Practical aspects of planning, building, and interpreting tissue microarrays: the Cooperative Prostate Cancer Tissue Resource experience.

This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.

The role of cell death in the growth of preneoplastic lesions: a Monte Carlo simulation model.

A variety of experimental and clinical examples of preneoplasia demonstrate that regression of early lesions is common. This paper examines the hypothesis that early lesions operate under the identical growth kinetics of 'late' lesions (neoplasms), but that kinetic features favouring continuous growth in established lesions tend to favour extinction of lesions composed of small numbers of cells. Growth simulations of early lesions were produced using the Monte Carlo method, a technique demanding intensive computations. With the advent of powerful personal computers, this technique is now widely available to biologists. Simulating growth under conditions of cell loss similar to those observed in established tumours, the model predicts that the great majority of initiated cell clusters are expected to reach extinction within a few cell doubling times, and most early (promoted) lesions would not likely progress to the size of a clinically detectable lesion within the life span of the host organism. These Monte Carlo simulations provide a model of initiated cell growth consistent with the recently demonstrated role of early lesion cell death in the development of human lymphomas and in transgenic mice expressing the bcl-2 oncogene. The model demonstrates that small increments in the intrinsic cell loss probability in even the earliest progenitors of malignancy can strongly influence the subsequent development of neoplasia from initiated foci.

Colonic metaplasia of ileostomies. Biological significance for ulcerative colitis patients following total colectomy.

Two patients who had undergone proctocolectomy for ulcerative colitis developed lesions in their ileal stoma that appeared to be inflammatory polyps morphologically similar to those encountered in the large intestine of ulcerative colitis patients. One of these patients eventually developed mucinous adenocarcinoma in the ileal stoma. The ileal mucosa adjacent to the neoplasm had morphologic features of large-bowel mucosa and was richly populated by sulfomucin-containing goblet cells, which are characteristic of large-bowel mucosa. Sulfomucin-containing goblet cells were also found in the inflammatory lesions biopsied from the ileal stomas of both patients, as well as from the adenocarcinoma found in one patient. These findings support the hypothesis that colonic metaplasia can occur in ileal stomas of ulcerative colitis patients. Furthermore, the metaplastic colonic tissue is the site of origin of lesions typically found in ulcerative colitis. Colonic metaplasia occurring in ileal stoma should be recognized by pathologists as a clinical entity. When colonic metaplasia is identified in the ileal stoma of an ulcerative colitis patient, biopsy surveillance of stomal mucosa is recommended.

5'-Nucleotidase activities in cultured rat liver epithelial and fibroblast cells.

Cell cultures of adult rat liver produced two distinct morphologic cell types: epithelial cells polygonal in shape and growing in nests of closely apposed cells, and fibroblast cells stellate in shape with little cell-cell contact at low density growth, but aligning in parallel arrays at high density. These two morphologic variants displayed dramatic differences in histochemically demonstrable 5'-nucleotidase activities. Fibroblast cells exhibited great activity throughout the cytoplasm with no concentration of activity in the cell membrane. The lesser activity in epithelial cells was concentrated on the cell membrane. The importance of this finding to the interpretation of data derived from experiments with whole liver homogenates is discussed.

Cell growth simulations predicting polyclonal origins for 'monoclonal' tumors.

Studies showing the clonal identity of various tumors have led to the belief that most tumors originate from a single cell. It is shown by Monte Carlo computer simulations that monoclonality can evolve from minor differences either in cell cycle time or in the probability of cell death in a polyclonal 'founder' population. If cells divide continuously without cell death (exponential clonal growth), a triclonal population with three starting cells (cell cycle times 0.9 days, 1 day and 1.1 days) converges to near-monoclonality in 100 generations. For cell cycle times of 0.9 days, 1.1 days and 1.1 days, and cell death probabilities of 0.45 and 0.46, populations tend toward monoclonality while the tumor is still small (less than 3 mm3).

Image analysis software for the detection of preneoplastic and early neoplastic lesions.

Preneoplastic lesions are usually small, and often appear as foci of atypical cells that blend into the surrounding normal tissue without producing a detectable tumor mass. Since these lesions seldom provide adequate tissue for biochemical studies, their detection often depends upon subtle distinctions in cytologic features. Image analysis permits pathologists to obtain quantitative measurements on cytologic and histologic preparations, so that visual impressions can be augmented by quantitative morphometry. Preneoplastic lesions have well-described morphometric features relating to nuclear area, texture, or shape. It is now feasible for every pathology department to capture images of pathologic material with equipment costing less than the price of a microscope. Captured image files can be analyzed using commercial software or software developed in several U.S. government agencies and made freely available to the public. Image analysis has been shown to improve the detection of preneoplastic cells. Recent improvements in the resolution of captured images, in the algorithms that measure preneoplastic descriptors, and in the ease and speed of transmission of images between laboratories, should increase our ability to detect and treat preneoplastic lesions.

Enhancement of mutagenesis during cell replication of cultured liver epithelial cells.

The susceptibility to mutagenesis of proliferating and non-proliferating mammalian cells was studied in cultured rat liver epithelial cells. Cells brought to growth quiescence by a non-toxic means were stimulated to proliferate and both types of cultures were exposed to methyl methanesulfonate (MMS). Cultures enriched in proliferating cells were more susceptible to both the toxic and mutagenic action of the mutagen than were quiescent cultures with a low level of proliferation.

Liver cell dysplasia: a DNA aneuploid lesion with distinct morphologic features.

Liver cell dysplasia is characterized by hepatocellular foci with nuclear atypia. It is often seen in cirrhosis and may be a precursor of hepatocellular carcinoma (HCC). To determine whether liver cell dysplasia is DNA aneuploid, 72 sections of 33 cirrhotic livers from the autopsy files of The Johns Hopkins Hospital were studied, and 14 foci of dysplasia from 13 cirrhotic livers were selected. Patients ranged in age from 32 to 70 years. Histologically, there were 10 foci of low-grade dysplasia and four foci of high-grade dysplasia. Nine HCCs served as positive controls; seven autopsy livers with no morphologic or clinical evidence of primary liver disease served as negative controls. One focus of HCC and one focus of dysplasia were unsatisfactory for analysis. Flow cytometric examination demonstrated subpopulations with DNA abnormality in four of nine (44%) foci of low-grade dysplasia, of which three were aneuploid. Three of four (75%) foci of high-grade dysplasia were aneuploid. Six of eight (75%) HCCs showed DNA abnormality, of which five were aneuploid. DNA aneuploidy was not present in the seven control livers; however, one showed DNA abnormality. We conclude that liver cell dysplasia is a morphologic entity that contains DNA aneuploid cells, a feature that supports the role of liver cell dysplasia in the evolution of HCC.

DNA analysis of cardiac myxomas: flow cytometry and image analysis.

Cardiac myxoma is the most common primary tumor of heart, but there is a longstanding controversy over whether it is a true neoplasm or a reactive lesion. We analyzed 24 cardiac myxomas from 22 patients: 22 by DNA flow cytometry and five by image analysis. Two myxomas were aneuploid; one of those analyzed by flow cytometry, and the other by image analysis. Proliferative fractions (S + G2/M) were high in three tumors from patients with multiple myxomas (mean, 15.9%; SD, 4.0%) as compared with 12 solitary uncomplicated myxomas (mean, 7.7%; SD, 6.0%). S-phase and proliferative fractions were low in embolic, recurrent, and solitary myxomas. The presence of aneuploidy in some myxomas supports a neoplastic origin for this tumor.

Performance analysis of manual and automated systemized nomenclature of medicine (SNOMED) coding.

Many pathology departments rely on the accuracy of computer-generated diagnostic coding for surgical specimens. At present, there are no published guidelines to assure the quality of coding devices. To assess the performance of systemized nomenclature of medicine (SNOMED) coding software, manual coding was compared with automated coding in 9353 consecutive surgical pathology reports at the Baltimore Veterans Affairs Medical Center. Manual SNOMED coding produced 13,454 morphologic codes comprising 519 distinct codes; 209 were unique codes (assigned to only one report apiece). Automated coding obtained 23,744 morphologic codes comprising 498 distinct codes, of which 129 were unique codes. Only 44 (.5%) instances were found in which automated coding missed key diagnoses on surgical case reports. Thus, automated coding compared favorably with manual coding. To achieve the maximum performance, departments should monitor the output from automatic coders. Modifications in reporting style, code dictionaries, and coding algorithms can lead to improved coding performance.

Automated review of blood donor screening test patterns at a regional blood center.

Regional blood centers recently increased the number of tests used to protect transfusion recipients from infectious disease. The Food and Drug Administration has noted that computer systems for managing these data have lost donor data or failed to recognize all deferrals. Of 118,396 consecutive donations, including 4,859 records with at least one positive test result, records were analyzed to determine the number and frequency of distinct combinations of test data. A modular precedence-logic expert system assigned donor deferrals and unit dispositions. Ten combinations of data accounted for 4,334 records (89%); the remaining 525 records (11%) were distributed among 85 combinations of test data. The expert system correctly assigned all records. Regional blood centers must interpret a growing number of test results, including donations with a complicated pattern of multiple positive screening test results. The distribution of these data is described and the expert system's ability to monitor deferrals and ensure database completeness is demonstrated.

Adenocarcinoma of the ileostomy: the latent risk of cancer after colectomy for ulcerative colitis and familial polyposis.

A case of a primary adenocarcinoma of an ileostomy is reported along with 15 other cases collected from the literature. These rare tumors are seen on the average 24 years after colectomy with ileostomy and in all cases are associated with a past history of ulcerative colitis or familial polyposis. Most of the reported cases of these tumors have appeared in the literature within the past 5 years, suggesting that there is a rising incidence of this disease corresponding to completion of a biologic latency period that began when the Brooke ileostomy was introduced for ulcerative colitis in 1951. In our case a mucinous adenocarcinoma occurred at the ileostomy site 34 years after colectomy. Adjacent to the tumor was mucosa showing colonic metaplasia and focal dysplasia. Subsequent biopsy specimens of the revised stoma showed inflammatory lesions morphologically suggestive of inflammatory (pseudo) polyps. The clinical and morphologic features in this case suggest that there is transition from ileal mucosa to colonic mucosa to colonic dysplasia to adenocarcinoma. Annual evaluation of the ileostomy for colonic metaplasia, inflammatory lesions consistent with ulcerative colitis and dysplasia, is recommended. In the presence of dysplasia, stomal revision is advised. Wide local excision is advised for adenocarcinoma.

U.S. Senate Bill 422: the Genetic Confidentiality and Nondiscrimination Act of 1997.

On March 11, 1997, Senator Pete Domenici introduced U.S. Senate Bill 422, the Genetic Confidentiality and Non-Discrimination Act of 1997. The bill specifies that "existing legal protections for genetic information are inadequate to ensure genetic privacy and to prevent genetic discrimination." The first stated purpose of the bill is "to define the circumstances under which DNA samples may be collected, stored, and analyzed and genetic information may be collected, stored, analyzed, and disclosed." The bill reinforces statutes already passed in 19 states that guarantee patients certain rights of control over their own personal genetic information (Congressional Record, S2141). This bill fundamentally changes the mechanism whereby molecular biologists acquire tissue, and defines new obligations between researchers and human subjects. Even if this bill is not passed in its present form, it appears inevitable that legislation with a similar intent will soon emerge. Any such legislation will carry the full weight and penalties of law. Because many current research practices may violate these proposed new regulations, researchers should understand the provisions of S. 422. Furthermore, it is crucial that researchers understand the bill now, before it becomes law, because this may be their last opportunity to lobby for any modifications of the bill.

DNA breakage by methyl methanesulfonate and its repair in brain and liver cells cultured from fetal rat and mouse.

DNA strand breakage and repair following methyl methanesulfonate (MMS) treatment of primary cell cultures from 14-day fetal Sprague-Dawley rat brain and liver and 12-day fetal C57BL/6 mouse brain and liver, were studied using alkaline sucrose density gradient analysis. Cells were incubated with MMS (7 mM or 14 mM) for 20 min and harvested for alkaline sucrose gradients 40 min or 24 h later. The extent of initial damage in fetal rat and fetal mouse cells was comparable. Fetal mouse brain and liver and rat liver showed nearly complete repair 24 h after treatment. However, fetal rat brain cells showed comparatively little repair after 24 h. The possible significance of a repair deficit in cultured rat fetal brain cells and the striking neurogenic organotropism of transplacentally administered direct-acting alkylating agents in the rat is discussed.

The function of gamma-glutamyl transpeptidase as a determinant in cell sensitivity to azaserine toxicity.

The enzyme gamma-glutamyl transpeptidase (GGT) is characteristically present at high levels in mammalian cells that are vulnerable in vivo to the selectively toxic and carcinogenic effects of the naturally occurring diazo amino acid L-azaserine. The possible role of GGT as a determinant of cellular sensitivity to azaserine toxicity was investigated. No correlation was found between GGT activity and the abilities of different cell lines or GGT-deficient cell strains of TuWi, a human nephroblastoma-derived line high in GGT, to accumulate azaserine. However, the thiols glutathione and cysteine were found to inhibit the toxicity of azaserine in cultures of TuWi. In addition, maleate lowered both intracellular and extracellular glutathione levels and enhanced sensitivity of TuWi cells to azaserine, while serine-borate, a potent inhibitor of GGT, increased extracellular glutathione levels and inhibited azaserine toxicity. Since extracellular glutathione accumulation, which may reflect the rate of cellular glutathione turnover, is increased in cultures of azaserine-resistant, GGT-deficient strains of TuWi, we propose that GGT enhances cellular sensitivity to azaserine primarily by increasing the rate of glutathione turnover, thus removing the glutathione from detoxification pathways.

Characterization of analog resistance and purine metabolism of adult rat-liver epithelial cell 8-azaguanine-resistant mutants.

Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied with both the concentration of AG used and the seeding density of the population under selection. Cells resistant to AG were also cross-resistant to 6-thioguanine and unable to grow in medium containing hypoxanthine, aminopterin and thymidine. Resistance was stable. AG resistance was due to a deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) activity which was not caused by an inhibitor. In the assay for HGPRTase, a substantial amount of product appeared as inosine (In) in addition to inosine monophosphate (IMP). Purine nucleoside phosphorylase will generate In from hypoxanthine and, indeed, the cells did possess this activity. However, several findings indicated that the In was derived from IMP by catabolism by 5'-nucleotidase (NTase): (1) IMP decreased as In increased and (2) the inhibitors of NTase, adenosine monophosphate and thymidine triphosphate, reduced the generation of In by over 90% without inhibiting purine nucleoside phosphorylase. The cells possessed substantial NTase activity, 35% of which was located in the cytosol along with 69% of HGPRTase. Several lines of evidence suggested that the NTase activity limited the amount of 8-azaguanylic acid presented to the cells by catabolising the nucleotide and, thereby, reducing the toxicity of available AG.

Endocardial schwannomas in rats. Their characterization by light and electron microscopy.

Thirty male Sprague-Dawley rats were given a single intravenous injection of methyl(acetoxymethyl)nitrosamine. Primary endocardial tumors developed in ten rats. By light and electron microscopy, the tumors had many of the typical features of schwannomas (neurinomas) in rats. Tumors were composed of masses of elongated cells surrounded by a fine reticulin network, occasionally forming Verocay bodies. Basement membrane was observed on the external surfaces of tumor cells, which had convoluted plasma membranes. To our knowledge, this is the first report of a spontaneous endocardial schwannoma in a rat. The literature addressing endocardial tumors in rodents and man is discussed, and criteria for distinguishing this neoplastic entity from other tumors and non-neoplastic endocardial proliferations are described.

Malignant fibrous histiocytoma. An unusual neoplasm of soft-tissue origin in the rat that is different from the human counterpart.

Malignant fibrous histiocytomas were found in eight F344 rats and four Sprague-Dawley rats. They appeared to originate in skin or peritoneum and to have metastasized to liver and lung. Tumor cells appeared to be histiocytic in the light microscopic studies and fibroblastic after ultrastructural evaluation. The cell of origin was uncertain.

Practice guidelines for autopsy pathology: autopsy reporting. Autopsy Committee of the College of American Pathologists.

The Autopsy Committee of the College of American Pathologists has prepared this revised guideline to reflect changes that have occurred in the reporting of autopsies since the original guideline was published in February 1995. It is intended to be an instrument to assist pathologists in the reporting of autopsies. The guideline is to be regarded as being primarily an educational tool. Application of these recommendations on autopsy reporting is to be made on the basis of the judgment of the pathologist engaged in a specific case.

A prototype Internet autopsy database. 1625 consecutive fetal and neonatal autopsy facesheets spanning 20 years.

OBJECTIVE: To demonstrate that cause-of-death statements can be generated by a computer algorithm from an autopsy database composed of diagnostic terms. DATA SOURCES: Over 49 000 autopsy facesheets contributed by over a dozen institutions were collected from a publicly accessible Internet autopsy database. This database is available at the following web site: http:@www.med.jhu.edu/pathology/iad.html STUDY SELECTION: To test the feasibility of creating and using a publicly available autopsy database, and to identify the technical and medicolegal problems that may arise with such a novel resource, a prototype study was designed by selecting autopsy facesheets from fetal and neonatal deaths. An algorithm was developed to determine the cause of death from the listing of anatomic diagnoses. DATA EXTRACTION: One thousand six hundred twenty-five fetal and neonatal autopsy facesheets were selected encompassing fetal and neonatal deaths occurring up to 28 days after birth. DATA SYNTHESIS: The algorithm determined causes of death from autopsy facesheet data in all cases. On review by an experienced pediatric pathologist, these automatically generated cause-of-death statements required no modification or only slight modification in over 90% of cases. CONCLUSIONS: A large multi-institutional autopsy database composed of demographic and diagnostic information has been deposited on the Internet. This information can be freely downloaded and used by any researcher without violating patient confidentiality. As a demonstration of one possible application of the database, fetal and neonatal autopsies generated cause-of-death statements using a computer algorithm. One can anticipate that the wealth of information contained in autopsy facesheets can be assembled into a database that will serve the public interest.