Morphological classification of oral leukoplakia (author's transl)

656 cases of oral leukoplakia were analysed according to macroscopic aspects, microscopic growth patterns and histologicalcytological differentiation, and the relationship to cancer of the oral cavity was studied. Homogeneous and speckled leukoplakia can be distinguished macroscopically, while flat (70%), papillary-endophytic (22%) and papillomatous-exophytic (8%) types can be distinguished by their growth pattern. Histological-cytological characteristics consist of epithelial hyperplasia (hyperkeratosis with ortho- or parakeratosis; akanthosis) and epithelial dysplasia (dyskeratosis, basal-cell hyperplasia, loss of polar arrangement of the basal cells, cell polymorphism, increased mitosis rate). No or little dysplasia was demonstrated in 74% of leukoplakias, moderate in 17% and marked in 6%. Carcinoma-in-situ, defined as high-grade dysplasia with additional loss of epithelial layering, was found in 3%. Precancerous leukoplakia (in almost 10% of cases, counting high-grade dysplasias and carcinoma-in-situ) must be delineated as a special group. Numerous correlations were found between dysplastic leukoplakias and oral cavity cancer as regards localisation, age and sex distribution. In the various leukoplakia forms there was an increased incidence of marked stroma reactions and of Candida colonisation with increased degrees of dysplasia.     

Localization of plasminogen activator inhibitor type 2 (PAI-2) in hair and nail: implications for terminal differentiation

To examine at which stage in the multistep process of head and neck tumorigenesis numerical chromosomal alterations can be detected by fluorescence in situ hybridization (FISH), biopsies and cell smear preparations of clinically healthy oral tissue, premalignant lesions (leukoplakias), and tumors were analyzed by FISH using chromosome-specific centromeric probes. Aberrations found in tumor biopsies and in tumor cell smears consisted of trisomy of chromosomes 1, 7, 10, and 17 and monosomy of chromosomes 1, 7, 9, 10, and 17. In five of eight dysplastic oral leukoplakia biopsies, aberrations were seen consisting of trisomy of chromosome 1, 7, and 17, and monosomy of chromosome 9. No aberrations were found in biopsies of hyperplastic lesions (n = 8), or in oral cell smears of persons at risk. Because numerical chromosomal aberrations seem to be highly specific for malignant cells, FISH may help to identify leukoplakias that have a high risk of malignant conversion.     

Phototherapy of cancer and atheromatous plaque with texaphyrins

The incidence of nonmelanoma skin cancer, including both squamous cell carcinoma and basal cell carcinoma, is a significant health problem in the United States. Actinic keratosis (AK), the precursor of cutaneous squamous cell carcinoma, is a major risk factor for nonmelanoma skin cancer. In addition, AKs are tissue targets for the identification of biomarkers for use in chemopreventive studies. The biomarker addressed in this study is epidermal cell proliferation, as quantitated by proliferating cell nuclear antigen (PCNA). Shave biopsies were obtained from AKs, tissue immediately adjacent to AKs, normal-appearing, upper-medial arm skin, and non-sun-exposed skin from 19 subjects. When any degree of PCNA staining was considered positive (semiquantitative 1-4 scale), there was a significant difference and a progressively increasing mean PCNA labeling index (LI) in the total epidermis (basal and suprabasal layers), beginning with non-sun-exposed buttock skin, with the lowest LI (2.5 + or - 1.6%), followed by upper-medial arm skin (12.3 + or - 7.4%; P = 0.0015), skin adjacent to AKs (19.2 + or - 12.2%; P = 0.0218), and finally, AKs with the highest LI (34.6 + or - 20.1%; P = 0.0017). This same pattern was observed when the epidermis was separated into basal and suprabasal layers, with the exception of a nonsignificant result for upper-medial arm skin compared with adjacent skin in the basal layer (P = 0.3981). PCNA LIs were also analyzed separately by staining intensity (i.e., scores of 1-4). The PCNA LI in skin with varying degrees of sun damage and/or histological atypia is a candidate surrogate end point biomarker for skin cancer chemoprevention studies.     

THe cost of radiotherapy. Piedmontese experience]

The blood supply of hepatocellular carcinoma (HCC) is primarily arterial. Recent studies reported differences of vascular, especially arterial, supply among low- and high-grade dysplastic nodules and HCC. We assessed arterialization using monoclonal antibody specific for smooth muscle actin as well as simultaneous changes in sinusoidal capillarization in cirrhotic nodules, dysplastic nodules, and HCC. We immunohistochemically stained 56 cirrhotic nodules, 20 low-grade dysplastic nodules, 27 high-grade dysplastic nodules, and 20 HCCs for alpha smooth muscle actin (to identify unpaired arteries (i.e., arteries not accompanied by bile ducts) and CD34 (indicating sinusoidal capillarization). Distribution and number of unpaired arteries and distribution of sinusoidal capillarization were graded semiquantitatively. Unpaired arteries were rare in cirrhotic nodules, significantly more common in dysplastic nodules of both types (p < 0.00001), and most common in HCC. Sinusoidal capillarization was least common in cirrhotic nodules, significantly more common in dysplastic nodules (p < 0.0035), and most common in HCC. No topographic relationship between unpaired arteries and sinusoidal capillarization was identified. These findings showed that (1) distributions of sinusoidal capillarization and unpaired arteries in dysplastic nodules are intermediate between those in cirrhotic nodules and HCC, supporting dysplastic nodules as premalignant lesions; (2) unpaired arteries are histologically useful for distinguishing dysplastic nodules from large cirrhotic nodules; and (3) areas of sinusoidal capillarization within dysplastic nodules are unrelated to location of arterialization.     

Electroencephalographical examination: special reference to the patients consulted from general practitioner

Expression of Proliferating cell nuclear antigen (PCNA) was evaluated in formalin-fixed, paraffin-embedded normal and abnormal cervical squamous epithelia using immunoperoxidase stains and PC10 monoclonal antibody to PCNA. PCNA was exclusively expressed in the parabasal and basal layers of normal ectocervix and a similar pattern was seen in nine of the 11 cases with squamous metaplasia. Examples of cervical dysplasia showed expression in higher layers of cervical epithelium, corresponding to the degree of dysplasia. Increased staining was seen in condylomas and markedly reduced staining with atrophy. The percentage of basal cells that stained increased progressively from atrophic to normal, to condylomatous, to dysplastic epithelia. Proliferative activity can be satisfactorily assessed in formalin-fixed cervical epithelia using PC10 PCNA antibody. This assessment can be of potential diagnostic use in difficult cases.     

Synthesis and biological evaluation of novel cryptophycin analogs with modification in the beta-alanine region

CENP-F is a newly characterized cell cycle-associated nuclear antigen that is expressed in low amounts in G0/G1 cells and that accumulates in the nuclear matrix during S phase with a maximal expression in G2/M cells. CENP-F can be analyzed by flow cytometry and used as a proliferation marker. In the present study, therefore, we characterized the expression of CENP-F in non-Hodgkin's lymphoma by immunohistochemical techniques to detect potential dysregulation of the protein or to establish CENP-F as a reliable proliferation marker. A polyclonal rabbit antibody reacting with CENP-F was prepared and used for immunohistochemical analyses after antigen retrieval. The rabbit antibody produced immunofluorescence patterns, flow cytometric profiles, and Western blot reactivity identical to those of the human autoantibody used in earlier studies. The percentage of CENP-F-positive and Ki-67-positive cells, as well as the labeling index, S-phase time, and potential doubling time, derived from in vivo iododeoxyuridine incorporation, were evaluated in 41 non-Hodgkin's lymphomas. Aggressive lymphomas showed higher CENP-F values than did indolent cases (10.1 vs. 3.4%). The percentage of CENP-F-positive cells correlated significantly to the S-phase fraction (r(s) = 0.68), the Ki-67 index (r(s) = 0.56) and the labeling index of iododeoxyuridine (r(s) = 0.47), as well as to S-phase time and potential doubling time (r(s) = 0.34 and -0.40). A lower fraction of CENP-F-positive cells was found, compared with the Ki-67 index (4.9 vs. 9.4%), supporting previous observations that CENP-F was expressed in a fraction of actively growing cells. These correlative data indicate that CENP-F expression defines a specific subpopulation of growing cells and that no clear evidence for dysregulation was found. Accordingly, CENP-F seems to be a useful proliferation marker for formalin-fixed and paraffin-embedded material.     

Decreased cyclin A2 and increased cyclin G1 levels coincide with loss of proliferative capacity in rat Leydig cells during pubertal development

Postnatal development of Leydig cells can be divided into three distinct stages of differentiation: initially they exist as mesenchymal-like progenitors (PLC) by day 21; subsequently, as immature Leydig cells (ILC) by day 35, they acquire steroidogenic organelle structure and enzyme activities but metabolize most of the testosterone they produce; finally, as adult Leydig cells (ALC) by day 90 they actively produce testosterone. The aims of the present study were to determine whether changes in proliferative capacity are associated with progressive differentiation of Leydig cells, and if the proliferative capacity of Leydig cells is controlled by known hormonal regulators of testosterone biosynthesis: LH, insulin-like growth factor I (IGF-I), androgen, and estradiol (E2). Isolated PLC, ILC, and ALC were cultured in DMEM/F-12 for 24 h followed by an additional 24 h in the presence of LH (1 ng/ml), IGF-I (70 ng/ml), 7alpha-methyl-19-nortestosterone (MENT, 50 nM), a synthetic androgen that is not metabolized by 5alpha-reductase, or E2 (50 nM). Proliferative capacity was measured by assaying [3H]thymidine incorporation and labeling index (LI). Messenger RNA (mRNA) and protein levels for cyclin A2 and G1, which are putative intracellular regulators of Leydig cell proliferation and differentiation, were measured by RT-PCR and immunoblotting, respectively. Thymidine incorporation was highest in PLC (9.24 +/- 0.21 cpm/10(3) cell, mean +/- SE), intermediate in ILC (1.74 +/- 0.07) and lowest in ALC (0.24 +/- 0.03). Similarly, LI was highest in PLC (13.42 +/- 0.30%, mean +/- SE), intermediate in ILC (1.95 +/- 0.08%), and undetectable in ALC. Cyclin A2 mRNA levels, normalized to ribosomal protein S16 (RPS16), were highest in PLC (2.76 +/- 0.21, mean +/- SE), intermediate in ILC (1.79 +/- 0.14), and lowest in ALC (0.40 +/- 0.06). In contrast, cyclin G1 mRNA levels were highest in ALC (1.32 +/- 0.16), intermediate in ILC (0.47 +/- 0.07), and lowest in PLC (0.12 +/- 0.02). The relative protein levels of cyclin A2 and G1 paralleled their mRNA levels. Increased proliferative capacity was observed in PLC and ILC, but not ALC, after treatment with either LH or IGF-I. Treatment with MENT increased proliferative capacity only in ILC and had no effect in any other group. Treatment with E2 decreased proliferative capacity in PLC but not in ILC or ALC. The changes in proliferative capacity after hormonal treatment paralleled cyclin A2 mRNA and were the inverse of cyclin G1 mRNA levels. We conclude that: 1) decreased cyclin A2 and increased cyclin G1 are associated with the withdrawal of the Leydig cell from the cell cycle; 2) the proliferative capacity of Leydig cells is regulated differentially by hormones and is progressively lost during postnatal differentiation.     

Ki-67 and p53 in T2 laryngeal cancer

OBJECTIVE: To study the relationship between the proliferative capacity, represented by the immunohistochemical labeling index (LI) of proliferation marker Ki-67, and the p53 status, as in theory an intact p53 cell cycle checkpoint system should result in a lower proliferative capacity. STUDY DESIGN: From a group of 128 patients with a T2 laryngeal carcinoma, presented from 1989 to 1993 at the University Hospital Utrecht, 20 patients with recurrent disease and 16 patients without recurrent disease were randomly selected. All patients received primary irradiation. METHODS: Denaturing gradient gel electrophoresis and immunohistochemistry determined the p53 status. MIB-1 staining was used to determine the Ki-67 LI. RESULTS: In 36% of specimens we found a p53 mutation with overexpression (LI, 31%). In 8% a p53 mutation without p53 overexpression was found (LI, 18%). Forty-two percent showed no mutation but, nevertheless, overexpression (LI, 35%). Neither mutation nor overexpression was found in 14% (LI, 38%). No correlation exists between p53 status and proliferative capacity of tumors (analysis of variance [ANOVA]; P = .104). The proliferation rate as established with Ki-67 LI positively correlates with response to radiotherapy (P = .006). CONCLUSIONS: 1. Overexpression of wild-type p53 protein does not result in cell cycle arrest measurable by a lower Ki-67 LI in comparison with cases overexpressing mutant type p53 protein. 2. A high Ki-67 LI correlates with a favorable response to radiotherapy.     

Reduced levels of the cell-cycle inhibitor p27Kip1 in epithelial dysplasia and carcinoma of the oral cavity

Recent studies have shown that the cyclin-dependent kinase (cdk) inhibitors play important roles in cell cycle progression in normal cells. Alterations in the cdk inhibitors also appear to be important in cancer development in a number of human tumors. p27Kip1 is a member of the CIP/KIP family of cdk inhibitors that negatively regulates cyclin-cdk complexes. Reduced levels of p27Kip1 protein have been identified in a number of human cancers, and in some cases reduced p27Kip1 is associated with an increase in proliferative fraction. In the present study, we examined p27Kip1 protein by immunohistochemistry in 10 normal and 36 dysplastic epithelia and in 8 squamous cell carcinomas from one anatomical site within the oral cavity, the floor of the mouth. Proliferative activity was assessed in serial sections by determining the expression of the cell cycle proteins Ki-67 and cyclin A. p27kip1 protein was significantly reduced in oral dysplasias and carcinomas compared with that in normal epithelial controls. In addition, there was a significant reduction in p27Kip1 protein between low- and high-grade dysplasias, suggesting that changes in p27Kip1 expression may be an early event in oral carcinogenesis. There was increasing expression of Ki-67 and cyclin A proteins with increasingly severe grades of dysplasia compared with normal controls. Although there was a strong correlation between Ki-67 and cyclin A scores (r2= 0.61) for all categories of disease, there was a weak negative correlation between Ki-67 and p27Kip1 levels (r2 = 0.29) and between cyclin A and p27Kip1 levels (r2 = 0.25). In conclusion, this study has found that a reduction in the proportion of cells expressing p27Kip1 protein is frequently associated with oral dysplasia and carcinoma from the floor of the mouth. Furthermore, reductions in p27Kip1 levels are associated with increased cell proliferation, although other changes likely contribute to altered cell kinetics during carcinogenesis at this site.     

Molecular and cellular biomarkers for field cancerization and multistep process in head and neck tumorigenesis

One way to explain the development of head and neck cancer is through the theories of field cancerization, i.e., the exposure of an entire field of tissue to repeated carcinogenic insult, and multistep process, i.e., development of multiple cancers in a predisposed filed through a series of recognizable stages. Recent molecular genetic studies of histologically normal and premalignant epithelia of high-risk subjects and studies of malignant tumors in aerodigestive tract epithelia have identified a continuum of accumulated specific genetic alterations that possibly occur during the clonal evolution of tumors, namely, during the multistep process. Second primary or multiple primary tumors arise in the same fields as independent clones, with similar but unique molecular genetic and/or cellular alterations. Consequently, the assessment of these genetic and phenotypic alterations has been integrated into clinical chemoprevention trials in an effort to identify biomarkers that are also risk predictors and intermediate end points. This review covers candidate biomarkers of the processes of field cancerization and multistep tumor development in aerodigestive tract epithelia, including general and specific genetic markers, proliferation markers, and squamous differentiation markers.     

Zonal variation of apoptosis and proliferation in the normal prostate and in benign prostatic hyperplasia

OBJECTIVE: To determine whether benign prostatic hyperplasia (BPH) results from an imbalance between cell proliferation and apoptosis, and the extent to which the rates of these opposing processes are altered with the expression of the anti-death oncoprotein bcl-2. MATERIALS AND METHODS: Ten prostate glands from normal men (mean age 43.7 years) were sampled according to McNeal's zonal anatomy, in addition to 30 prostate adenomas obtained from prostatectomy specimens from symptomatic patients (mean age 61.4 years). Tissue samples were fixed in formalin and embedded in paraffin. Proliferation and bcl-2 expression were assessed by immunostaining using Mib-1 and anti-bcl-2 antibodies, while apoptotic bodies were specifically stained using in situ nick translation. The percentage of positive cells was determined by optical microscopy. RESULTS: In normal epithelium, the rates of proliferation and apoptosis were increased in the peripheral zone (Mib-1 1.7%, apoptotic bodies 3.3%) compared with the central (0.2% vs 1.4%) and transition (0.1% vs 1.8%) zones. Proliferation was significantly greater in BPH than in normal prostate tissue (3.7%), contrasting with a stable rate of apoptosis (1.4%). In the normal prostate, bcl-2 was expressed by glandular and basal cells in the peripheral zone. In the central zone, bcl-2 was overexpressed in basal cells and in most glandular cells of the intraluminal ridges. Bcl-2 expression in the transition zone was limited to dispersed basal cells. In BPH, bcl-2 was strongly expressed by basal cells in mature glandular formations and in most cells of young small nodules. CONCLUSION: BPH may result from both an increase of proliferation within the basal compartment and a failure of apoptosis to counterbalance basal cell proliferation. Increased expression of bcl-2 may participate in this process by blocking apoptosis.     

Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification

Cyclin D1 proto-oncogene is a key regulator of the mammalian cell-cycle acting at the restriction point in late G1. Amplification of the cyclin D1 locus, located on chromosome 11q13, as well as cyclin D1 protein overexpression have been reported in several human malignancies. The purpose of this study was to evaluate cyclin D1 gene copy status and protein expression during the multistep process of head and neck tumorigenesis, using a combination of fluorescence in situ hybridization and immunohistochemistry techniques. From 29 selected patients presenting with head and neck squamous carcinoma and whose tumor cytospins had been previously screened for presence (16 cases) or absence (13 cases) of amplification at the 11q13 band, we analysed 46 paraffin-embedded tissue specimens that demonstrated, besides the primary tumor, the presence of contiguous adjacent normal tissue and/or premalignant lesions. Of the 16 amplified cases, nine demonstrated a continuous progression from premalignant to invasive carcinoma and seven (77.7%) of these cases showed cyclin D1 gene amplification in premalignant lesions prior to development of invasive carcinoma. Increased cyclin D1 protein expression was observed in all 16 amplified tumors and five of the 13 (38.4%) non-amplified tumors. Interestingly, dysregulated cyclin D1 expression was also found in the premalignant lesions adjacent to all 16 amplified tumors, and it appeared to precede cyclin D1 gene amplification. In contrast no dysregulated expression was detected in the premalignant lesions of the non-amplified tumors. In conclusion, these findings provide strong evidence for early dysregulation of cyclin D1 expression during the tumorigenesis process and suggest that dysregulated increased expression precedes and possibly enables gene amplification.     

Autoimmunity to the cell cycle-dependent centromere protein p330d/CENP-F in disorders associated with cell proliferation

p330d/CENP-F is a novel proliferation-associated and cell cycle-dependent centromere autoantigen which appears to play a very important role in mitotic progression. As an initial step in exploring the clinical and biological significance of autoantibodies to this protein, we evaluated the clinical histories of 26 patients producing these antibodies. The antibodies were detected by both indirect immunofluorescence microscopy (IIF) and Western blotting. All the sera contained anti-p330d/CENP-F IgG antibodies, with an average titer by IIF of 1:6,917 (range 1:160 to 1:20,480). Most of the patients had disorders associated with abnormal or increased cell proliferation at the time the anti-p330d/CENP-F antibodies were detected. These included cancers of various types (14), chronic liver disease (3), chronic rejection of renal allografts (2), and Crohn's disease (1). The average IIF titer of the anti-p330d/CENP-F antibodies in the patients with cancer, 1:10,103, was significantly higher than the average titer in non-cancer patients, 1:3,200 (P = 0.008). Autoimmunity to p330d/CENP-F appeared not to be associated with rheumatic diseases, in particular scleroderma, since only three of the 26 patients had rheumatic disease and the antibodies were not detected by IIF in a group of 351 patients with scleroderma and related disorders. Our findings, although retrospective and limited to a relatively small number of patients, point to the hypothesis that autoimmunity to p330d/CENP-F could be related to events involving increased or abnormal cell proliferation.     

Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS)

BACKGROUND: Steroid 5 alpha-reductase is essential for the intracellular accumulation of dihydrotestosterone (DHT), which mediates androgen effects on target tissue. METHODS: In the present study, we describe the differential expression and cellular localization of 5 alpha-reductase 1 and 2 isoenzymes in the human prostate, and untreated and hormone-resistant prostatic carcinomas. The secretory epithelium of normal and hyperplastic glands showed strong nuclear 5 alpha-reductase 1 reactivity. Accordingly, the DHT forming 5 alpha-reductase process in secretory luminal cell types may be mediated predominantly by the type 1 isoenzyme. The androgen-independent basal cell layer variably expressed type 1 and 2 isoenzymes in nuclear and cytoplasmatic compartments. This suggests that circulating androgens are involved to control the basal cell layer, which represents the proliferative compartment of the human prostate. RESULTS: When compared with benign prostate tissue, increased 5 alpha-reductase reactivity was detected in prostate cancer, particularly in high-grade tumors and androgen-insensitive states of the disease. In cancerous lesions, the type 1 isoenzyme tended to shift to the cytoplasm, while the nuclear staining remained unchanged or slightly increased. Referring to the type 2 isoenzyme, increased cytoplasmatic and nuclear enzyme activity was detected in malignant cells when compared with adjacent benign prostate tissue. Even endocrine differentiated tumor cells that consistently lacked the nuclear androgen receptor variably expressed 5 alpha-reductase immunoreactivity. CONCLUSIONS: Although the functional significance of the differential subcellular localization of type 1 and 2 isoenzymes is currently unknown, the present data suggest that prostate cancer retains the DHT forming 5 alpha-reductase process in high-grade lesions and recurrent disease. Accordingly, circulating androgens may be still significant in these hormone-refractory malignancies.     

Temporal variation in cellular proliferation during recornification of mouse tail skin

The influence of the time of injury on subsequent epidermal regeneration is unknown. Epidermal cell proliferation of tail skin in C57BL/6J mice in response to tape stripping was followed for 7 days by radiolabelled thymidine incorporation and autoradiography. The homeostatic labelling index (LI) of the basal epidermis of unmanipulated, unwounded (control) animals was 7.6% and did not vary depending on the time of day. Tape stripping increased the LI of epidermal basal cells 110% above control values 24 h after injury. Labelling indexes of epidermal basal cells in the skin adjacent to the wounded area were 7.0%. Basal cell DNA synthesis stimulated by wounding exhibited a distinct temporal variation at 24 h postinjury, with tail skin wounded at 12.00 h found to be 275% greater than control values and elevated 78% from LIs recorded at any other time point. This temporal spike was due to the time of day at which wounding occurred rather than the time point when the LI was determined. Mice wounded at 12.00 h and terminated 27 h later (15.00 h) had LIs that were 52% greater than wounds created at 09.00 h and examined at 12.00 h the following day. Higher levels of DNA synthesis in tail skin injured at 12.00 h compared to wounding at 09.00 h was detected 12-48 h after injury. Furthermore, DNA synthesis in wounds created at 12.00 h returned to baseline levels 1-2 days earlier than tail skin wounded at 09.00 h. Investigation of other strains of mice detected differences in radiolabelling of epidermal basal cells 24 h after tape stripping at 12.00 h or 09.00 h in CD-1 and BALB/cJ mice, but not in the C3H/HeJ strain. These results indicate: (a) there is no diurnal variation in the LI of mouse tail skin under normal homeostatic conditions (b) tape stripping is a potent stimulator of basal cell turnover in the epidermis (c) the time of wounding determines the magnitude of the increase in the LI of basal cells following injury, and (d) the proliferative response to wounding of the tail is dependent on the strain of mouse.     

Growth and cellular proliferation of antral follicles throughout the follicular phase of the estrous cycle in Meishan gilts

Meishan gilts were ovariectomized 2 h after an i.v. injection of 5'-bromo-2'-deoxyuridine (BrdU, a thymidine analogue; 5 mg/kg body weight) on Days 15-19 of the estrous cycle or 24-30 h after observed estrus (post LH, PLH). All antral follicles > or = 3 mm from one ovary were fixed in Carnoy's solution. Granulosa and thecal cell labeling indexes (LI; percentage of nuclei staining for BrdU) as well as LI of cells within the basal, middle, and antral thirds of the granulosa cell layer were estimated for each follicle. In addition, antral and granulosa cell layer volume, granulosa cell layer thickness, granulosa cell density, number of granulosa cells, and number of S-phase cells per hour were estimated for each follicle. Mean follicular diameter increased linearly (p < 0.01) from Day 15 to PLH, with a growth rate of 0.77 mm/day. Granulosa and thecal cell LI decreased (p < 0.01) from Day 15 to PLH; however, granulosa cell LI was greater (p < 0.01) than thecal cell LI on Days 15 and 16 but less (p < 0.05) than thecal cell LI on Day 19. Follicles collected from PLH gilts contained no labeled granulosa cells. Cells within the basal third of the granulosa cell layer contained fewer (p < 0.01) labeled nuclei than did cells within the middle or antral thirds. In addition, LI within the basal and middle thirds of the granulosa cell layer decreased (p < 0.01) from Days 15 to 18 and from Days 15 to 17, respectively, whereas LI within the antral third remained constant from Days 15 to 18. Granulosa cell layer thickness was greatest (p < 0.01) on Day 15, then decreased (p < 0.01) and was similar from Day 16 to PLH. Granulosa cell density was similar from Days 15 to 19, then decreased (p < 0.01) for PLH gilts. Antral and granulosa cell layer volumes increased linearly (p < 0.01) from Days 15 to 19 and Day 15 to PLH, respectively, resulting in 2.8 and 1.9 volume doublings and doubling times of 1.4 and 2.7 days, respectively. Number of granulosa cells per follicle increased linearly (p < 0.01) from Day 15 to PLH, resulting in 1.5 cell doublings and a doubling time of 3.3 days. Number of S-phase cells per follicle per hour was similar from Days 15 to 18 and then decreased (p > 0.01) from Day 18 to PLH. In summary, the percentages of proliferating granulosa and thecal cells decreased throughout the final stages of antral follicular development. Differentiation of granulosa cells occurred from the basal to the antral area as follicles matured. We proposed that, during the latter stages of follicular development, the rapid increase in follicular diameter resulted primarily from expansion of the antral cavity, whereas increases in the granulosa cell layer volume and number of granulosa cells per follicle maintained a constant granulosa cell layer thickness.     

Clonal analysis of macronodules in cirrhosis

Several arguments suggest that most hepatocellular carcinomas (HCCs) occurring in human cirrhotic livers arise from large hepatocellular nodules or macronodules. Except for nodules with obvious features of HCC, there exist no consistent criteria enabling the differentiation between benign regenerative and neoplastic, potentially malignant macronodules. Surrogate markers able to accurately discriminate those lesions that will evolve toward a HCC are required. In this study, we investigated the clonality of 26 macronodules isolated from eight cases of explanted cirrhotic livers in women by analyzing X-chromosome inactivation, as indicated by the methylation status of the human androgen receptor gene (HUMARA). For each macronodule, a large set of pathological features was evaluated and used to classify the macronodules into four groups: entirely benign-looking nodule (type 1), low-grade dysplastic nodule (type 2), high-grade dysplastic nodule (type 3), and HCC (type 4). Clonal analysis showed that 14 macronodules (54%) were monoclonal and 12 (46%) were polyclonal. Monoclonality was detected in 5 of 11 (45%) nodules from groups of entirely benign-looking and low-grade dysplastic nodules (types 1 and 2) and in 9 of 15 (60%) nodules from the group of high-grade dysplastic nodule and HCC (types 3 and 4). Neither the etiology of cirrhosis nor the size or histological classification of macronodules was correlated with the clonal status. In conclusion, clonal analysis of macronodules enables the differentiation between mono- and polyclonal macronodules in cirrhosis. Because monoclonal macronodules are prone to evolve to HCC, the determination of the clonal status of a macronodule could provide additional information for evaluating the prognosis of these lesions.     

Dysplasia in Barrett's esophagus: diagnosis, surveillance and treatment

Barrett's esophagus is a premalignant metaplastic change in the lining of the distal esophagus. It represents a peculiar form of healing which occurs in response to chronic gastroesophageal reflux disease. The condition should be considered in all patients undergoing endoscopy for symptoms of reflux disease and is confirmed when any biopsy shows the presence of specialized intestinal metaplasia irrespective of the macroscopic appearance of the distal esophagus. Endoscopic surveillance with multiple biopsy sampling of the esophageal mucosa is indicated for all medically fit patients with Barrett's esophagus. The diagnosis of dysplastic change within this abnormal mucosa requires histological examination of the biopsies by 2 independent but experienced pathologists. Identification of high-grade dysplasia heralds the development of invasive cancer and offers the physician an opportunity to intervene. Despite extensive endoscopic sampling of the esophageal mucosa the differentiation between high-grade dysplasia and invasive adenocarcinoma is unreliable. Esophagectomy remains the treatment of choice for patients with high-grade dysplasia since adenocarcinoma of the esophagus carries such a poor prognosis.     

The prevalence of bcl-2, p53, and Ki-67 immunoreactivity in transitional cell bladder carcinomas and their clinicopathologic correlates

The aim of this study was to investigate the expression of bcl-2, p53 oncoproteins, and Ki-67 antigen in a series of transitional cell bladder carcinomas and its relation to the traditional prognostic indicators and patient's survival. One hundred six cases with transitional cell carcinoma (TCC) were examined for detection of bcl-2, p53 proteins, and Ki-67 antigen (MIB1 antibody). Bcl-2 immunohistochemical positivity was observed in 52% of TCCs and in 57% of low-grade and 44% of high-grade TCCs. Bcl-2 was also detected in normal urothelium and dysplastic lesions with basal cell expression, and negative staining was observed in carcinomas in situ. Tumor stage showed a significant inverse correlation with overall bcl-2 positivity. The loss of bcl-2 protein expression in higher-stage TCCs was statistically significant (Pt = .01). p53 protein was overexpressed in 50% of TCCs and more frequently in invasive and in carcinomas in situ than in superficial TCCs (Pt = .03). In contrast, detection of p53 was not observed in normal and dysplastic urothelium. p53 positivity was related to the degree of differentiation and to the stage of the disease (Pf = .01 and Pt = .03, respectively). Concerning Ki-67 antigen, its expression was found in 57.5% of TCCs. There was a strong overall correlation of Ki-67 with tumor stage (Pt = .002) and grade (Pf = .002). Univariate statistical analysis showed that the expression of p53 and Ki-67 was significantly correlated to poor prognosis (P = .02, P = .02, respectively). On multivariate analysis, none of these markers but only stage and grade were significantly correlated to prognosis (P = .02, P = .02, respectively). These findings suggest that overexpression of bcl-2 protein may be an early event in tumorigenesis. Tumors with loss of bcl-2 positivity and overexpression of p53 and Ki-67 had an unfavorable prognosis; however, in multivariate analysis, they had no independent prognostic value.     

The role of p53, MDM2 and c-erb B-2 oncoproteins, epidermal growth factor receptor and proliferation markers in the prognosis of urinary bladder cancer

The immunohistological expression of p53 and MDM2 oncoproteins was examined in paraffin embedded tissue from 106 patients with transitional cell carcinoma of the urinary bladder and was related to various clinicopathological features, the expression of proliferation associated markers (proliferating cell nuclear antigen - PCNA - and Ki-67), c-erb B-2 oncoprotein and epidermal growth factor receptor (EGFR), as well as to survival. MDM2 immunoreactivity was seen in 38% of our cases, and in 14% was accompanied by p53 positive immunohistochemistry. The rate of p53 positivity was associated with grade, stage and papillary status, whereas MDM2 immunopositivity increased with grade and stage (Ta VS T1), and MDM2 labeling index (LI) with stage. MDM2 expression was related to p53 expression and less strongly to proliferation rate (Ki-67 LI). The simultaneous p53 and MDM2 expression was more frequently observed in higher grade and stage tumours. C-erb B-2, EGFR and proliferation marker expression increased with grade, stage and non-papillary configuration. In univariate analysis high grade, solid growth pattern, advanced T-category, cystectomy, EGFR and Ki-67 expression were linked to shorter overall survival but only Ki-67 LI, along with T-category and type of therapy, had independent prognostic value. C-erb B-2 expression and stage were the two independent predictors of disease-free survival and Ki-67 LI and EGFR LI the independent predictors of post-relapse survival. For patients with superficial tumors PCNA LI emerged as the single independent determinator of survival. p53 and MDM2 expression did not appear to have any significant impact on survival, although the simultaneous expression of p53 and MDM2 turned out to be a highly significant parameter of shortened overall survival in univariate analysis.