Total Labeling Index Research Articles

Abstract 3185: Use of whole slide digital imaging for determination of caPCNA and Ki-67 labeling index in normal breast reductions and DCIS: Comparison of the two biomarkers using the Aperio image-based positive pixel algorithm

Abstract While mitotic rates as well as proliferation indices have been reported as significantly different among normal breast and different grades of breast cancer, a reproducible cut-off value has not emerged. The goal of this study was to determine whether more reproducible algorithms using whole slide digital images (WSDI) can define better cut-off values for the normal breast vs. DCIS. Design: Whole tissue sections from 10 normal breast reductions and 10 cases of DCIS were evaluated by immunohistochemistry for caPCNA and Ki67 (Mib-1). Having identified an isoform of PCNA that was uniquely expressed by cancer cells (caPCNA) and distinct from the isoform expressed by non-malignant and normal cells (nmPCNA), antibody selectively binding to caPCNA was developed and used for these studies. Stained slides were scanned using the Aperio (Vista, California) automated whole slide scanning system (Scanscope CS) and were viewed using ImageScopeTM. Single fields of view from each WSDI measuring ∼420,000 µ2 and representing the highest density of caPCNA and Ki-67 positive nuclei were selected for analysis. Different areas were analyzed in a subset of slides to determine the variability in each case, and the total nuclear labeling index was generated using the Aperio positive pixel algorithm. Results: We analyzed the labeling indices in 10 normal breast reductions and 10 DCIS cases. Both caPCNA and Ki-67 were nuclear specific with minimal cytoplasmic staining. The mean labeling indices for the Aperio automated positive pixel counts were caPCNA (0.15) and Ki-67 (0.12) for normal breast and caPCNA (0.52) and Ki-67 (0.29) for DCIS. The correlation coefficients between automated counts were 0.653 for normal breast and 0.616 for DCIS, with an overall correlation coefficient of 0.708 for the entire series. Conclusions: Our analysis of both biomarkers yielded a similarly increasing labeling index for DCIS cases when compared to their expression in normal breast expression. The positive pixel algorithm method has a sampling bias due to subjective selection of the area being counted. In summary, the present results show similar findings for caPCNA and Ki-67 in normal breast reductions with no evidence of breast disease and in DCIS clinical cases. Comparing caPCNA with Ki-67, and unlike traditional PCNA, caPCNA can potentially better differentiate between normal breast and DCIS, and may be a useful biomarker for early stage cancer diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2011-3185

Role of Necl‐5 in the pathophysiology of colorectal lesions induced by dimethylhydrazine and/or dextran sodium sulphate

Necl-5 is an immunoglobulin-like molecule that was originally identified as a poliovirus receptor. Although Necl-5 expression is often up-regulated in cancer cells, its pathophysiological significance in the development of cancer remains unclear. We investigated the roles of Necl-5 in the development of colitis-associated neoplasia. Necl-5-deficient mice were generated and treated with dimethylhydrazine (DMH) and/or dextran sodium sulphate (DSS) to induce colitis and its associated neoplasias. Colon tissues were examined for histology, Ki-67 expression by immunohistochemistry and K-ras gene mutation. Colon tumours occurred significantly less frequently in heterozygous (Necl-5(+/-)) or homozygous Necl-5-deficient (Necl-5(-/-)) mice than in wild-type (WT) mice with DMH/DSS treatment. Total ulcer index and inflammatory cell infiltration were significantly lower in Necl-5(-/-) mice than in WT mice with DSS alone or DMH/DSS treatment. Colon tumours in both WT and Necl-5(-/-) mice showed high cell proliferation ability but lacked K-ras mutation. The total Ki-67 labelling index in non-neoplastic colon epithelium was significantly higher in WT (45.9 +/- 0.94) than in Necl-5(+/-) (34.3 +/- 1.40) or Necl-5(-/-) (27.7 +/- 1.15) mice with DMH/DSS treatment (p < 0.001). Necl-5 plays a role in the development of colitis-associated cancer by up-regulating colonic mucosal cell proliferation.

Proliferation indices of invasive breast carcinomas after in vivo 5-bromo-2'-deoxyuridine labelling: a flow cytometric study of 75 tumours.

In vivo labelling of human breast tumours with 5-bromo-2'-deoxyuridine and analysis of proliferation by multiparameter flow cytometry is a new tool by which the labelling index, S phase duration (TS) and potential doubling time (Tpot) of the tumour can be estimated. Tumour specimens from 75 patients with invasive breast carcinoma were studied. Six tumours could not be analysed, and the TS and Tpot values could not be calculated for a further 18. The median labelling index (n = 69) was 4.2 per cent. The median TS (n = 51) was 8.7 h and the median Tpot (n = 51) was 8.2 days. The median intra-assay coefficient of variation for determination of the labelling index was 0.29 in five tumours. There were no significant differences in the total labelling index, TS or Tpot when patients were stratified according to lymph node status, tumour size, tumour grade or menopausal status.

Cell proliferation kinetics are abnormal in transitional mucosa adjacent to colorectal carcinoma.

The transitional mucosa adjacent to colorectal carcinoma shows characteristic morphological and histochemical abnormalities that may be indicative of premalignant changes. Sixteen colorectal carcinomas were studied to analyse the proliferative activity and its distribution pattern in tissue samples from transitional mucosa located within 2 cm of the cancer and from uninvolved mucosa 10 cm from the cancer. Proliferative activity was assessed using a monoclonal antibody to proliferating cell nuclear antigen (PCNA). The total labelling index (percentage of labelled cells in a column) of transitional mucosa was not significantly different from that of uninvolved mucosa and normal control mucosa but it was significantly higher in cancer tissue (P < 0.001). An upward shift of the compartment of proliferating epithelial cells towards the bowel lumen was seen in transitional mucosa. The labelling index of the middle compartment (compartment 3) of the crypt in transitional mucosa was significantly higher than that in uninvolved mucosa and control mucosa (P < 0.001). Unstable cytokinetic change may already have started in apparently normal transitional mucosa. Assessment of cell proliferation kinetics by PCNA immunostaining may be useful in screening for heightened risk of the development of colorectal cancer.

Proliferation characteristics of human colorectal carcinomas measured in vivo

The cell proliferation kinetics of 100 human colonic and rectal adenocarcinomas have been studied in vivo by bromodeoxyuridine infusion and multiparameter flow cytometry. A total of 97 patients, three with synchronous tumours, consented to receive a single bolus dose of 250 mg between 2.4 and 16 h before curative or palliative surgery. By this method, the ploidy pattern, the total and aneuploid labelling indices (LI), the S phase duration (Ts) and the potential doubling time (Tpot) can be estimated. Of the tumours 48 were diploid and 52 were aneuploid. The mean and median total LI of 100 tumours were 9.0 per cent (range 0.7-22.2 per cent). The mean aneuploid LI was 12.1 per cent (median 12.0 per cent, range 2.0-25.5 per cent), and was significantly higher than the total LI (P = 0.01). The labelling index alone is not a sufficient indicator of proliferation, because the Ts also varies within and between tumours. The intertumour range of the Ts varied from 4.0 to 28.6 h. The mean was 14.1 h and the median was 13.1 h. The mean Tpot was 5.9 days (median 3.9 days) with a range of 1.7-21.4 days. No correlation was found between any kinetic parameters and the Dukes' classification or histological classification. The correlation between proliferation and prognosis will be established in due course.

Crypt organization in the progression to colorectal neoplasia: Computerized three dimensional (3D) crypt reconstruction and quantitative analysis of bcl-2, bas, mitosis and apoptosis within the crypt

AIMS: Alterations in epithelial proliferation and apoptosis in colonic mucosa are associated with an increased risk of colon cancer. Deregulation of bcl-2 and bax are important factors in the control of cell turnover. The aim of this study was to analyze patterns of bcl-2 and bax expression and in mitosis and apoptosis in the progression to colorectal neoplasia, and to reconstruct colorectal crypts to visually demonstrate this deregulation. METHODS: 12 patients with a sigmoid adenomatous polyp and 12 low risk controls were studied. Mucosal biopsies were taken from the polyp, tissue adjacent to and distant from the polyp and from the sigmoid colon of controls. Apoptotic and mitotic figures, and cells expressing bcl-2 or bax were counted, and labeling indices calculated for each within each crypt. Results are expressed as total labeling index (TLI) and labeling index (L1) for each of the five compartments (cpts) in which colonic crypts were divided for mitotic index (MI), apoptotic index (AI), and bd-2 and bax expression. Sample individual crypts that were complete within serially sections were idemified. The appropriate cells were marked, the images captured and stored, and then whole crypts were reconstructed in 3D using Analyze3.1 to assess the intra-erypt relationships of each variable. RESULTS: There was no significant difference between TLI and LI for bcl-2 and bax for controls vs. distant mucosa. The TL1 and L1 were significantly higher for polyp vs adjacent vs. distant mucosa for both bcl-2 (cpts 1-3, p<O.02) and bax (cpts 2-5, p<0.01). There was a significam increase in MI in polyp vs. distant mucosa (cpts 1-3, p<0.05). No difference in AI was seen between distant and adjacent mucosa, but there was a trend to a reduction in AI in polyp mucosa vs. all other sites. A total of 56 crypts were available for reconstruction. In reconstructed crypts, deregulation of bcl-2 and bax, increased mitosis, and the presence of apoptotic bodies in the basal cpts of polyps where bd-2 expression was highest, was easily visualized in still images and video sequences. CONCLUSIONS: Mitosis, expression ofbcl-2 and bax, and apoptosis maybecome deregulated within the colorectal crypt in the progression to colorectal neoplasia. In addition to quantitative analysis, computerized reconstruction with still images or video sequences of whole crypts can be useful in advancing our understanding of this process.

Variability of cell proliferation in the proximal and distal colon of normal rats and rats with dimethylhydrazine induced carcinogenesis.

To investigate the patterns of cell proliferation in proximal and distal colons in normal rats and rats with 1,2-dimethylhydrazine (DMH) induced carcinogenesis using the thymidine analogue bromodeoxyuridine. Colonic crypt cell proliferation was immunohistochemically detected using the anti-bromodeoxyuridine Bu20a monoclonal antibody. Marked regional differences were found in both groups. Total labelling index (LI) and proliferative zone size in both normal (8.65+/-0.34 vs 7.2+/-0.45, 27.74+/-1.07 vs 16.75+/-1.45) and DMH groups (13.13+/-0.46 vs 11.55+/-0.45, 39.60+/-1.32 vs 35.52+/-1.58) were significantly higher in distal than in proximal colon (P<0.05), although the number of cells per proximal crypt was greater (31.45+/-0.20 vs 34.45 +/-0.39, 42.68+/-0.53 vs 49.09+/-0.65, P<0.0001). Crypt length, total LI and proliferative zone size all increased in both proximal and distal regions of DMH rats compared to normal controls (P<0.0001). In DMH-treated rat colon a shift of labelled cells to higher crypt cell positions was demonstrated distally whilst a bi-directional shift was evident proximally (P<0.05). Our results show that changes in cell proliferation patterns, as assessed by bromodeoxyuridine uptake, can act as a reliable intermediate marker of colonic cancer formation. Observed differences between proliferation patterns in distal and proximal colon may be associated with the higher incidence of tumors in the distal colon.

Severe imbalance of cell proliferation and apoptosis in the left colon and in the rectosigmoid tract in subjects with a history of large adenomas

BACKGROUNDAlterations in epithelial proliferation and apoptosis in colonic mucosa are associated with an increased risk of colon cancer. It is unclear if these alterations represent a generalised “field defect”.AIMSTo analyse...

Effects of Calcium and Vitamin Supplementation on Colon Cell Proliferation in Colorectal Cancer

Calcium and antioxidant vitamins, such as A, C, and E, have been shown to reduce colorectal epithelial proliferation and thereby to act as possible chemoprotective agents in colorectal cancer. We investigated the effects of an intervention with calcium and vitamins on cell proliferation in the colonic mucosa of patients operated on for colorectal cancer. Patients with resected colorectal cancer Dukes' stage B-C were randomized to receive daily 30,000 IU of axerophthol palmitate (vitamin A) plus 1 g ascorbic acid (vitamin C) plus 70 mg of dl-alpha-tocopherol acetate (vitamin E) and 2 g natural calcium daily or indistinguishable placebo for 6 months. At the time of surgery and after 6 and 12 months of treatment, cell kinetics of normal colonic mucosa were assessed by using proliferating cell nuclear antigen (PCNA). Ninety patients were enrolled and 77 were assessable: 34 in the treatment group and 43 in the placebo group. A significant reduction of mean total PCNA labeling index (PCNALI) was evident in both groups after 6 months (vitamins/calcium, from 16.11 ± 2.43 to 10.71 ± 2.81; placebo, from 17.30 ± 2.63 to 12.53 ± 3.40). The difference in the percentage of reduction of mean PCNALI between baseline and after 6 months was not statistically significant in the treatment and placebo groups: 34% and 28%, respectively. A second control, 6 months after discontinuation of vitamin and calcium supplementation, showed a further decrease of mean total PCNALI in both groups, but this was not statistically significant. Our randomized trial showed that calcium and vitamin supplementation does not reduce cell kinetics of colon epithelium. Furthermore, this study suggests the need for extreme caution in the interpretation and publication of studies on chemoprotectants in colon cancer without a control group.

Tumour cell kinetics as a prognostic factor in squamous cell carcinoma of the cervix treated with radiotherapy

Purpose: Proliferative rate and DNA ploidy status were evaluated by flow cytometry in cervical cancer patients, prior to radiotherapy, to assess their importance as prognostic factors to predict survival rates. Material and methods: Between 1987 and 1995, a total of 260 patients with squamous cell carcinoma (SCC) of the cervix, FIGO stages IB–IIIB were analysed. Tumour samples were incubated with bromodeoxyuridine (BrdUrd) in vitro to measure their total labelling index (totLI) and LI (totLI for diploid and anLI for aneuploid tumours). Proliferation was also assessed by S-phase fraction (SPF) analysis of the DNA profile. Patients had intracavitary therapy (three applications, each of 16 Gy to point A) and XRT of 40–50 Gy given over 4–5 weeks. Results: The cervical tumours were characterized by a high proliferation rate which varied within each clinical stage of disease. The totLI ranged from 1.1 to 33.1% with median value of 9.6% whilst the LI ranged from 1.1 to 37.1% with a median value of 10.9%. Univariate analysis identified patient’s age ( cut- offpoint≤50& greater; years ) and to a lesser extent proliferation (cut-off point, median totLI=9.6%) as significant prognostic factors for 5-year survival. The median survival time for younger patients (≤50 years) with tumours of low proliferation (totLI≤9.6%) tumours was 17.5 months compared with 56 months in the faster proliferating tumours ( P=0.0354). In the older patient sub-group, proliferation rate had no influence on survival. The median LI value was not a useful parameter in survival. Cox multivariate analysis showed that patient age (≤50 years) and low proliferation of the tumour cells (totLI≤9.6) were unfavourable prognostic factors for cervical cancers treated with radiotherapy. DNA ploidy was not significant in this series. Conclusions: These data suggest that further improvements in therapy might be gained by selection of alternative treatments strategies such as neoadjuvant chemotherapy or radiation sensitizers in younger patients with more slowly proliferating tumours.

Colonic epithelial cell proliferation in hereditary non-polyposis colorectal cancer

Background—Despite the recent discovery of four genes responsible for up to 90% of all cases of hereditary non-polyposis colorectal cancer (HNPCC), there will still be families in whom predictive testing...

The flow cytometric analysis of total p53 protein content and proliferation indices in colorectal cancer, in relation to clinical outcome

This study was undertaken to assess the value of flow cytometric measurements of total p53 protein content and proliferation indices derived from in vivo halogenated pyrimidine labelling. Two series of colorectal cancer specimens were studied for which clinical outcome data were recorded. A series of 84 archival, ethanol-fixed, bromodeoxyuridine (BrdUrd) labelled colorectal tumours were analysed by flow cytometry for their total and cell cycle phase p53 protein content using the pAb1801 monoclonal antibody. A second series of 33 freshly obtained tumours was used for assay evaluation and for comparison with the archival material. In the archival series (n=84), the median p53-pAb1801 LI was 81.9% (range: 11.1-99.8%). In only three tumours could significant amounts of p53 protein not be detected. The median phase specific p53-pAb1801 LI in G0/G1 was 71.6%, in S was 95.5%, and in G2/M was 98.5%. In the series of fresh tumours (n=33), the median p53-pAb1801 labelling index (LI) was 94.6% (range: 17.9-99.9%). Only two tumours failed to express significant amounts of p53 protein. There was no significant difference in the generally high levels of p53 protein content between the fresh and archival series. Life-table analysis of the patients in the archival series failed to demonstrate a statistical difference in life expectancy in relation to Dukes' stage when tumours were stratified by the median total p53 labelling index. In this study, p53 content and proliferative indices measured by flow cytometry do not have independent predictive value over Dukes' grading in determining the outcome of colorectal cancer. Flow cytometry is confirmed as a practical tool for multi-parametric and cell cycle analysis of oncoprotein expression in human tumour biopsies.

Calcium regulation of colonic crypt cell kinetics: Evidence for a direct effect in mice

Background & Aims: Oral calcium supplementation is believed to decrease colonic hyperproliferation through neutralization of fatty acids and bile acids. In the present study, the effect of oral calcium, given with low-fat diets, in the early stages of colorectal carcinogenesis is evaluated. Methods: In experiment A, mice received normal or low-calcium diets and were killed at 25 weeks. In experiment B, mice were fed the same diets but were submitted to six weekly injections of dimethylhydrazine and were killed at 10, 16, and 21 weeks. Cell proliferation was evaluated using bromodeoxyuridine immunohistochemistry. Results: In experiment A, mice fed low-calcium diets showed a significant upward shift of the proliferative compartment ( P = 0.04) (phase 2 defect) in the absence of hyperproliferation. In experiment B, besides a phase 2 defect, dimethylhydrazine-induced hyperproliferation was also significantly enhanced in animals fed low-calcium diets (phase 1 defect) as shown by an increased number of labeled cells per column and total labeling index ( P = 0.01). Conclusions: Low-calcium diets induce an upward shift of the main proliferative compartment, which reflects an increased risk for malignant transformation. This effect was observed with a low-fat diet, suggesting a direct mechanism, rather than the usual indirect one, documented with high-fat diets.

Colonic cell proliferation in two different ethnic groups with contrasting incidence of colon cancer: is there a difference in carcinogenesis?

Most studies on colorectal carcinogenesis suggest a field defect, preceding overt development of cancer. The low incidence of adenomatous polyps in the African population, however, suggests that there may be an alternative route for cancer development. The aim of the study was to discover if the difference in incidence of colorectal cancer in Africans compared with the white population is reflected in a different pattern of cell proliferation. Histological normal mucosa from 30 patients (15 white South African (W), 15 South African Africans (A)) with confirmed colon cancer were examined. Proliferating cells were detected using the Ki-67 antigen. In addition, cell proliferation data were obtained, from 30 age matched controls (15 Africans, 15 white South Africans), without colorectal disease. The African controls were significantly younger (mean (SD) (A: 42 (20), W: 66 (13), p < 0.05)) than the white controls. The second control group had a significantly higher mean (SD) total labelling index (W: 11 (3), A: 6 (4), p < 0.05). In addition the proliferative pattern of the white group without evidence of colorectal cancer showed a comparatively large amount of dividing cells in compartment 2, compared with African controls (mean (SD) (W: 21 (8), A: 9 (8), p < 0.05)). Mucosa from Africans with cancer showed a proliferative pattern with the same increased total labelling index (A: 15 (5), W: 16 (6), p = NS, phase II proliferative lesion) and an even more pronounced upward expansion (phase I proliferative lesion) compared with white cancer patients. This suggests that the mechanism of colorectal carcinogenesis is similar in Africans and the white population. The lack of clinical evidence of the adenoma-carcinoma sequence, and the incidence of cancer at a comparatively young age in Africans may be explained by the fact that colorectal cancer in this ethnic group behaves more aggressively and that adenomatous polyps are rapidly converted into overt cancer before detection.

Both cdc2 and cdk2 promote S phase initiation in Xenopus egg extracts.

Xenopus egg extracts induce S phase DNA replication in added sperm pronuclei in a highly regulated manner, similar to events in vivo. Removal of cyclin-dependant kinases (cdks) or cdk2 from these extracts using affinity matrices severely inhibits initiation of S phase. We have used p13suc1 beads to remove both cdk2 and cdc2 proteins from egg extracts and developed a method to replace either protein alone to assess their capacity to initiate DNA replication. Re-addition of either cdk2 or cdc2 proteins to depleted extracts, through translation of their respective mRNAs, restimulated replication, judged by both total synthesis and labelling index. An ATP-binding-site mutant cdk2 mRNA (cdk2.R33) failed to stimulate replication and inhibited S phase initiation in mock-depleted extracts. Both human and Xenopus cdc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the stimulation induced requires cdc2 catalytic activity, though not its mitotically active form. Rescue of replication by p34cdc2 is also observed in extracts depleted of cdks with a cdk2 antibody, which still retain much of their endogenous cdc2 protein. We conclude that newly synthesised p34cdc2, but not the inherited 'old' form, can induce S phase and in this form may overlap in function with p33cdk2.

Zonal heterogeneity of peroxisomal enzymes in rat liver: Differential induction by three divergent hypolipidemic drugs

The hepatic zonation of peroxisomes and four of their matrical enzyme proteins (catalase, acyl coenzyme A oxidase, multifunctional protein, thiolase) has been investigated in normal male rats and after treatment with three divergent hypolipidemic drugs by means of automatic image analysis and quantitative immunoelectron microscopy. The induction of peroxisomal enzymes was confirmed by Western blotting and enzyme activity determinations in liver homogenates and in highly purified peroxisome fractions. In control animals the volume density of peroxisomes and the concentrations of all four enzymes were comparable across the liver acinus. The treatment with all three compounds induced stronger proliferation of peroxisomes in zone 3 than in zone 1 hepatocytes, and significant differences between the different compounds were detectable only in pericentral hepatocytes. The immunolabeling density reflecting the enzyme protein concentration in the peroxisomal matrix was unchanged or reduced for catalase but showed significant elevations for beta-oxidation enzymes in treated animals. It did not increase, however, in parallel with the augmentation of the volume density but showed a different pattern for each enzyme, depending on the acinar location and the type of compound used, suggesting a complex fine regulation for each protein. The total labeling index, reflecting the total amount of each protein, was for almost all enzymes higher in perivenous hepatocytes, indicating that they respond more intensely to hypolipidemic drugs than periportal cells. The elevations of the peroxisomal enzymes in zone 3 of the acinus may contribute to the perturbation of the sterol and bile acid metabolism associated with hypolipidemic therapy.

Prospective evaluation of cell kinetics in head and neck squamous carcinoma: the relationship to tumour factors and survival.

Tumour growth rates were measured in 105 patients using in vivo incorporation of bromodeoxyuridine (BrdU) and investigated for any relationship to tumour factors or survival. The median labelling index (LI) was 8.7%, the duration of S-phase (Ts) was 14 h and the potential doubling time (Tpot) was 5.9 days. The labelling index in aneuploid tumours was significantly higher than that in diploid tumours. However the total labelling index (TLI) did not differ significantly between aneuploid and diploid tumours, and so it would seem likely that the difference in LI is due to the dilutional effect of benign tissue upon the calculation of LI in diploid tumours. The total labelling index, duration of S-phase and potential doubling time were not related to the tumour factors examined (site, T stage, N stage, stage grouping). Interim survival analysis was carried out and there was no difference in survival between those patients with high values for TLI, Ts, and Tpot and those with low values.

Circadian variations of epithelial cell proliferation in human rectal crypts

Background/Aims: Evidence (almost exclusively from animal studies) suggests that proliferation within the colorectal mucosa undergoes circadian variations. The epithelial cells that line the human colorectal crypt occupy definite positions along the longitudinal axis according to their proliferative potential and degree of differentiation. Thus, circadian rhymicity was investigated in humans to locate the areas along the longitudinal crypt axis in which diurnal fluctuation might occur. Methods: Rectal mucosal biopsy specimens were obtained every 4 hours for a 24-hour span from each of 23 subjects (8 healthy volunteers and 15 with histories of sporadic adenomatous polyps). [3H]thymidine histoautoradiography was used to determine ratios of S-phase to total cells (total labeling index) in the crypt. Glands were also divided into 5 equal compartments from base (compartment 1) to mouth (compartment 5), and labeling indices were calculated for each. Results: Important temporal variations were confined to compartment 2 (F = 5.15, P = 0.0003) and total labeling indices (F = 3.57, P = 0.005). Despite individual variations, proliferation was generally higher at night and lower during afternoon. Upper-crypt proliferative rates (compartments 4 and 5) remained decidedly stable (F = 0.5, P = NS). Normal subjects and patients with polyps displayed similar circadian behaviors. Conclusions: Circadian fluctuation in proliferation is confined to the area of the crypt normally associated with replication. Upper-crypt indices, including those that were higher than normal (a colon-cancer risk marker) are stable over 24 hours. These findings should be useful in planning chemoprevention trials and chemotherapeutic regimens.

Correlation of epithelial proliferation and squamous esophageal histology in 1185 biopsies from Linxian, China.

Epithelial proliferation is an active area of research in gastrointestinal cancer, but only a few studies have examined the relationship of esophageal epithelial proliferation and squamous histologic findings in populations with high rates of squamous esophageal cancer. In order to study this correlation, tritiated thymidine labeling was performed on 1185 esophageal biopsies from 745 residents of Linxian, China, a county with some of the highest esophageal-cancer rates in the world. Total labeling index (TLI = total labeled cells/total cells counted) was used to measure the amount of proliferation, and the proportion of labeled cells found in cell layers 4 to 10 (labeled cell fraction 4 plus, LF4+ = labeled cells in layers 4-10/total labeled cells) was used to measure the vertical distribution of proliferation. Of the biopsies, 979 were histologically normal, 51 showed acanthosis, 35 showed esophagitis, 116 showed squamous dysplasia, and 6 showed invasive squamous cancer. The mean values of both proliferation variables, stratified by histologic diagnosis, showed the following relationships: normal = acanthosis < esophagitis = dysplasia < cancer. The ranges of proliferation values overlapped extensively in all biopsy categories, so that measuring proliferation could not substitute for histologic diagnosis. It remains to be seen whether proliferation values, histologic diagnoses, or some combination of these methods is most predictive of subsequent esophageal cancer.

ASSESSMENT OF CELL-PROLIFERATION KINETICS IN FAMILIAL ADENOMATOUS POLYPOSIS BY PCNA IMMUNOSTAINING

Cell proliferation was assessed using immunostaining for proliferative cell nuclear antigen in 10 patients with familial adenomatous polyposis (10 normal-appearing mucosa, 19 adenoma and 3 adenocarcinoma specimens) and 6 control subjects (rectosigmoid mucosal biopsies). The total labeling index (percentage ratio of the number of labeled cells to the total number of cells in a column) was 31.70+/-9.32% (mean+/-SD) in adenocarcinoma, 27.95+/-4.24% in adenoma (p<0.001 versus normal-appearing mucosa), 19.54+/-3.46% in normal-appearing mucosa (p<0.01 versus control mucosa) and 13.73+/-5.19% in control mucosa. Adenomas showed hyperproliferation and upward expansion of the proliferative zone. When adenomas were classified by size, their diameter was related to the proliferative activity (r=0.73, p<0.01). The normal-appearing mucosa already showed changes in cell proliferation. In an animal study, a linear correlation between the labeling index for proliferative nuclear cell antigen and that for bromodeoxyuridine was demonstrated in rat colonic mucosa (r=0.96, p<0.001). These results suggest that immunostaining for proliferative cell nuclear antigen can be used as a convenient and precise method of studying cell proliferation kinetics and that the increase of the proliferative activity of adenomas in proportion to their size provides some support for the adenoma-carcinoma sequence.