Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer.

Lung carcinogenesis is assumed to be a multistep process, but detailed understanding of the sequential morphological and molecular changes preceding invasive lung cancer remains elusive. To better understand early lung carcinogenesis, we initiated a program of fluorescence bronchoscopy in smokers at high risk for lung cancer. In the bronchial biopsies from these subjects, we observed a unique lesion consisting of capillary blood vessels closely juxtaposed to and projecting into metaplastic or dysplastic squamous bronchial epithelium, angiogenic squamous dysplasia (ASD). Serial sections of the capillary projections confirmed that they represent intramucosal capillary loops. Microvessel density in ASD was elevated in comparison to normal mucosa (P = 0.0003) but not in comparison to other forms of hyperplasia or dysplasia. ASD thus represents a qualitatively distinct form of angiogenesis in which there is architectural rearrangement of the capillary microvasculature. Genetic analysis of surface epithelium in a random subset of lesions revealed loss of heterozygosity at chromosome 3p in 53% of ASD lesions. No confirmed p53 mutations were identified. Compared with normal epithelium, proliferative activity was markedly elevated in ASD lesions. ASD occurred in 54 of 158 (34%) high-risk smokers without carcinoma and in 6 of 10 patients with squamous carcinoma who underwent fluorescence bronchoscopy. One early-stage invasive carcinoma was noteworthy for the occurrence of ASD juxtaposed to invasive tumor. Seventy-seven (59%) of the ASD lesions were detected by abnormal fluorescence alone. Twenty bronchial sites (11 patients) were rebiopsied 1 year after the initial diagnosis. At nine (45%) of these sites, the lesion was found to persist. The lesion was not present in biopsies from 16 normal nonsmoker control subjects. The presence of this lesion in high-risk smokers suggests that aberrant patterns of microvascularization may occur at an early stage of bronchial carcinogenesis.     

The association between epithelial proliferation and disease progression in the oral mucosa

The purpose of this study was to examine the possible association between epithelial proliferation and disease progression in the oral mucosa. Archival specimens of normal oral mucosa (n = 12), dysplasia (n = 17) and squamous cell carcinoma (n = 18) were sectioned and proliferating cells visualised by staining with Ki-67 antibody. The proliferative index of the epithelium (PI) was determined by total cell counts and point counting. Similar results were obtained using either method. Comparison of the three groups of tissues by one-way analysis of variance showed a significant increase in PI with increasing lesion severity (p < 0.001). The PI of both dysplasia and carcinoma groups was significantly higher than that of normal oral mucosa (p < 0.001). However, the difference between dysplasia and carcinoma groups was not significant. PI was not associated with tobacco or alcohol consumption. We therefore conclude that Ki-67 expression is an early marker of disease progression in the oral mucosa but, on its own, is not a good indicator of neoplastic transformation.     

Neoangiogenesis: A putative marker of malignancy in non-small-cell-lung-cancer (NSCLC) development

Several studies have documented a relevant prognostic role of microvessel count (MC) in non-small-cell lung carcinomas (NSCLC). However, no evidence has been reported about the involvement of neo-angiogenesis in the development of bronchial cancers. The aim of this study was to analyze microvessel density both in normal and in pathological features of the bronchial tree detected concomitantly with carcinomas. In a group of 34 patients resected for NSCLC, 48 bronchial lesions (hyperplasia, squamous metaplasia, moderate dysplasia and in situ carcinoma) were identified. In addition, 20 samples of normal bronchial epithelium from the same patients were analyzed. A monoclonal antibody was used in order to identify microvessels in the most intense areas of neovascularization from the bronchial specimens. MC was also analyzed in invasive components. An increased number of microvessels was observed from normal to dysplastic epithelium, including in situ carcinoma. Mean MC was significantly lower in normal, hyperplastic and squamous metaplastic epithelium than in dysplastic epithelium and in situ carcinoma. In particular, no differences were observed between normal and hyperplastic/metaplastic components, whereas a statistically significant difference appeared between the latter and dysplastic lesions. Moderate dysplasia and in situ carcinoma showed a number of microvessels in the lamina propria of their mucosa which were not significantly different from the invasive component, whereas hyperplastic/metaplastic lesions presented a much lower number of microvessels than invasive cancer. From these data it appears that normal bronchial epithelium and lesions associated with cancers of the bronchial tree show neovascularization in their stromal component. Hyperplasia and squamous metaplasia, unlike dysplasia and in situ carcinoma, show a low microvessel count, and they cannot represent precursor or incipient changes in the bronchial epithelium before the fully developed in situ stage has also been reached. © 1996 Wiley-Liss, Inc.     

Biological features of bronchial squamous dysplasia followed up by autofluorescence bronchoscopy

Some dysplasias in the bronchial epithelium are thought to be precancerous lesions that can develop into squamous cell carcinomas. In this investigation, we assessed the biological behavior of bronchial squamous dysplasia in order to define which dysplasias have the potential to progress to squamous cell carcinoma. Using autofluorescence bronchoscopy, we followed up periodically localized dysplasias and examined for correlation between histological outcome and smoking status during the follow-up period, telomerase activity, Ki-67 labeling index, and p53 immunoreactivity of initial biopsy specimens. Ninety-nine dysplasias from 50 participants mainly with sputum cytology suspicious or positive for malignancy were followed up. Of 99 dysplasias, 3 dysplasias progressed to squamous cell carcinoma, 41 dysplasias remained as dysplasia, 6 dysplasias changed to metaplasia, 14 dysplasias changed to hyperplasia, and 35 dysplasias regressed to bronchitis or normal bronchial epithelium. There were no significant associations between histological outcome and smoking status. Mean initial telomerase activity and Ki-67 labeling index values in the dysplasias increased in proportion to the severity of the histological outcome at the second biopsy. There was also a significant difference between p53-positive and p53-negative dysplasia in terms of histological outcome at the second biopsy. Our results suggested that dysplasias with high telomerase activity, increased Ki-67 labeling index, and p53-positivity tended to remain as dysplasia and might have the potential to progress to squamous cell carcinoma. Patients with dysplastic lesions with these characteristics should be carefully followed up.     

Modulation of neoangiogenesis in bronchial preneoplastic lesions.

We have previously demonstrated that vascular count significantly increases in the preneoplastic lesions of the bronchial tree, starting from very low levels in the normal epithelium to a significantly higher number of microvessels in moderate dysplastic lesions and in situ carcinomas. Vascular endothelial growth factor (VEGF) protein expression has shown to be strictly associated with neovascularization both in human cancer and in various type of preinvasive lesions. A number of studies have demonstrated that mutant p53 is involved in the regulation of angiogenesis, and immunohistochemical detection of the p53 protein is associated with p53 gene mutations. In this study we looked for possible correlation between p53 protein detection, VEGF expression and vascular count in a series of preneoplastic and neoplastic lesions of the bronchial tree in order to investigate the angiogenic pattern and its genetic control in the early steps of bronchial cancer development. Twenty-four retrospective bronchial lesions with different grades of dysplasia and a case of normal bronchial epithelium were analysed. Surgical specimens removed from patients either confirmed, or suspect for lung carcinoma were stained immunohistochemically for CD34, VEGF, and p53. There were significant increases in microvascular density (MVD), VEGF, and p53 expression from normal bronchial epithelium through moderate dysplasia to in situ carcinoma to invasive cancer and these factors were significantly associated with moderate dysplastic lesions. A statistically significant difference was observed in MVD between hyperplastic-metaplastic, moderate dysplastic lesions and in situ carcinoma. A similar pattern was also observed for VEGF and p53 protein expression but no significant difference was observed between moderate dysplastic lesions and in situ carcinoma with regard to VEGF protein expression. The association between MVD, VEGF expression, p53 mutations and preinvasive lesions of the bronchial tree suggests that neoangiogenesis is early in non-small cell lung cancer (NSCLC) development and that p53 may have an important role in promoting angiogenesis in this human model of carcinogenesis.     

Molecular follow-up of preneoplastic lesions in bronchial epithelium of former Chernobyl clean-up workers

Ionizing radiation is a potent lung carcinogen, but the precise molecular damage associated with it is still unknown. In this study we investigated cancer-related molecular abnormalities including K-ras (codon 12) mutation, p16(INK4A) promoter hypermethylation and microsatellite alterations at seven chromosomal regions in successive biopsies obtained from former Chernobyl cleanup workers in comparison with smokers and nonsmokers who have never had radiation exposure. Our results indicate that prolonged persistence of inhaled radioactive particles is associated with appearance of allelic loss at 3p12, 3p14.2 (FHIT), 3p21, 3p22-24 (hMLH1) and 9p21 (p16INK4A) in bronchial epithelium of former Chernobyl clean-up workers. The prevalence of 3p14.2 allelic loss was associated with decreased expression of the FHIT mRNA in their bronchial epithelium in comparison with control group of smokers. During several years of our monitoring samples of epithelium were collected from the same area of bronchial tree. In epithelium exposed to carcinogens (tobacco smoke and/or radioactivity) the total number of molecular abnormalities was significantly higher in dysplasia and in morphologically normal foci progressed later to dysplasia than in these samples which never showed evidence of such progression. Our findings indicate that extensive cancer-related molecular abnormalities sequentially occur in radiation damaged bronchial epithelium of former Chernobyl clean-up workers.     

Apoptosis and cell proliferation in medullary carcinoma of the breast: a comparative study between medullary and non-medullary carcinoma using the TUNEL method and immunohistochemistry

BACKGROUND AND OBJECTIVES: Medullary carcinoma of the breast has generally been considered to result in better prognosis than ordinary invasive ductal carcinoma, which would seem to be discrepant when one considers its anaplastic histology and high mitotic rate. We attempted to elucidate the prognostic implications of apoptosis and cell proliferation in medullary carcinoma of the breast. METHODS: Formalin-fixed, paraffin-embedded specimens of 50 cases of typical medullary carcinoma (MC) of the breast and those of 50 control cases of non-medullary invasive ductal carcinoma (N-MC), which were matched to the MC cases in both age and TNM classification, were investigated utilizing the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemistry for p53, bcl-2, and Ki-67. RESULTS: Mean values of the apoptotic index (AI), the proliferative index (PI), and the ratio of AI to PI (AI/PI) were significantly higher in MC than in N-MC (P < 0.0001). MC exhibited significantly lower positivity for bcl-2 than N-MC (P = 0.00003), while there was no significant difference in p53 positivity between MC and N-MC. CONCLUSIONS: A high frequency of apoptosis may be related to a favorable prognosis in MC, even though it demonstrates a high proliferative activity, exhibiting a rapid cell turnover.     

Expression of Ki-67 and p53 in cutaneous free flaps used to reconstruct soft tissue defects following resection of oral squamous cell carcinoma

Radial forearm free flaps are used routinely to reconstruct oro-facial tissues following resection of oral squamous cell carcinoma. Surprisingly, there is little information regarding their behaviour following engraftment. The present report is a clinico-pathological study of 10 patients who had incisional biopsies of cutaneous free flaps after the presence of a white patch or erythema raised clinical suspicion. Tissues were stained with haematoxylin and eosin, diastase-periodic acid-Schiff reagent and labelled immunohistochemically for Ki-67 and p53. Four of 10 specimens showed severe epithelial dysplasia within the graft, which was contiguous with dysplasia in the adjacent oral mucosa; the remaining grafts had features typical of candidosis (n=4) or hyperkeratosis (n=2). Grafts with dysplasia had a significantly higher Ki-67 labelling index than lesions in the 'non-dysplastic' group. There were no significant differences in the Ki-67 labelling index between areas of dysplasia in the graft and areas of dysplasia in the adjacent oral epithelium. p53 staining was present in all strata of the epithelium in the dysplastic grafts and adjacent dysplastic mucosa, but was absent or weakly expressed in the stratum basale of grafts showing reactive changes only. None of the dysplastic lesions progressed to carcinoma despite a mean follow-up period of 32 months; one patient developed a recurrent mucosal tumour at the resection margin. These observations indicate that cutaneous free flaps grafted to a site of field cancerisation can develop severe epithelial dysplasia with concomitant deregulation of proliferation and increased p53 expression. Such changes raise the possibility that these lesions have the potential for malignant transformation.     

Multiple high-grade bronchial dysplasia and squamous cell carcinoma: concordant and discordant mutations.

Loss of heterozygosity (LOH) involving chromosomes 3p, 5q, 9p, or 17p and aberrant expression or mutation of p53 are reported previously in selected bronchial dysplasias and squamous cell cancers (SCCs). Yet, comprehensive analyses of LOH patterns at these chromosomal sites and of p53 alterations are not reported for histologically normal bronchial epithelium, high-grade bronchial dysplasia, and SCC present in the same pulmonary resections. Whether concordant or discordant genetic changes are detected in these bronchial tissues, especially when multiple high-grade dysplastic bronchial lesions are present, was studied. Genomic DNA was microdissected from eight pulmonary SCCs and high-grade dysplastic lesions that were associated with SCC. In four cases, two independent high-grade dysplastic bronchial lesions were identified. When available, histologically normal bronchial epithelium was microdissected. Germ-line genomic DNA was isolated from normal lymph nodes. LOH was assessed for 15 microsatellite markers on chromosomes 3p, 5q, 9p, or 17p, sites frequently deleted in lung cancers. Immunohistochemical p53 expression was studied and correlated with p53 DNA sequence analyses. Progressive LOH for these markers was found when SCCs were compared with high-grade dysplasia and histologically normal bronchial epithelium present in the same resections. Histologically normal bronchial specimens had LOH in up to 27% of informative markers. High-grade dysplastic lesions exhibited LOH for 18-45% and SCC had LOH for 18-73% of the markers. Common regions of LOH were found in some dysplasias compared with SCCs. In other dysplasias, discordance was found relative to SCCs, especially for p53 mutations. In cases with a single or second high-grade dysplasia associated with SCC, heterogeneity in LOH markers was detected. These concordant and discordant changes were consistent with convergent and divergent clonal selection pathways in pulmonary squamous cell carcinogenesis. Some histologically normal bronchial epithelial tissues had genetic changes more similar to those in the SCCs than in dysplastic lesions. DNA loss or mutations accumulate in SCC, but discordant genetic changes can exist in the same carcinogen-exposed bronchial tissues. These findings have implications for lung cancer prevention trials.     

p53 Overexpression in Oral Mucosa in Relation to Shisha Smoking in Syria and Lebanon

Introduction: Shisha (waterpipe) smoking is becoming a very prevalent form of tobacco consumption in the Middleeast and use is growing over the world. Smoking-related malignancies have a high genome-wide burden of mutations,including examples in the gene encoding p53. Aims: To investigate alterations in p53 immunohistochemical expressionin the normal, pre-malignant, malignant oral mucosa in relation to Shisha smoking habits. Materials and Methods:A total of 105 paraffin embedded tissue sections of OSCCs (52 smokers,53 non-smokers), 96 of premalignant lesions(48 smokers,48 non-smokers) and 60 normal oral mucosa. Some 30 patients with a history of Shisha smoking dailyfor more than 5 years were also investigated for mutant expression of p53. Tissue samples were considered positivefor p53 staining when any positive cells of epithelial origin could be detected. Results: The majority (74.3%) of oralsquamous cell carcinomas showed positive staining for p53 expression (83.1% and 65.5% with Shisha smokers andnon-smokers, respectively). In the 96 premalignant lesions, about 23% from non-smokers and 41.7% from smokersshowed p53 positivity. In normal epithelium, P53 positive cells were noted in 6.6% of non-smokers and 16.6% ofsmokers. Positive correlations with Shisha smoking were evident for the following groups: WDOSCC, MDOSCC,mild dysplasia G1, moderate dysplasia G2 and in normal mucosa using Student’s t- test, P valueThese results strongly suggest that p53 mutations are associated with Shisha smoking in OSCC, pre-malignant lesionsand normal mucosa of the oral cavity.     

Bronchial micronuclei as a marker of an early stage of carcinogenesis in the human tracheobronchial epithelium

In the bronchial epithelium, smoking initiates a multistep process that first appears histologically as premalignant squamous metaplasia/dysplasia, a biological predecessor of squamous-cell lung cancer. Reflecting chromosomal damage from a carcinogenic insult, micronuclei may reveal earlier events in the carcinogenic sequence. We prospectively evaluated and correlated micronucleus count, histology (index of metaplasia) and smoking exposure in 35 consecutive subjects (9 active smokers, 10 previous smokers and 16 never-smokers) undergoing diagnostic bronchoscopy. Samples for micronuclei and histological evaluation were taken from the main carinal mucosa in each subject for site-specific comparisons. The median and mean micronucleus counts per 1,000 cells were significantly higher in active smokers than in non-smokers (subjects who had never smoked and previous smokers): median counts were 3.7 vs. 1.4, p = 0.03; mean counts were 4.7 vs. 1.9, p = 0.01. There was no significant difference, however, in micronucleus counts between subjects who had never smoked and previous smokers. Bronchial metaplasia and smoking history were not associated. Our findings suggest that micronuclei are a readily quantitated, early intermediate-endpoint marker for detecting tobacco-initiated tracheobronchial carcinogenesis.     

Differential expressions of cyclin-dependent kinase inhibitors (p27 and p21) and their relation to p53 and Ki-67 in oral squamous tumorigenesis

The cyclin-dependent kinase inhibitors p27Kip1 and p21WAF1/Cip1 play important roles in cell-cycle regulation. Although alterations of these genes have been linked to tumorigenesis of several human carcinomas, their involvement in head and neck squamous tumorigenesis is rarely investigated. To determine the role of these genes in the evolution of squamous carcinoma of the head and neck we evaluated their protein expression by immunohistochemistry in non-dysplastic squamous epithelium, premalignant lesions and oral squamous carcinomas. The p53 gene and Ki-67 expressions were correlated with traditional clinicopathologic variables. Our study shows that in histologically non-dysplastic squamous epithelium, p27 expression was noted mainly in superficial differentiated cells, whereas p21, p53 and Ki-67 staining was observed in basal and suprabasal cells. In dysplasia, divergent expression between p27 and p21 was observed: p27 precipitously decreased and p21, p53, and Ki-67 increased with histologic progression. In squamous carcinomas, p27 was mainly expressed in well differentiated tumor cell nests, while the expressions of p21, p53, and Ki-67 were variable in the poorly differentiated tumor areas. A significant inverse relationship between p27 expression and those of p21, p53, and Ki-67 was observed, but no significant association between any of these markers and clinicopathologic factors was noted in this cohort. Our study indicates that: i) down-regulation of p27 and up-regulation of p21 are associated with early progression of HNSC, ii) p21 expression correlates positively with proliferation while p27 correlates positively with cell differentiation and iii) concurrent p27 and p21 expression analysis may allow for better assessment of HNSC progression.     

Overexpression of cyclins D1 and E is frequent in bronchial preneoplasia and precedes squamous cell carcinoma development.

Increased protein expression of the G1 cyclins D1 and E is reported in invasive non-small cell lung carcinoma. However, during transformation of the bronchial epithelium, overexpression of these species occurs, and their relationship to aberrant expression of p53 and retinoblastoma (Rb) has not been described previously. To determine the expression of these cell cycle regulators during the development of invasive squamous cell carcinoma (SCC) of the lung, the immunohistochemical expression patterns in normal bronchial epithelium (n = 36), squamous metaplasia (SM; n = 28), and epithelial atypia (n = 34) were compared with that in low-grade dysplasia (LGD; n = 17), high-grade bronchial dysplasia (HGD; n = 30), and SCC (n = 36). Monoclonal anti-p53 Pab1801, polyclonal anti-cyclin D1 DCS6, monoclonal anti-cyclin E HE12, and monoclonal anti-Rb OP-66 antibodies were used. Cyclin D1 was not expressed in normal bronchial epithelium but was detected in 7% of SMs, 15% of atypias; 18% of LGDs, 47% of HGDs, and 42% of SCCs. Cyclin E was not detected in normal epithelium (n = 24), SM (n = 16), or LGD (n = 12), but it was found in 9% of atypias (2 of 22), 33% of HGDs (7 of 21), and 54% of SCCs (13 of 24). p53 was not expressed in normal epithelium, SM, and LGD, but it was overexpressed in 6% of atypias, 53% of HGDs, and 61% of SCCs. Abnormal Rb expression was found only in 2 of 36 cases of SCC. A total of 91% of HGDs and 92% of SCCs exhibited overexpression of at least one of the p53, cyclin D1, or cyclin E species. However, no link was observed between overexpression of p53 and the overexpressed G1 cyclins in preneoplastic lesions. Overexpression of cyclin D1, cyclin E, and p53 occurs frequently and independently in pulmonary SCC and is detected in lesions before the development of invasive carcinoma. In contrast, altered Rb expression is a late and infrequent event in squamous cell carcinogenesis.     

食管黏膜上皮癌变过程中p27、cyclinD1和bcl-2蛋白的表达及其意义

目的　探讨p2 7、cyclinD1和bcl 2基因在食管鳞癌发生发展中的意义和相互间的作用。方法　采用免疫组化染色检测p2 7、cy clinD1和bcl 2蛋白在正常食管黏膜、非典型增生及浸润癌各 64例中的表达。结果　p2 7在正常食管黏膜表达率最高 (90 6% ) ,随食管黏膜上皮病变的发展表达率呈下降趋势 ,而cyclinD1和bcl 2在食管鳞癌的发生发展中表达呈上升趋势 ,p2 7、cyclinD1和bcl 2的表达率在非典型增生组、浸润癌组与正常组比较均有显著性差异 (P <0 0 1) ;相关分析显示食管鳞癌组织中p2 7与cyclinD1呈负向表达 ;p2 7、cyclinD1和bcl 2的表达与食管癌的淋巴结转移密切相关 (P <0 0 5 )。结论　食管癌的发生发展是多因素作用的结果 ,p2 7、cyclinD1和bcl 2的表达与食管癌的临床病理指标有关 ,可作为食管鳞癌早期诊断和判断预后的重要分子指标。     

Increased telomerase activity and elevated hTERT mRNA expression during multistage carcinogenesis of squamous cell carcinoma of the lung

BACKGROUND: Telomerase activation is believed to play a critical role in the immortalization of cells and carcinogenesis. Telomerase activity is undetectable in normal somatic cells (except for those cells undergoing proliferation) but is expressed in the majority of human tumors including lung carcinoma. The expression of hTERT mRNA has been found to be correlated with telomerase activity. In the current study, the authors analyzed telomerase activity and hTERT mRNA expression in preinvasive bronchial lesions using biopsy specimens obtained by fluorescence bronchoscopy. METHODS: The authors studied 150 bronchial biopsy specimens obtained by fluorescence bronchoscopy. The intensity of telomerase activity was determined by the fluorescence-based telomeric repeat amplification protocol method in 74 bronchial biopsy specimens (22 normal bronchial epithelium or bronchitis cases, 15 squamous metaplasia cases, 23 dysplasia cases, and 14 squamous cell carcinoma cases), and the level of hTERT mRNA was analyzed in another 76 specimens (24 normal bronchial epithelium or bronchitis cases, 15 squamous metaplasia cases, 20 dysplasia cases, and 17 squamous cell carcinoma cases) by real-time polymerase chain reaction. RESULTS: The mean values (+/- the standard deviation [SD]) of telomerase activity in normal bronchial epithelium or bronchitis, squamous metaplasia, dysplasia, and squamous cell carcinoma cases were 6.2 +/- 7.5, 13.9 +/- 14.8, 18.5 +/- 20.8, and 54.5 +/- 22.3 U/microg protein, respectively. The upper limit of telomerase activity in normal bronchial epithelium or bronchitis was 21 U/microg protein (mean + 2SD). It is interesting to note that, 5 of 15 squamous metaplasia biopsies (33%), 8 of 23 dysplasia biopsies (35%), and all squamous cell carcinoma biopsies (100%) exhibited levels of telomerase activity that were > 21 U/microg protein. The mean levels of hTERT mRNA in normal bronchial epithelium or bronchitis, squamous metaplasia, dysplasia, and squamous cell carcinoma cases were 891 +/- 840, 1936 +/- 1704, 3019 +/- 2607, and 12965 +/- 18008 copies/microg total RNA, respectively. Telomerase activity and hTERT mRNA expression were found to increase in proportion to the severity of histologic change from normal bronchial epithelium or bronchitis to squamous cell carcinoma. CONCLUSIONS: These results suggest that an increase in telomerase activity and hTERT mRNA expression are features of the early stages of the development of squamous cell carcinoma of the lung, with strong telomerase activity and hTERT mRNA expression being prominent during the latter stages.     

Long-term impact of smoking on lung epithelial proliferation in current and former smokers.

BACKGROUND: Lung cancer risk remains elevated for many years after quitting smoking. To assess using proliferation indices in bronchial tissues as an intermediate endpoint biomarker in lung cancer chemoprevention trials, we determined the relationship between the extent, intensity, and cessation of tobacco smoking and proliferative changes in bronchial epithelial biopsy specimens. METHODS: Bronchial biopsy specimens were obtained from up to six epithelial sites in 120 current smokers (median pack-years, 42) and 207 former smokers (median pack-years, 40; median quit-years, 8.1). Sections from the paraffin-embedded specimens were stained with hematoxylin--eosin to determine the metaplasia index and with an antibody to Ki-67 to determine the proliferative (labeling) index for the basal and parabasal (Ki-67 PLI) layers. All statistical tests were two-sided. RESULTS: Biopsy sites with metaplasia had statistically significantly higher Ki-67-labeling indices than those without metaplasia (P<.001) in both current and former smokers. Increased proliferation was observed in multiple biopsy sites, with the average Ki-67 PLI of the subject strongly correlating with the metaplasia index (r =.72 for current smokers; P<.001), even in sites without metaplasia (r =.23 for current smokers; P<.001). In current smokers, the Ki-67 PLI was associated with the number of packs smoked/day (P =.02) but not with smoking years or pack-years. In subjects who had quit smoking, the Ki-67 PLI dropped statistically significantly within 1 year (P =.008) but remained detectable for more than 20 years, even in the absence of squamous metaplasia. CONCLUSION: Smoking appears to elicit a dose-related proliferative response in the bronchial epithelia of active smokers. Although the proliferative response decreased gradually in former smokers, a subset of individuals had detectable proliferation for many years and may benefit from targeted chemoprevention. Bronchial epithelial proliferation, measured by Ki-67, may provide a useful biomarker in the assessment of lung cancer risk and in the response to chemopreventive interventions.     

N-(4-hydroxyphenyl)retinamide in the chemoprevention of squamous metaplasia and dysplasia of the bronchial epithelium.

Lung cancer remains the number one cause of cancer-related deaths in the United States. To reduce the mortality associated with this disease, individuals at risk must be identified prior to the development of lung cancer, and effective prevention strategies must be developed. One such strategy is to use retinoids like N-(4-hydroxyphenyl)retinamide (4-HPR), which has been found to possess chemopreventive activities in preclinical studies. In this study, 139 smokers were registered and 82 were randomized onto a double-blinded, placebo-controlled chemoprevention trial of 4-HPR administered p.o. (200 mg once daily). Of these, 70 participants were eligible for response evaluation. Biopsies were obtained at six predetermined sites in the bronchial tree from participants before and at the completion of 6 months of treatment. 4-HPR treatment had no measurable effect on histopathology (squamous metaplasia and dysplasia) in the bronchial epithelium of current smokers. 4-HPR was detected (104.5+/-64.0 ng/ml, mean +/- SD) in the serum of participants, supporting its potential bioavailability. Serum retinol levels decreased markedly (44% of placebo-treated patients) as a consequence of 4-HPR treatment. Notably, the mRNA level of retinoic acid receptor beta, which is typically increased by retinoid treatment, did not change in the bronchial epithelium of 4-HPR-treated participants. Clonal populations of bronchial epithelial cells were detected by analysis of loss of heterozygosity at putative tumor suppressor loci on chromosomes 3p, 9p, and 17p, and these changes were not altered by 4-HPR treatment. In conclusion, at this dose and schedule, 4-HPR was not effective in reversing squamous metaplasia, dysplasia, or genetic and phenotypic abnormalities in the bronchial epithelium of smokers.     

3-Methylcholanthrene triggers the differentiation of alveolar tumor cells from canine bronchial basal cells and an altered p53 gene promotes their clonal expansion

Alveolar type II cells arising in canine bronchial autografts following exposure to 3-methylcholanthrene (MCA) give rise to carcinomas of varying glandular and squamous growth patterns. To study the role of the tumor suppressor gene p53 in this process, sections from progressive lesions were immunostained for p53 protein; microdissected regions were screened for p53 mutations. Adjacent sections were examined for type II cell expression [cuboid cell shape, large roundish nucleus, cytoplasmic staining for surfactant protein A (SP-A)] and proliferating cell nuclear antigen expression. Evidence for an altered p53 function (nuclear staining, missense mutations) was found in most carcinomas of all histologic types and in all grades of bronchial dysplasia, but not in hyperplastic or normal bronchial epithelium. It was primarily associated with the hyperplastic type II cell populations present in the basal zone of the lesions. In addition, we found SP-A staining in hyperplastic (but not in normal) bronchial basal cells. These data suggest that MCA initiates type II cell differentiation through phenotypic selection (basal cells). Inactivation of the p53 gene promotes the clonal expansion of the type II cells into discernible populations of (squamous or glandular) alveolar tumor cells. This in vivo study is the first to show that p53 is involved in a specific pathway leading to bronchogenic carcinoma.     

p53 immunoexpression: An aid to conventional methods in the screening of precursor lesions of squamous esophageal cancer in patients at high-risk?

INTRODUCTION: Squamous cell carcinoma of the esophagus (SCCE) is diagnosed late and carries a poor prognosis. Lugol chromoendoscopy (LC) has being shown a useful tool in the management of patients at high risk for SCCE. Biomarkers such as p53 protein expression may be present in the esophageal mucosa long before esophageal symptoms or lesions appear and may aid in early diagnosis. This study was carried out to investigate the p53 immunoexpression in esophageal mucosa of smokers and alcohol consumers and study its relationship with different degrees of histological findings and the role of LC to detect areas that express p53. METHODS: Group 1: One hundred and eighty-two asymptomatic subjects at high risk for SCCE (consumption of more than 80 g of ethanol and 10 cigarettes/day for at least 10 years). Group 2: Twenty healthy volunteers who neither smoked nor consumed alcohol. Both groups underwent upper GI endoscopy plus LC, with biopsies of the esophageal mucosa. Expression of p53 protein was compared to histological findings. RESULTS: Group 1: There was 25/182 (14%) Lugol's unstained areas. p53 protein was expressed in a stepwise fashion according to the severity of the histological findings: normal mucosa (12/103 or 12%), mild esophagitis (6/43 or 14%), moderate esophagitis (4/18 or 22%), severe esophagitis (1/3 or 33%), low-grade dysplasia (4/11 or 36%), high-grade dysplasia (2/2 or 100%) and squamous cell carcinoma (2/2 or 100%) (p=0.001). Nine in 25 (36%) patients with Lugol's unstained areas and 22/157 (14%) with normal appearing Lugol's stained mucosa expressed p53. Group 2: There was no Lugol unstained areas. The histological analysis and immunohistochemistry for p53 were normal with the exception of two patients that presented mild esophagitis and expressed p53. Unstained areas were 3.5 times (95% CI: 1.2-9.6) more likely to express p53 then stained ones. Alcoholics/smokers were 1.9 (95% CI: 0.4-8) times more likely to express p53 than non-alcoholics/non smokers. CONCLUSIONS: In this study, we find an association between histological alterations, p53 expression and Lugol's unstained areas. It may point to a higher risk for SCCE. Smokers and alcohol drinkers with normal mucosa or chronic esophagitis that express p53 protein may represent an unrecognized sub-group of individuals that may benefit from surveillance or intervention.