Chester J. Herman

Tissue-specific markers in flow cytometry of urological cancers: cytokeratins in bladder carcinoma

Thirty‐eight transitional‐cell carcinomas (TCC) were analyzed by flow cytometry (FCM) using propidium iodide for DNA analysis and antibodies to cytokeratin by indirect immunofluorescence. By means of two‐dimensional FCM analysis, cytokeratin‐positive tumor cells could be analysed separately from cytokeratin‐negative stromal and inflammatory cells. This resulted in an 18% increase in sensitivity of FCM detection of aneuploidy (10/38 samples with one‐parameter DNA analysis versus 15/38 samples with two‐parameter DNA and cytokeratin analysis). In addition, S‐phase could be determined in the 15 aneuploid samples by means of two‐parameter analysis where this was not possible using only DNA content because of the overlap of diploid and aneuploid populations. FCM analysis allowed quantification of the percentage of tumor cells expressing cytokeratin 18 which has previously been shown to correlate quantitatively with higher grade, higher stage TCC. The quantitative measurement of tumor‐cell expression of cytokeratin 18 by FCM analysis appears to provide additional information of potential prognostic value, independent of tumor‐cell ploidy and proliferative fractions.

Antibodies to cytokeratin and vimentin in testicular tumour diagnosis

Thirteen primary and metastatic testicular germ cell tumours, including classical and anaplastic seminomas, and non-seminomatous testicular tumours were examined for their intermediate filament protein (IFP) types. The seminomas were shown to react with a monoclonal and a polyclonal antibody to bovine lens vimentin, while non-seminomatous germ cell tumours were strongly positive for a polyclonal and a monoclonal antibody to cytokeratin. In one case of seminoma with elevated serum levels ofβHCG andαFP, cytokeratin positive tumour cells were found. In the case of teratocarcinoma, several components of the tumour could be distinguished using a combination of antisera in double-label immunofluorescence microscopy. The glandular component of this tumour was positive with the polyclonal antikeratin, but also with the monoclonal cytokeratin antibody specific for glandular epithelia (RGE 53). However, the squamous component was negative with this latter antibody. Strikingly, the spindle cell component showed focal positivity for vimentin, with coexpression of cytokeratin and vimentin in some cells. Our data show that antibodies to cytokeratin and vimentin can be helpful in the diagnosis of testicular germ cell tumours, especially in the differentiation between seminomas and non-seminomatous tumours.

Survival of spermatogonial stem cells in the rat after split dose irradiation during LH-RH analogue treament

A rat model has been created in which a single injection of an LH-RH analogue depot preparation (Zoladex, ICI 118630) produced a temporary interruption of the pituitary-gonadal axis. This effect applied during irradiation was investigated as a possible mechanism to protect the testis from radiation damage. A local testicular irradiation dose of 6.0 Gy was given either as a single dose or as a fractionated (2 X 3.0 Gy) dose at different time intervals ranging from 8 to 72 h. Stem cell survival was measured 11 weeks after irradiation by means of the repopulation index and the number of haploid cells (spermatids) measured by flow cytometry. Serum gonadotrophins and testosterone concentrations were measured to evaluate hormonal recovery. No significant differences were observed between serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone and the duration of the fractionation interval. Stem cell survival was higher following fractionated irradiation in comparison with the single dose. For the 8 h interval an increase in recovery ratio was found, amounting to a factor of 5 of the single dose value. The fluctuating pattern of the recovery curves indicated changes in radiosensitivity of stem cells. The combination of hormonal inhibition of spermatogenesis and fractionated irradiation led to a decrease in the absolute numbers of stem cells. However, the stem cell recovery curves were identical to those seen without hormonal inhibition. It was concluded that hormonal pretreatment with Zoladex during split dose irradiation had no protective effect on stem cell survival.

Artificial vaginas: Possible sources of epithelialization

Biopsy specimens from surgically created peritoneium-lined vaginas in 23 patients with complete vaginal agenesis showed squamous metaplastic epithelium lining the vaginal canal. In nine of the patients, histologic or cytologic evidence, or both, suggested that the metaplastic squamous epithelium could be derived from pre-existing glandular epithelium - sebaceous, paramesonephric (mullerian), or urogenital sinus. The histologic and cytologic features in six patients with paramesonephric-like gland elements in the mucosa and submucosa of the neovagina were similar to the features seen in milder forms of vaginal adenosis.

Applications of a human tumour clonogenic cell culture system in gynaecological oncology: review and personal experience

Current and future clinical applications of the Human Tumour Clonogenic Cell Culture (HTC3) system are presented. Advanced gynaecological cancers, especially ovarian, endometrial and cervical carcinomas, require extensive systemic chemotherapy. The HTC3 system seems to be an adequate instrument for individualized chemosensitivity testing in order to choose proper cytostatic treatments; that is, maximum tumour cell kill with minimal side-effects. Furthermore, this system helps in assessing the response after treatment by detection of remaining clonogenic, and thus viable, tumour cells. Other clinical applications of the system include grading of the tumour and recognition of the histologic type. Thus the HTC3 provides a potential tool in diagnosis, treatment and follow-up of gynaecologic malignancies.

Effects of Testicular Irradiation on Stem Cell Survival, Hormonal Environment and Spermatogenic Cells in Wistar Rats

Adult male Wistar rats were exposed to 3.0 Gy local testicular irradiation. Testes of irradiated and non-irradiated rats were examined histologically and flow cytometrically, at several intervals up to 78 days after irradiation. Concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone determined at these intervals were not different from those of controls. The survival of stem cells were measured 11 weeks after irradiation (with doses varying from 1.0 Gy to 6.0 Gy at 0.6 Gy/min) by means of the repopulation index and by the number of haploid cells (spermatids). Correlation between both methods and relation to stem cell survival were discussed. The dose response curves yielded D0 values of stem cell survival of 2.33 +/- 0.06 Gy (repopulation index) and 2.08 +/- 0.08 Gy (number of haploid cells). The D0 value of the rat was not much different from that found in mice. It was concluded that the used parameters can offer insight when studying hormonal substances during irradiation.