Kohsuke Sasaki

DNA ploidy analysis by laser scanning cytometry (LSC) in colorectal cancers and comparison with flow cytometry

We evaluated laser scanning cytometry (LSC) by comparing nuclear DNA ploidy determined by LSC and by flow cytometry (FCM) in 77 samples of human colorectal cancer from 48 patients. Both methods revealed an aneuploid peak in 30 (62.5%) of the cases, although two samples that were aneuploid by LSC were diploid by FCM and two others were diploid by LSC and aneuploid by FCM. The concordance rate for nuclear DNA ploidy was 91.7% in the 48 patients and 87.0% for the 77 samples. The DNA index was also highly correlated between two methods (r2 = 0.97, P < 0.001). We concluded that LSC provides DNA histograms equivalent to FCM for surgical specimens and has potential clinical application in pathology.

Intracellular localization of cyclin B1 during the cell cycle in glioma cells.

We investigated cyclin B1 expression during the cell cycle in human glioma cells cultured under asynchronous growing condition by two cytometry techniques: flow cytometry (FCM) and laser scanning cytometry (LSC). FCM analysis revealed the specific accumulation of cyclin B1 in G2/M phase with a wide intercellular variation (Dunphy WG: Trend Cell Biol 4:202-207, 1994). It is noteworthy that LSC, which is characterized by rapid quantitative analysis followed by imaging, allows morphological observation of the intracellular distribution of cyclin B1 as a function of cell cycle position cell by cell (Hunter T: Cell 75:839-841, 1993). Cyclin B1 was virtually undetectable in cells from G0/G1 phase to mid S phase, but became visible in the cytoplasm in late S phase. As cells proceeded within G2 phase, the level of cyclin B1 rapidly increased in the perinuclear region of the cytoplasm, but cyclin B1 was still faintly present in the nucleus. Cyclin B1 appeared in the nucleus at the mitotic phase. Then the nuclear membrane was disrupted and cyclin B1 was distributed evenly in the cell. The level of cyclin B1 was maximum in metaphase. However, it abruptly degraded at the end of metaphase, and subsequently G1 cells were cyclin B1 negative.

Measurement of proliferating cell nuclear antigen (PCNA) and its clinical application in oral cancers.

The PCNA score was measured in oral squamous cell carcinoma (SCC), and its relationship to other cell proliferation markers, Ki-67 score, S-phase fraction (SPF), and AgNORs counts was investigated. The PCNA score ranged from 0.4% to 43.5% with an average value of 22.8%, the Ki-67 score ranged from 4.9% to 40% with an average of 24.1%, and the SPF ranged from 0.4% to 32.5% with an average of 12.4%, while AgNORs counts ranged from 2.53/nucleus to 7.03/nucleus with an average of 4.74/nucleus. These four parameters were closely interrelated. There was a significant difference in PCNA score between malignant and nonmalignant lesions, suggesting a difference in growth activity. The mean PCNA score decreased significantly from 20.0% to 8.0% after cancer chemotherapy. The response of cancer cells to anticancer agents may be estimated by consecutive measurement of PCNA, since the PCNA score dropped after treatment in cases showing a favorable prognosis.

Intratumoral heterogeneity in DNA ploidy of esophageal squamous cell carcinomas

Intratumoral heterogeneity in DNA ploidy was investigated in 23 cases of squamous cell carcinoma of the esophagus. Nuclear DNA content was determined for multiple samples taken from the same tumor, using a flow cytometric technique. The incidence of DNA aneuploidy was 87% in this series, and DNA indices ranged from 0.78 to 2.64 but most of them fell within values between 1 and 2. Of these cases ten (43.5%) showed intratumoral heterogeneity in DNA ploidy; in addition to a diploid population, one to four heterogeneous aneuploid subpopulations were discernible in the same tumor. However, morphologic variation was minimal within the same tumor. DNA index seen in metastatic lesions was identical with one of those in the primary lesion. Mechanisms responsible for intratumoral difference in DNA ploidy are also discussed from the aspect of tumor progression. Cancer 68:2403–2406, 1991.

The significance of PCNA and p53 protein in some oral tumors

The expression of PCNA and p53 protein was evaluated in a total of 75 cases of benign and malignant lesions of the oral cavity, comprising 50 squamous cell carcinomas (SCCs), 14 leukoplakias, and 11 pleomorphic adenomas. The DNA histogram of 20 SCCs was measured by flow cytometry. p53-positive cells were frequently seen in SCCs, but were rare in leukoplakias and pleomorphic adenomas. The PCNA labeling index (LI) was higher in SCCs than in other benign lesions. The expression rate of p53 protein was markedly elevated in SCCs obtained from smoking patients, when compared to nonsmoking patients. DNA ploidy did not show a close relationship with PCNA and p53 expression. The mean value of PCNA LI for 22 cases carrying positive p53 protein was 52.3%, which was higher than that of p53 protein negative cases (35.7%). The Kaplan-Meier survival curve of the patients who were negative for p53 was significantly more favorable than for patients who were positive for p53 (P < 0.01, Cox-Mantel test). These results suggest that PCNA and p53 LI are markers for the malignant potential of the oral mucosa, and are a useful indicator suggesting a poor prognosis.

Detection of numerical chromosomal aberrations in malignant melanomas using fluorescence in situ hybridization

To evaluate the numerical chromosomal aberration i malignant melanoma, we have applied fluorescence in situ hybridization (FISH) with repetitive DNA probes specific for chromosomes 1, 6, 7, 9, 10, and 17 on 24 fresh malignant melanomas (primary: 14, metastatic: 8).

Monosomy of chromosome 18 detected by fluorescence in situ hybridization in colorectal tumors

Background.At least two different evolutional pathways of colorectal cancer, namely the adenoma‐carcinoma sequence and de novo carcinogenesis, have been indicated. However, whether there is a difference between them in affected chromosomes and genes has not yet been elucidated. Chromosomal examination is expected to provide a clue to an answer to this question. In this study, the relation of aberrations in chromosome 18 to type of colorectal cancer was examined.

Immunohistochemical detection of p21waf1/cip1/sdi1 and p53 proteins in formalin-fixed, paraffin-embedded tissue sections of colorectal carcinoma

Wild-type p53 protein is a critical participant in G1 cell cycle arrest through the induction of waf1/cip1/sdi1 gene product p21, but mutant p53 proteins have lost transcriptional activity. To know whether the view obtained from studies using cultured cells is adaptable to human solid tumors, the authors immunohistochemically stained p21 and p53 in formalin-fixed, paraffin-embedded tissue sections from 67 cases of colorectal carcinoma. Of these, 54 cancers showed positive staining of p53, whereas p21-positive cells were identified in 46 tumors. However, the proportion of cells positive for these proteins varied considerably among cases, and moreover wide variation in p21 immunoreactivity was noted within the same tumor. Although there was an inverse relationship between p21 and p53 staining in tumors, namely, p21-positive cells were p53 negative and vice versa, an intermingling of tumor cells expressing concomitantly both genes was noted in 34 (51%) of 67 tumors. In these tumors, both proteins were usually moderately stained, but tumor cells intensely coexpressing both were found in one sample. This study supports the notion that although p21 expression is regulated by p53 under physiological conditions, it can be regulated by an additional pathway(s) in solid malignant tumors.

DNA ploidy heterogeneity in early and advanced gastric cancers.

To evaluate the clinical utility of flow cytometric DNA analysis in gastric cancers, four or more fresh tissue specimens were systematically taken from gastric cancers in 127 consecutive patients including 68 early cancers. DNA ploidy and its variation in individual tumors were determined, and the data were related to clinicopathologic findings. DNA aneuploidy was detected frequently (84.3%) irrespective of tumor progression and correlated significantly with histologic grade (G1-2 [89.6%] vs. G3-4 [76.0%], P < 0.05). DNA ploidy heterogeneity was found in 67.7% of tumors and correlated with invasion depth (mucosa [40.5%] vs. submucosa-serosa [81.2%], P < 0.001), regional lymph node metastases (negative [58.4%] vs. positive [82.0%], P < 0.01), and stage grouping (I [58.8%] vs. II-IV [86.0%], P < 0.01). The maximum DNA index of a tumor correlated significantly with invasion depth (mucosa [1.16, median] vs. submucosa [1.82], P < 0.01) and lymph node metastases (negative [1.22] vs. positive [1.86], P < 0.001). The DNA index of the subpopulation that was the most widely distributed within the tumor was significantly associated with lymph node metastases (negative [1.14, median] vs. positive [1.44], P < 0.001) and histologic grade (G1-2 [1.37] vs. G3-4 [1.12], P < 0.001). More than 80% of the diploid and/or single aneuploid stemline tumors were stage I, whereas more than half of diploid and multiple aneuploid stemline tumors were stage IV. Variation in DNA ploidy rather than presence of DNA aneuploidy correlates best with progression of gastric cancer.

Measurement of Bromodeoxyuridine Labeling Index, Ki-67 Score and Ag- NOR Count in Breast Carcinomas

Nuclear DNA content and three cell proliferation markers, the bromodeoxyuridine (BrdUrd) labeling index, Ki-67 score and Ag-nucleolar organizer region (Ag-NOR) counts, were measured in 21 cases of breast carcinoma, and then relationships among them were investigated. The BrdUrd labeling indices and Ki-67 scores were strongly correlated with one another (r = 0.90, p less than 0.001). They were also slightly correlated with the Ag-NOR counts (p less than 0.05). Although growth activity varied greatly from case to case, even within the same histologic grade in breast carcinoma, high histologic grade carcinomas were frequently associated with high values of both the BrdUrd labeling index and Ki-67 score, as compared with low histologic grade ones. The DNA ploidy level (DNA index) was independent of these cell proliferation markers. In the present series, 17 of 21 cases (81%) were aneuploid. All cases without DNA aneuploidy fell into a group of low histologic grade, while 7 of 11 low grade tumors were aneuploid. All three cell proliferation markers were low in 2 of the 4 cases without DNA aneuploidy. It may be possible to identify cases with favorable prognosis in a group of low grade carcinomas on the grounds of diploid DNA content and low values of these markers.