Biomedical subjects
Publications and source records attributed to L G Koss.
Computer-assisted image analysis was used to demonstrate in exponentially proliferating human tumor cells the uneven postmitotic apportionment of several oncogene-encoded proteins (ras p21; erbB-2 p185; fos p55; myc p62). This observation may provide the explanation for the high degree of heterogeneity of postmitotic cells and the asynchrony in cell cycle traverse of cultured cells.
The key events leading to a better understanding of the natural history of urothelial tumors of the bladder are summarized. These were: the recognition of flat carcinoma in situ and related lesions (intraurothelial neoplasia) as principal sources of invasive cancer; identification of the unique structure of the urothelium; analysis of DNA content and the recognition of two pathways of urothelial tumors. A brief comment on the current status of immunologic and molecular genetic markers is appended.
This report concerns the study of Ha-ras gene mutations and ras p21 expression in primary tumors of the urinary bladder. Polymerase chain reaction-based techniques and computerized image analysis were used. The data obtained were related to tumor grade, DNA ploidy, and tumor invasion. A point mutation (G-->T) at Ha-ras codon 12 was found in 30 of 67 tumors. The mutation frequency was greater in grade III (65%) than in grade II (44%) tumors; no mutations were observed in grade I tumors. The mutation was observed more often in aneuploid (58%) than in diploid (28%) tumors. No other substitution at codon 12 was seen and no codon 61 mutation was detected. The tumors were also tested for the A-->G mutation at position 2719 of Ha-ras intron D. Concurrent codon 12 and intron D mutations were identified in seven high-grade aneuploid tumors; six were invasive. The levels of the ras gene product p21 were approximately 10 times higher in tumors with intron D mutation than in those without. These findings confirm on human bladder tumors the observations of the effect of synchronous exon-intron mutations reported on the bladder cancer cell line T24. Our results are the first demonstration of Ha-ras intron D alterations in human tumor tissues and suggest that concurrent mutations at codon 12 and intron D of this gene within the same tumor may contribute to the aggressive behavior of human bladder carcinomas.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The DNA ploidy and cell-cycle distribution of three separate fresh tissue samples of 60 colorectal adenocarcinomas were analyzed by flow cytometry. DNA ploidy was concordant among the three samples in 38 cases (63.3%). In the remaining 22 cases (36.6%), the DNA histograms of two of the three multiple samples were similar; however, the ploidy of the third sample was discordant. No relationship was observed between Dukes' stage and histologic grade with concordance or discordance among samples. Thus, in about one third of the colonic carcinomas, a single sample showing either a diploid or diploid-cycling DNA histogram would not detect aneuploid DNA patterns. Comparison of scrapes and fine-needle aspirates of tumors as alternative sampling methods of tumors for DNA ploidy analysis indicated a strong association with the tumor ploidy (84% and 96%, respectively) only when the ploidy of the multiple samples was concordant. In about 25% of the cases, tumor scrapes and fine-needle aspirates did not correlate with the "most abnormal" ploidy observed in one of the three tissue samples. The data suggest that single or even double tissue samples may not show aneuploid DNA patterns in a substantial proportion of colorectal cancers.
A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter- and intra-laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter- and intra-laboratory differences. Results were evaluated by a two-way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra- and inter-laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)
A survey of over 225 recent studies examining the relationship between the flow cytometric DNA analysis of solid tumors and clinical prognosis indicates that criteria used to classify DNA histograms are variable and often inconsistent with the recommendations proposed by the Convention on Nomenclature for DNA Cytometry. Numerous reports not only lack unambiguous descriptions of the histogram features used to differentiate diploid from aneuploid DNA distributions, but also inadequately describe the technical aspects of data acquisition, standardization, and inclusion or exclusion of subpopulations by gating. In many cases, the coefficient of variation of the diploid and aneuploid G0/1 peaks, which would allow an assessment of histogram quality, is not reported. Because of the differences in DNA histogram interpretation, extrapolation of the results among laboratories may be difficult and is probably not reliable. This review summarizes the criteria that have been used to classify the DNA histograms and illustrates the effects of these different classifiers on DNA ploidy analysis and clinical conclusions.
This report concerns the study of the relationship between protein expression and the cell cycle in exponentially proliferating benign and malignant human prostate epithelial cells in short-term cultures. Multiparameter flow cytometric measurements were performed to correlate the expression of prostate-specific acid phosphatase, epithelial membrane antigen and epitectin with cell cycle progression. The expression of the three proteins was heterogeneous in G1 cells. The early post-mitotic cells exhibited the lowest levels when compared with late G1 cells, wherein the expression was many times greater. There was no further increase as the cells progressed through S and G2 + M. These findings, corroborating prior observations in other systems, suggest the possibility that the levels of the proteins studied increase during the G1 phase of the cell cycle and drop during or immediately after cytokinesis. As an alternate explanation, the heterogeneity of protein expression characteristic of G1 cells may be due, at least in part, to an asymmetric apportionment of cell constituents at mitosis.
We report a method of in situ hybridization (ISH) of 10-year-old archival cervical smears with a cocktail of nick-translated human papillomavirus (HPV) DNA types 6, 11, 16, 18, and 31. The method, which does not require destaining, results in excellent preservation of morphological detail with only 2% cell loss. Methods of smear treatment and detection of the biotinylated probe with a multistep avidin-biotin-immunoperoxidase method are described. Biotinylated PBR 322 plasmid and biotinylated human DNA were used as negative and positive controls in each run. Twenty-nine of 50 smears (58%) showing changes consistent with CIN I-II were positive for HPV. Fourteen corresponding cervical biopsies were also studied by ISH, seven corresponding to HPV-positive smears and seven to HPV-negative smears. HPV DNA was demonstrated in six of seven biopsies (87%) from the positive group but none could be demonstrated in the negative group. We conclude that retrospective study can be performed on routine alcohol-fixed, Papanicolaou-stained cervical smears with biotinylated HPV probes with excellent cell preservation, minimal cell loss, and high degrees of specificity.
Explore the source record for details and available documents.
With the use of fresh cell suspensions, the DNA ploidy of 11 benign and 27 malignant spindle cell soft-tissue lesions was determined by flow cytometry (37 cases) and/or image cytometry (35 cases). Of the 27 malignant lesions, 10 were low- or intermediate-grade sarcomas, and 17 were high-grade sarcomas. In the malignant lesions, the DNA ploidy was correlated with the histologic grade and the clinical outcome. Of the 11 benign lesions, six had a diploid DNA ploidy pattern, and five were nondiploid by either flow cytometry or image cytometry. All benign cases had a favorable outocome regardless of ploidy. Eight of the 10 low- or intermediate-grade malignant lesions were diploid, whereas two were nondiploid. Of the 17 high-grade sarcomas, 15 were nondiploid by either method of measurement and nine by both. Of the 10 diploid malignant tumors, only one had an unfavorable outcome (malignant mesothelioma with biopsy only), whereas of the 17 malignant lesions that were nondiploid, five had no evidence of recurrent disease, three cases recurred, eight patients died of disease, and one patient died of an unrelated cause. We concluded that (1) ploidy does not differentiate benign from malignant spindle cell soft-tissue tumors, (2) the nondiploid DNA pattern is more common in high-grade than in low- or intermediate-grade sarcomas, and (3) there is a statistically significant relationship between nondiploidy and a worse clinical outcome in sarcomas.
Explore the source record for details and available documents.
It has been reported that deoxyribonuclease (DNAse) treatment does not destroy viral DNA, but it does digest native nuclear DNA. To determine what effect, if any, papillomavirus infection has on DNA ploidy values of genitourinary condylomas, DNA was measured with and without DNAse exposure in seven urethral condylomas, shown by prior in situ hybridization to contain abundant human papillomavirus types 6 and 11. Normal human skin was used as a negative control. Consecutive paraffin-embedded tissue sections were stained according to Feulgen before and after DNAse treatment. The DNA was measured by image analysis. In control tissue, DNAse obliterated DNA, and the Feulgen reaction was negative. In six of seven condylomas the DNA content was reduced, but a measurable Feulgen reaction was still present in isolated cells. In the seventh case there were no significant changes in the histograms. This observation strongly suggests that the presence of human papillomavirus has a significant effect on measurements of DNA ploidy in genital condylomas and, by implication, possibly also in other tissues containing the virus. Possible mechanisms are discussed.
Explore the source record for details and available documents.
The use of antibodies permits the study of oncogene product expression in cells and tissues. However, quantitation of the levels of expression in immunohistochemical preparations is beset by difficulties, and the available scoring system provide semiquantitative data at best. Here we describe the use of computer-assisted image analysis for determination of oncoprotein levels in a model system and compare the results with those generated by flow cytometric analysis. The oncogene products measured are located in the nucleus (c-myc p62 and c-fos p55), the inner surface of the membrane (c-ras p21), and both sides of the membrane (c-erbB-2 p185). In each instance, both analytic modalities yielded concordant results. Our data indicate that computer-assisted image analysis is a useful tool for quantitating cell components in immunohistochemical preparations.
The role of human papillomaviruses (HPV) as possible agents in the genesis of cervical cancer is reviewed. Several types of HPV (mainly 6, 11, 16, 18, 31, and 33) have been shown to be associated with precancerous processes and cancer of the uterine cervix. HPV also induces pathognomonic abnormality of squamous cells, known as koilocytosis, that may precede or accompany various manifestations of cancerogenesis including invasive cancer. Still, recent studies suggest that HPV infection is quite common in normal people and may prove to be ubiquitous. Hence, the activation of the virus and its ability to interact with cervical epithelium is likely to be due to "patient factors" rather than the presence of the virus per se. The possible mechanisms of virus-epithelial interaction and of the factors that may put a woman at risk for cervical carcinoma are discussed.
The expression of the ras gene product p21 in normal gastric mucosa, early gastric carcinoma of diffuse (gastric) and intestinal types, and in adjacent mucosal abnormalities is reported. The analysis was performed on paraffin sections by an immunohistochemical assay using the mouse monoclonal antibody RAP-5 and the rat monoclonal antibody Y13-259. Expression of ras p21 was assessed by staining intensity and percentage of positively stained cells. In comparison to normal gastric mucosa of non-cancer patients, p21 was overexpressed in nearly all early carcinomas of both types and in the dysplastic and/or metaplastic mucosal alterations accompanying intestinal type of gastric cancer. Increased p21 expression was also observed in the normal-appearing mucosa adjacent to early carcinomas of diffuse type, but not in the morphologically normal gastric epithelium adjacent to the intestinal type. The results of this investigation suggest that ras p21 overexpression may be related to early events of human gastric carcinogenesis. The study supports the notion of different pathways in the development of diffuse (gastric) and intestinal types of gastric carcinomas.