The value of the reprocessing method of paraffin-embedded biopsies for transmission electron microscopy.

Transmission electron microscopy (TEM) implies an elaborate preparation protocol that includes: fixation in glutaraldehyde followed by osmium tetraoxide postfixation, specimen dehydration, infiltration, resin embedding, ultrathin sectioning and staining with heavy metal salts. The aim of TEM is to examine the ultrastructure of specimens in ways that cannot be examined using other equipments or techniques. In some cases, when the requirement for TEM were made after tissue collection, useful information can be obtained from reprocessing the formalin-fixed, wax-embedded tissue used for light microscopy.

Expression of cytokeratin 20 and CD44 protein in upper urinary tract transitional cell carcinoma: cytologic-histologic correlation.

OBJECTIVE: To explore the potential utility of immunostaining for CK20 and CD44 protein isoforms in evaluating cases of upper urinary tract transitional cell carcinoma (UTTCC). STUDY DESIGN: Of 105 consecutive patients diagnosed cytologically with UTTCC, 33 subsequently underwent open surgical procedures. Cytologic samples from these patients retrieved by aspiration and biopsy, and corresponding surgical specimens were graded and staged using World Health Organization/International Society of Urologic Pathologists criteria. Immunostaining for CK20, CD44 standard (CD44s) and CD44v6 isoform (CD44-v6) was performed on all available cytologic and surgical materials. Expression levels and distributions of these markers were correlated semiquantitatively with grade and stage. RESULTS: Cytologically assigned grade correlated with final histologic grade in 19 of 31 cases examined (61%). However, tumor invasion was not accurately assessable in cytologic samples from the majority of these cases. Statistically significant correlations of both increasing tumor grade and stage with abnormal CK20 expression were found. In addition, a significant relationship between focal CD44 isoform expression loss and tumor grade was identified. However, CD44 isoform expression loss did not significantly correlate with increasing tumor stage. CONCLUSION: Although cytologic tumor grading of UTTCC was accurate, invasion could not be adequately assessed. As an adjunct to morphologic analysis, immunostaining for CK20 and CD44 may aid in the clinical evaluation of UTTCC tumor stage and biologic behavior prior to definitive therapy.

DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry.

OBJECTIVE: To evaluate a rapid and simple method for DNA content analysis of urinary tract epithelial tumors with laser scanning cytometry (LSC). STUDY DESIGN: The subjects were 25 patients (37 specimens) who underwent surgery for urinary tract epithelial tumors. Tissue specimens of such tumors were frozen immediately after tumor resection and stored at -80 degrees C until used. Touch preparations were made and fixed in ethanol at room temperature. The cell nucleus was stained with propidium iodide solution containing RNase, and DNA ploidy was analyzed by LSC. Nuclear debris and overlapping nuclei were gated out by special statistical filters. In LSC, a normal diploid reference peak was determined by observing lymphocytes morphologically on the computer display of the instrument and/or under the microscope. RESULTS: DNA ploidy could be evaluated in all tumor tissues. The time it took from preparing the tumor specimen to the last measurement was about 40 minutes at the shortest, and measurement of all the specimens was completed within one hour. The coefficient of variation was 2.8-7.8% (mean, 4.4%). All eight specimens (100%) at grade 1 (G1) were DNA diploid, but 20% and 85.7% of the G2 and G3 cells, respectively, were DNA aneuploid. In total, 15 of the 37 specimens were DNA aneuploid. All 17 pTa-pT1 specimens (100%) were DNA diploid, but 100% and 50% of the T2 and T3 tumors, respectively, were DNA aneuploid. CONCLUSION: One can now supplement a morphologic diagnosis with useful information using LSC of touch preparations of tumors obtained at surgery or of imprints of archived, frozen specimens. LSC provides excellent DNA histograms for surgical specimens and has great potential for clinical application in pathology.

Needle aspiration cytology of metastatic high-grade transitional-cell carcinomas of the urinary tract.

We reviewed 4 cases of high-grade transitional-cell carcinoma (TCC) of the urinary tract with solitary pulmonary metastases that were studied by transthoracic needle aspiration biopsy cytology. There were two grade II and two grade III TCCs. The two grade II tumors yielded, in needle aspirates, syncytial tumor-cell clusters showing ill-defined, granular cytoplasm and slightly pleomorphic nuclei with inconspicuous nucleoli. In one case the tumor-cell cluster showed a focal acinar arrangement, mimicking cells of an adenocarcinoma. In both cases the electron microscopy (EM) study of aspirated tumor cells revealed epithelial cells with well-formed cell junctions, intracytoplasmic vesicles, apical short microvilli, and focal interdigitation of lateral cell membranes, suggesting a urothelial neoplasm. The two grade III TCCs yielded, in needle aspirates, pleomorphic malignant cells singly and in small clusters, showing well-defined, granular cytoplasm and pleomorphic nuclei containing prominent nucleoli, suggesting a poorly differentiated adenocarcinoma or an anaplastic large-cell carcinoma. By EM examination the aspirated tumor cells from one case revealed well-formed cell junctions, intracytoplasmic vesicles, poorly formed microvilli, and focal interdigitation of lateral cell membranes, suggesting a urothelial differentiation. In the other case the tumor cells were pleomorphic cells with occasional cell junctions and no ultrastructural features as seen in the other 3 cases of TCC. The tumor cells from the two grade II TCCs showed strong immunopositive reaction with keratin 7 antibody and weakly positive reaction with carcinoembryonic antigen antibody (CEAA), while those of the two grade III TCCs displayed only a weak and focal immunopositive staining with keratin 7 antibody and strong reaction with CEAA.

Clonal chromosome changes in non-neoplastic ureters.

Cytogenetic analysis was performed on 23 samples from non-neoplastic ureters. Clonal chromosome abnormalities were found in eight. They were: loss of Y chromosome, as a single abnormality (five cases) or associated with trisomy 10 and 20 (one case) or with trisomy 2 (one case); and duplication of Y chromosome (one case). Different numerical and structural sporadic abnormalities were found in nine cases. Immunohistochemical analysis and direct observation using the inverted microscope showed that the cells were mainly of the fibroblastic type. FISH analysis with chromosome 7 alpha-satellite probes failed to detect the presence of trisomy 7 in three epithelial cases tested.

Sarcomatoid carcinoma: an ultrastructural study with light microscopic and immunohistochemical correlation of 10 cases from various anatomic sites.

The histogenesis of sarcomatoid carcinoma has been an intriguing topic for pathologists for many years, and considerable evidence has accumulated in the fields of tissue culture, electron microscopy, and immunohistochemistry to support the concept that the sarcomatous cells derive by way of "divergent differentiation" (metaplasia) from the carcinomatous elements. We have studied a group of 10 cases of these tumors from various organs, using detailed ultrastructural analysis as well as light microscopic and immunohistochemical correlation. We found that there is an ultrastructural spectrum of differentiation from epithelial to mesenchymal type features and that the malignant spindle cells may be purely epithelial (3 cases), purely mesenchymal (3 cases), or a mixture of both (4 cases). Furthermore, individual cells may show biphasia, having desmosomes and tonofibrils as well as well developed rough endoplasmic reticulum and filaments with dense bodies. Electron microscopic and immunohistochemical results do not always correlate, illustrating the prudence of using several keratin antibodies, including wide-spectrum ones, and of performing electron microscopic examination on these tumors.

Sarcomatoid carcinoma of the urinary tract.

Sarcomatoid carcinoma is a rare variant of malignant tumor arising from the urinary tract. This tumor had been termed carcinosarcoma because of its carcinomatous and sarcomatous components. There is still some confusion in the terminology between true carcinosarcoma and sarcomatoid carcinoma; however, the latter is now regarded as primarily a malignant epithelial tumor with pseudosarcomatous transformation. Four cases of sarcomatoid carcinoma arising from the urinary tract are reported. The patients were a 77 year old female, and three males aged 62, 69 and 80 years. All but the eldest patient complained of gross hematuria. Surgical removal was performed in the younger three cases, and an autopsy was done in the remaining case. All the tumors were macroscopically polypoid. Histopathologic examination revealed fasciculated spindle-cell tumors with myxoid stroma or malignant fibrous histiocytoma-like spindle cell tumors. The epithelial nature was proven in these sarcomatous cells by immunohistochemical and/or electron-microscopic examinations. Only a small amount of squamous cell carcinoma components was also evident in the latter three cases. Although the younger three patients were alive at 44, 23 and 39 months' follow-up, respectively, constant careful monitoring is recommended.

Application of scanning electron microscopy and X-ray analysis to urinary tract cancer in animals and humans.

Scanning electron microscopy (SEM) has proven useful in various aspects of urinary bladder carcinogenesis research and these are reviewed as they pertain to our research involving sodium saccharin in the rat. Sodium saccharin-carcinogenesis in rats requires administration at high doses beginning at birth or earlier. Administration beginning at ages of 5 weeks or later results in much lower incidences of bladder tumors. Methods were developed for examining the rat fetal and neonatal bladder to further evaluate effects at these critical ages. Several significant differences were found by SEM between the fetal bladder compared to the adult. The typical polygonal superficial cells of the bladder with asymmetric unit membrane were present before birth, but the slow turnover rate of the adult bladder did not occur until 3-4 weeks of age. Sodium saccharin causes increased proliferation rates and hyperplasia of the urothelium which is dose-dependent. SEM was found to be more sensitive than either light microscopy or labeling indices to detect the earliest lesions induced by sodium saccharin. More recently, amorphous and crystalline material in the urine of rats fed high doses of sodium saccharin were detected by SEM examinations which contained silicon as well as calcium, phosphate, and magnesium as detected by energy dispersive X-ray analysis (EDS) with the SEM. These parameters may be relevant to differences between rats and humans and pertain to extrapolations regarding risk assessment.

The nested variant of transitional cell carcinoma: a neoplasm resembling proliferation of Brunn's nests.

This report describes four cases in which a malignant transitional cell neoplasm initially manifested as small nests and abortive tubules infiltrating the lamina propria. Many of the tumor cells were only slightly atypical but careful examination revealed at least some significantly anaplastic cells in every case, the degree of cellular atypia tending to parallel the depth of invasion. Despite the initial impression of a benign lesion resembling a proliferation of Brunn's nests, these carcinomas tended to be persistent and aggressive.

Ultrastructural localization of c-erbB-2 gene product in transitional cell carcinoma of the urinary tract.

The c-erbB-2 gene product was demonstrated immunoelectron microscopically in human transitional cell carcinomas of the urinary tract. Two antibodies specific for the c-erbB-2 gene product were used: a polyclonal antibody directed to the intracellular domain and a monoclonal antibody recognizing an epitope in the extracellular domain. The immunoreaction with the polyclonal antibody was observed on cell membranes and the cytoplasmic area beneath membranes with particularly strong reaction on microvilli. The immune reaction with the monoclonal antibody was confined to cell membranes with a linear pattern. Amorphous deposits in the intercellular spaces were also immunoreactive with the monoclonal antibody directed to an extracellular epitope, suggesting that a part of the c-erbB-2 gene product may be released from the cancer cell membranes into the extracellular space.

Predictive value of DNA measurements in bladder washings. Comparison of flow cytometry, image cytophotometry, and cytology in patients with a past history of urothelial tumors.

Comparative DNA ploidy measurements were carried out by flow cytometry and by image analysis on cells in 71 bladder washing specimens from 50 patients with past histories of bladder tumors. Among the specimens classified as diploid or questionable by flow cytometry, 14 showed the presence of aneuploid DNA values documented by image analysis. In 18 of the 50 patients, recurrent tumors were observed during a relatively brief period of follow-up. In 15 of them the DNA pattern was aneuploid and in three it was questionable. In nine of the 15 patients, both methods of DNA analysis disclosed aneuploidy, but in six patients aneuploidy was detected by image analysis only. A combination of DNA aneuploidy, whether observed by flow cytometry, image analysis, or both, and of positive or suspicious urine cytology is highly predictive of recurrence of high grade bladder tumors. Image analysis of DNA content in bladder washings adds information of clinical value above and beyond that obtained by flow cytometry.

Oat cell carcinoma of the urinary tract. An immunohistochemical and electron microscopic study.

Four oat cell carcinomas of the urinary tract (three of the bladder and one of the ureteropelvic junction) were studied using immunohistochemical and electron microscopic methods to evaluate the possible histogenesis of these tumors. Ultrastructurally, the oat cell component of the tumors had neurosecretory-type granules and positive immunoreaction for neuron-specific enolase, indicating the endocrine nature of the tumors. Three tumors were mixed with a non-oat cell component: adenocarcinoma and/or transitional carcinoma or carcinoid tumor. There are no previous reports of mixed oat cell and adenocarcinoma or carcinoid tumor of the urinary tract. This report is also the first of an oat cell carcinoma originating in the pelvic ureteric junction.

Clonogenic assay and in vitro chemosensitivity testing of human urologic malignancies.

Over the past year, we have attempted to grow both primary and metastatic urologic malignancies using a recently developed human tumor cloning system. Formation of colonies in vitro occurred in 125 of 164 primary tumors (76%), including 34 of 47 uroepithelial cancer specimens, 45 of 50 renal cell cancer specimens, 24 of 33 prostatic cancer specimens, and 22 of 34 testicular cancer specimens. A large percentage of metastatic cancers have also been successfully cultured. Growth sufficient for chemosensitivity testing ranged from 43% of the uroepithelial cancers cultured to 64% of the renal cell cancer specimens cultured. When in vitro chemosensitivity testing was performed, the in vitro chemosensitivity results show a striking similarity to clinical response rates for the same agents used for these tumors. Overall the human tumor cloning system appears to be a reasonable model for the study of human urologic malignancies.

Evaluation of growth rate of human transitional cell tumors according to sex chromatin (Barr bodies) content.

In 53 women with urothelial tumors of the bladder, renal pelvis and ureter of different in growth stages and grades of differentiation, a negative correlation among the value of the mitotic index and sex chromatin content was found. With a decrease in the number of nuclei containing sex chromatin bodies we have noted not only a proportional mitotic index increase but also a regular connected decrease of histological differentiation and a deeper invasion of the tumor. The significance of this simple test consists in the fact that one may judge the rate of tumor growth by the sex chromatin content in a small volume of biopsy material. A table of mitotic index values depending upon the quantity of nuclei with sex chromatin bodies in transitional cell tumors is presented.

Scanning electron microscopy in the study of normal, inflamed and neoplastic human urothelium.

Seventy-two samples of human urothelium have been examined by scanning electron microscopy (SEM) and the appearances compared with those of conventional light microscopy. Six main SEM cell types were recognised. None was diagnostic of malignancy; in particular, the presence of surface pleomorphic microvilli was not pathognomonic. However, the relation of the SEM appearance to malignancy and the accepted histological grades and clinical stages of malignancy was such as to warrant further examination.

The ultrastructure of surfaces of positively identified cells in the human urinary sediment. A correlative light and scanning electron microscopic study.

Surfaces of cells in the urinary sediment, postively identified by light microscopy (LM) were studied by scanning electron microscopy (SEM). About 35% of benign urothelial cells were characterized by the presence of short, stubby microvilli (MV). About 60% of such cells had surface microridges, and the remaining cells had surface blebs. Squamous epithelial cells were characterized primarily by microridges or by a mixture of microridges with short microvilli. Urothelial cancer cells had various surface characteristics. About 30% of these cells displayed plemorphic MV, about 10% of these cells showed short, regular MV, and the remainder appeared to be poorly preserved and had smooth surfaces, either with MV remnants or points of membrane rupture (holes or pits). The correlation between SEM surface features and the LM appearance of urothelial cancer cells was poor. Many of the most characteristic cancer cells on LM had no distinguishing SEM features. It is concluded that the application of SEM for the diagnosis of urothelial carcinoma in urine is of uncertain benefit, pending further studies.

Transitional cell carcinoma in the domestic cat.

In 17 cases of transitional cell carcinoma in cats the most common site for primary neoplasia was the urinary bladder; metastasis was recorded in five of the cases. Nine of the cases have been reported previously; the eight additional carcinomas from the files of the New York State College of Veterinary Medicine were highly invasive and had a desmoplastic response. According to WHO guidelines they were classified as nonpapillary, infiltrating; papillary, infiltrating; and variants of transitional cell carcinoma.