J. L. M. Beck

In interphase cells the proliferation-associated antigen recognized by monoclonal antibody Ki-67 is almost exclusively located in the nucleoli. When cells at several stages of mitosis were examined for the localization of the Ki-67 antigen, a striking redistribution could be observed. During prophase the distinct nucleolar Ki-67 fluorescence changed to a bright irregular meshwork throughout the nucleoplasm. At metaphase the antigen appeared to be distributed in a reticulate structure surrounding the condensed chromosomes, while at late telophase a punctated staining of the entire nucleoplasm was observed, which preceded the typical nucleolar localization pattern in each of the two daughter cells. Immunolabelling with Ki-67 of metaphase chromosome spreads revealed a circumferential staining of the individual chromosomes. The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells. When mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold. Quantification of the Ki-67 fluorescence signal using flow cytometry revealed the highest staining intensities in mitotic cells. Furthermore, it was shown that nutritionally deprived cells became negative for Ki-67.

The nuclear DNA content of 53 transitional cell carcinomas (TCCs) of the urinary bladder, as determined by flow cytometry (FCM), was compared with chromosome ploidy as detected by nonradioactive in situ hybridization (ISH). For this purpose, probes for repetitive DNA targets in the (peri) centromeric region of chromosomes 1 and 18 were used. Hybridization results with both probes of 35 TCCs, which had a DNA index of approximately 1.0 as concluded from FCM, showed evident chromosome 1 aberrations in approximately 25% of the tumors, and in a few cases an aberration for chromosome 18 was detected. Comparison of the ISH spot numbers for both chromosomes showed in most cases a higher number for chromosome 1 than for chromosome 18. ISH on 18 cases of TCCs, which showed a single peak in FCM with a DNA-index of 1.2 to 3.2, exhibited a profound heterogeneity. In these TCCs the ratio between chromosomes 1 and 18 varied over a wide range, resulting in cases showing more hybridization signals for chromosome 1 than for chromosome 18 or the opposite. Furthermore, using ISH minor cell populations showing polyploidization and giant cells containing numerous ISH signals could occasionally be detected. Results showed that interphase cytogenetics by ISH enable a fast screening of numerical chromosome aberrations and detection of different cell populations within one tumor, which was apparently homogeneous according to FCM.

This study presents the results of cytophotometric (CPM) and flow cytometric (FCM) DNA ploidy measurements in cervical intraepithelial neoplasias grade III (CIN III) with and without synchronous invasive squamous cell carcinoma. Hysterectomy and biopsy material from 21 patients 35 years of age or younger and from 18 patients age 50 years or older was studied. The DNA analysis was performed in nuclei isolated from specific areas of paraffin-embedded tissue. There were significant differences in the distribution of DNA patterns between the two age groups. About 80% of CIN III lesions in women 50 years of age or older, with or without a coexisting invasive cancer were aneuploid. In the group of younger women a diploid DNA pattern was found in about 60% of CIN III with concomitant invasive cancer. In the absence of an invasive cancer, CIN III lesions were mostly polyploid. The DNA pattern of invasive cancers was generally identical with the adjacent CIN, thus suggesting that the two lesions were related. Although the prognostic value of DNA ploidy measurements in cervical intraepithelial lesions in women in these two age groups has to be further evaluated, these results are at considerable variance with previously published data on DNA values in CIN and invasive carcinoma. In four CIN III lesions without invasive cancer, in women of the group of 35 years of age or younger, human papilloma virus common antigen could be demonstrated by immunochemical procedure. In three of these cases a polyploid DNA pattern was present; the fourth case showed a bimodal aneuploid pattern.

Both image and flow DNA cytometry were performed in isolated nuclei from paraffin-embedded tumor tissue of patients with small cell carcinoma of the lung (SCCL). In 14 patients tissue was obtained by surgery from the primary tumor. From 14 patients tissue was taken by autopsy. From two patients tissue obtained by both surgery and later autopsy were available. From the autopsy patients tissue was taken only from the primary tumor (n = 6), from a metastasis (n = 1) and from the primary tumor and distant metastases (n = 7). Twelve of the tumors obtained by surgery were diploid, and two multiploid (two stem lines present). This was found both with image and flow cytometry. The group of patients could clearly be subdivided in short survivors (less than 9 months, n = 6) and long survivors (greater than 16 months, n = 8); since in both groups one multiploid and the remainder diploid cases were present, ploidy did not seem to be a good prognosticator for survival. In most (n = 26) of the tissues measured from the autopsy patients, again, a good correlation between image and flow DNA cytometry was obtained, the histograms being either (near) diploid or multiploid. In six cases, however, flow cytometry showed multiploidy whereas image showed aneuploidy (one single peak clearly deviating from diploidy). This discrepancy is caused because normal diploid (nonneoplastic) cells in the preparations could not be discarded from the flow cytometry measurements. Using the image cytometry data of the primary tumors, five diploid, three aneuploid, and four multiploid tumors were found. In five of the seven patients of whom tissue was obtained from the primary tumor and multiple metastases, differences between the histograms were found, mostly showing two malignant cell populations in one tissue and only one of them in another. Of one of the two patients of whom tissue was obtained by surgery and later autopsy, a change in histogram pattern was observed. It is concluded that although there is a high similarity between image and flow DNA cytometry, for an optimal interpretation of the histogram pattern, image measurements are more reliable. Ploidy determination does not seem to be of use in prediction of survival, and care should be taken in interpreting DNA histograms of metastases in SCCL patients because of the variability in histogram pattern.