An immunocytochemical study of isolated human retinal Müller cells in culture.

BACKGROUND: We report a modified method for the isolation and propagation of adult human Müller cells in culture. METHODS: The retina of postmortem human eyes was mechanically dissociated and cultured. Using immunocytochemical techniques, these cells were stained with monoclonal antibodies specific for Müller cells, glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS) and keratin. Transmission electron microscopy (TEM) was also performed. RESULTS: The dissociated and cultured cells expressed vimentin and GS, but not GFAP. At least 85% of these cells stained with a Müller cell-specific monoclonal antibody. Using TEM, flat cells containing 13-nm intermediate filaments and glycogen were identified. CONCLUSION: Human retinal Müller cells can be isolated and propagated in culture. Purified cell cultures are required for controlled studies of the normal physiology and pathologic responses of Müller cells.

Comparative genomic hybridization analysis of archival formalin-fixed paraffin-embedded uveal melanomas.

Comparative genomic hybridization (CGH) was used to analyze seven autologous uveal melanomas with both formalin-fixed, paraffin-embedded and fresh-frozen specimens. In addition, DNA from two archival formalin-fixed tumors more than 45 years old were also analyzed. The most frequent genetic changes were loss of chromosome 3; increase in copy number of 6p and loss of 6q; and increase in copy number of 8q. A comparison of CGH data from the fresh-frozen tumors and their autologous formalin-fixed tumors revealed a correlation coefficient of 0.83. Comparative genomic hybridization (CGH) analysis of 45-year-old specimens identified genetic changes similar to those found in more recent tumors including loss of chromosome 3 and increase in copy numbers of 6p and 8q. The results indicate that there is a good agreement between data obtained from formalin-fixed and fresh-frozen specimens using CGH. Furthermore, the results demonstrate the applicability of this technique in analyzing archival formalin-fixed tumors that were previously not accessible to cytogenetic analysis.

p53 gene and cell cycling in uveal melanoma.

PURPOSE: To determine whether alterations of p53, a tumor suppressor gene, were present in uveal melanoma, and to characterize further the nature of those changes. METHODS: Immunohistochemical analysis with a monoclonal antibody was used to determine whether alterations of p53 were present in 35 enucleated archival uveal melanomas. Further characterization was done by comparing the p53 gene and cell cycling status by using bromodeoxyuridine staining. The alterations in p53 were characterized using polymerase chain reaction single-strand conformational polymorphism analysis and sequencing. RESULTS: Four of 35 uveal melanomas showed low levels (0.5% to 5.0%) of positive immunostaining for altered p53 in tumor cell nuclei using monoclonal antibody DO-7. These four tumors had the three highest and the 14th highest bromodeoxyuridine labeling indices, ranging from 1.3% to 7.0%. Polymerase chain reaction single-strand conformational polymorphism analysis of p53 exons 5 to 8 was performed on three p53-positive and six p53-negative tumors, and no altered motility shifts were detected. Sequencing of one of the positive staining specimens confirmed no mutations in exons 5 through 8 in the p53 gene. Double immunohistochemical labeling for both bromodeoxyuridine and p53 in one tumor showed that most of p53-positive cells were in S phase. CONCLUSIONS: Mutation of p53 is an uncommon event in uveal melanomas. Nuclear accumulation of p53 protein was found in three of the four tumors with the highest levels of cell cycling.

Comparative analysis of proliferating cell nuclear antigen, bromodeoxyuridine, and mitotic index in uveal melanoma.

PURPOSE: Recent production of a monoclonal antibody, PC10, against proliferating cell nuclear antigen (PCNA) makes it possible to evaluate cell cycling in formalin-fixed tissues. In this study, the authors quantitatively evaluated the relationship between PCNA expression and two other measures of cell cycling, bromodeoxyuridine labeling index (BrdU LI) and mitotic index (MI), in archival uveal melanomas. The authors also examined the relative prognostic importance of each measure. METHODS: Serial sections from 35 formalin-fixed, paraffin-embedded uveal melanomas were immunostained with PC10 and BrdU antibody using a standard avidin-biotin-peroxidase method. A quantitative scoring system was used to evaluate the fraction of cells that were positive for PCNA, BrdU, and mitotic figures in the regions of high cycling. The LIs of the different markers were compared, and their prognostic importance was evaluated. RESULTS: The median PCNA LI was 3.05% compared to the median BrdU LI of 0.94% and the median MI of 0.034%. The PCNA LI was more variable in replicate sections than either the MI or the BrdU LI. The correlation between PCNA LI and BrdU LI was 0.58, between PCNA LI and MI it was 0.46, and between BrdU LI and MI it was 0.81. The relative risk of tumor-related mortality per doubling of BrdU LI was 2.35, and of MI it was 2.34. Although these were significant, PCNA LI of 1.08 was not. CONCLUSIONS: Proliferating cell nuclear antigen immunostaining did not demonstrate a strong relationship with either BrdU LI or MI. Unlike MI and BrdU LI, PCNA LI was not correlated with tumor-related mortality. Caution is warranted in the interpretation of PCNA immunostaining in uveal melanomas.

Cell cycling and prognosis in uveal melanoma.

Uveal melanoma cell cycling, quantified by bromodeoxyuridine (BrdUrd)-labeling index and mitotic index, is predictive of tumor-related mortality. Serial sections from 36 formalin-fixed melanoma specimens were labeled with BrdUrd and stained with hematoxylin and eosin. All tumors were assessed for the area of highest cell cycling activity and counts for mitotic figures and BrdUrd labeling were performed in these areas in a masked manner. The BrdUrd labeling index and mitotic index were calculated and analyzed in relation to tumor-related mortality and histopathological criteria (largest tumor diameter, cell type, extra-scleral extension, ocular location). Cox multivariate analysis estimated an increased relative risk of tumor-related mortality of 2. 32 (95% confidence interval, 1.22-4.41) per doubling of BrdUrd labeling index and 2.41 (95% confidence interval, 1.29-4.49) per doubling mitotic index. Larger tumors, nonspindle cell tumors, and anterior-located tumors tended to have higher cycling rates.

Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.

Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples.

Comparative genomic hybridization in the detection of DNA copy number abnormalities in uveal melanoma.

Genomic instability appears to play an important role in the development, growth, invasiveness, and eventual metastasis of the neoplastic cell. We have used a powerful new technique, comparative genomic hybridization, to evaluate genetic alterations in 10 fresh frozen uveal melanomas. Comparative genomic hybridization utilizes dual fluorescence in situ hybridization to characterize chromosome deletions and duplications, allowing for simultaneous evaluation of the entire human genome. Several consistent chromosomal abnormalities were detected. This study confirmed previous findings obtained using standard cytogenetic techniques but demonstrated an increased incidence in abnormalities of chromosomes 3 and 8; there was loss of chromosome 3 and duplication of 8q. In addition, we identified, although less frequently, other recurrent abnormal regions including alterations on chromosomes 6p, 7q, 9p, and 13q.

Multiple HPV infection: microanatomy by in situ hybridization and immunohistochemistry.

Specific human papillomavirus (HPV) types have been shown to be associated with proliferative epithelial lesions with variable biological consequences in infected patients. Simultaneous infection by more than one HPV type has been infrequently reported, and its clinical significance is unknown. We have examined four biopsies of cervical and vulvar tissue, each with evidence of infection by two different HPVs. Using both in situ hybridization and immunohistochemical techniques, we determined the cellular distribution of the viral infections. Using biotinylated type-specific probes and stringent conditions we were able to demonstrate that in each case the two HPVs occupied distinct, non-overlapping foci within the lesions. The condylomatous tissues contained DNA from HPV types that are associated with high-grade neoplasia and invasive cancer (16 and 18), as well as types commonly associated with benign proliferative lesions. Immunohistochemical analysis of the lesions with antibody to bovine papillomavirus capsid antigen failed to detect HPV in regions shown by in situ hybridization to contain HPV 16 and 18 DNA, whereas type 6 and 11 infected areas were readily identified. These results provide indirect evidence of viral interference between HPV types and indicate that interference may limit the number of HPV types that produce active infections within a single cell.