observed. The findings obtained by in situ hybridization and immunohistochemistry correlated significantly. Our study demonstrates the suitability of in situ hybridization with MLH1 and MSH2 probes for paraffin-embedded tissue. Tissue microarrays can be used successfully in both in situ hybridization and immunohistochemistry to analyze the expression of DNA mismatch-repair genes.

Cytokeratin positivity in paraffin-embedded malignant melanomas: comparative study of KL1, A4 and Lu5 antibodies.

The unclear role of cytokeratin (CK) in the progression and diagnostics of malignant melanomas stimulated us to compare the reactivity of three antibodies directed to CK in 109 paraffin-embedded melanomas. By far the majority of melanomas did not express cytokeratin even at the<1% level, only vimentin. In about 6% of melanomas it was possible to find CK expression ranging between 3 and 40% of melanoma cells. There was a correlation between CK expression and pT-stage. Cytokeratin-expressing tumours were found in the more advanced pT-stages. The independent prognostic values of none of the three CK antibodies investigated could be shown.

Analysis of adenomatous polyposis coli gene expression, APC locus-microsatellite instability and APC promoter methylation in the progression of melanocytic tumours.

Adenomatous polyposis coli gene (APC) defects have been demonstrated for the first time in familial adenomatous polyposis. Recent reports indicate that the APC gene is an intermediary between cell adhesion molecules and the cytoskeleton and that it may function as a gatekeeper of colonic epithelial proliferation. The objective of this study was to analyse APC's presence in lentigos, primary melanomas and melanoma metastases. By immunohistochemistry, APC was demonstrated in all lentigos, in 75 out of 88 primary melanomas and in 16 out of 28 melanoma lymphatic metastases. The percentage of immunolabelled tumour cells (APC index) in lentigos ranged between 5 and 69%, in primary melanomas between 0 and 98% and in melanoma metastases between 0 and 52%. Statistically significant differences between lentigos and primary melanomas and between lentigos and metastases in APC expression were found. In a multivariate analysis, APC showed an independent prognostic impact. Analysis of microsatellite instability in the APC locus was performed on 29 melanomas. Microsatellite instability was found in 5/29 melanomas and loss of heterozygosity in 1/29 melanomas. Promoter methylation of APC was found in 6/10 APC-negative primary melanomas and in 9/10 APC-negative melanoma lymphatic metastases investigated. We conclude about important role of APC alterations for melanoma progression.

Comparison of DNA ploidy status and DNA ploidy-related parameters in malignant melanoma tissue microarrays and full sections.

A new high-throughput tissue-arraying technique, now frequently used in tumor pathology, requires standardization of methods of DNA analysis, previously applied in full histological sections. The main objectives of this study were to evaluate DNA ploidy status and DNA ploidy-related parameters using the CAS200 image analyzer in malignant melanoma tissue microarrays and to compare them with full histological sections. Comparison of DNA ploidy-related parameters, including percentage of diploid cells, percentage of aneuploid cells between 2c and 4c, percentage of tetraploid cells, percentage of aneuploid cells between 4c and 8c, percentage of octaploid cells, percentage of 16-ploid cells, and 5c exceeding rate, did not reveal any significant differences between malignant melanoma tissue microarrays and full sections. The DNA ploidy status according to Auer differed in 1 out of 59 cases investigated. Our study demonstrated that it is possible to evaluate DNA ploidy status and DNA ploidy-related parameters in tissue microarrays, which is of practical relevance to tumor pathology.

Analysis of the p53-hMDM2-p21 (WAF1/CIP1) Cell Cycle Regulation Pathway in Malignant Fibrous Histiocytomas.

This study was undertaken to analyze patterns of expression of critical cell cycle regulators (CCR) involved in the p53 pathway in malignant fibrous histiocytomas (MFH). Protein expression was assessed using immunohistochemistry analyzing p53, hMDM2 and p21 (WAF1/CIP1) phenotypes. p53- and hMDM2-positive phenotypes were found to be associated with low p21 levels (p<0.01). Positive hMDM2 phenotype did not correlate with any hMDM2 mutations, which in our tumor collective were not found. High-grade MFH differed from MFH grade I and II concerning higher p53 and lower p21 levels, while hMDM2 expression was independent of grade. Inclusion of categorized values into a Cox regression study proved the independent prognostic relevance of p53, hMDM2 and p21 phenotypes.

Application of new ploidy-related parameters for the diagnosis of ovarian tumours.

Differential diagnostics of borderline ovarian tumours and ovarian carcinomas is generally based on morphological criteria, which are not always sufficient for final diagnosis. Therefore, we investigated the practical diagnostic application of the CAS200 image analyzer and new ploidy-related parameters in a series of 68 borderline tumours and 42 low-grade carcinomas of the ovary. Highly significant differences between borderline and malignant lesions were found for the percentage of diploid cells (p = 0.0001), the percentage of aneuploid cells between 4c and 8c (p = 0.0001), the percentage of octaploid cells (p = 0.0001), as well as for the 5c exceeding rate (p = 0.0001). The difference concerning the ratio of tetraploid cells also reached the level of significance (p = 0.0320). We suggest that new ploidy-related parameters evaluated by the CAS200 image analyzer can be helpful in ovarian lesions with unclear morphology.