A follow-up study of progression from dysplasia to squamous cell carcinoma with immunohistochemical examination of p53 protein overexpression in the bronchi of ex-chromate workers.

Squamous cell carcinoma (SCC) of the bronchus is considered to develop from preneoplastic ''dysplasia'', but reports of sequential observation of this dysplasia-carcinoma sequence in humans are very few. We followed four dysplastic lesions found in the bronchi of three ex-chromate workers by bronchoscopy and biopsy and found that all of them progressed to SCC. Of the four lesions, three were severe dysplasias at the first biopsy which progressed to SCCs in 7-13 months. The last one was a slight dysplasia at the first biopsy and showed progression of the atypia to carcinoma in 6 years and 10 months. An immunohistochemical analysis of the chronological change in p53 protein expression in these lesions and in normal ciliated epithelium taken from the surroundings was conducted in each case. Overexpression of p53 protein was observed in two of the severe dysplasias and the one slight dysplasia, as well as their eventual SCCs. However, no such change was apparent in one case of severe dysplasia or its eventual SCC. Normal epithelium was consistently negative. Our results provide direct proof of the dysplasia-carcinoma sequence and suggest that alteration in the expression of p53 protein might be an important early event which persists. Therefore, the immunohistochemical detection of p53 overexpression in biopsy specimens of bronchial epithelium might be useful for evaluation of preneoplastic lesions in high-risk group individuals and for early diagnosis of bronchial cancer.     

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.     

p53 Gene mutation and genetic instability in superficial multifocal esophageal squamous cell carcinoma

Tumor multicentricity is occasionally observed in esophageal squamous cell carcinoma (SCC). We studied five surgically resected superficial multifocal esophageal SCCs for p53 gene mutation and genetic instability, using DNA extracted from microdissected areas. A total of 38 target areas (TAs) were analyzed in SCC, dysplasia, basal cell hyperplasia (BCH) and normal squamous epithelium. Analysis of the replication error (RER) at 10 microsatellite loci showed microsatellite instability in all TAs, as well as in normal squamous epithelium. p53 gene mutation was identified in 28.9% (11/38 TAs). All cases showed a common missense mutation in exon 8 at codon 273 (CGT-->CAT, Arg-->His), which was DNA contact mutation in the S10 beta strand. In association with microsatellite alterations, 7 of 9 TAs with p53 mutation in exon 8 at codon 273 also showed loss of heterozygosity (LOH) of p53 gene. LOH of p53 gene was detected in 83.8% (31/37 TAs). LOH at D2S123 on 2p16 near MSH2 gene and at D3S1611 on 3p22 near MLH1 gene was detected in 65.4% (17/26) and 71.4% (10/14) TAs, respectively. Frequencies of LOH at p53 and D2S123 were similar in non-cancerous areas and SCCs. LOH of p53 and D2S123 were found in 50% (5/10 TAs) of non-cancerous areas and 60% (9/15 TAs) of SCCs. Our results suggest that genetic instability induces esophageal tumor multicentricity, and that p53 gene contact mutation together with LOH are early events of the multistage carcinogenesis of multifocal primary esophageal SCC.     

p53 and chromosome 3 abnormalities, characteristic of malignant lung tumours, are detectable in preinvasive lesions of the bronchus.

Bronchial epithelial dysplasia is believed to precede invasive squamous cell carcinoma of the lung. Six paired dysplasia and tumour samples were distinguished histologically in sections of formalin-fixed paraffin-embedded lung tissue from patients with lung cancer. Additionally, samples of dysplastic bronchial epithelium were obtained from patients without lung tumours. Microdissection of the unstained sections provided dysplastic and tumour samples from which DNA was prepared for comparison with the patients' constitutional genotype, using polymerase chain reaction-based restriction fragment length polymorphism analysis. All six samples of tumour and the paired adjacent samples of bronchial dysplasia showed loss of heterozygosity (LOH) at loci on the short arm of chromosome 3. Five of the six cases showed involvement of the p53 gene as assessed by LOH at the AccII site within the gene, and by immunoreactivity to CM-1, an antibody which recognizes the mutated form of the p53 protein in paraffin-embedded material. Of the dysplastic samples, obtained from patients without invasive tumours, all three showed LOH at 3p; one sample showed LOH at the AccII polymorphic locus within the p53 gene, and another sample, uninformative at this locus, stained positively with this antibody. These results indicate that somatic genetic changes are present in preinvasive lesions in the bronchus.     

p53 expression in normal and dysplastic bronchial epithelium and in lung carcinomas.

Bronchial epithelial dysplasia is thought to be a premalignant stage in the evolution of lung cancers. Using the CM-1 polyclonal antibody, we have examined the expression of the p53 protein in a larger series of bronchial dysplasias (n = 60) than hitherto investigated. The p53 protein was detected in 14% of mild, 25% of moderate and 59% of severe dysplasias; increased p53 expression correlated with the severity of dysplasia. p53-positive dysplasias had greater PCNA indices than p53-negative dysplasias. p53 expression in dysplastic tissues was compared with that in two groups of histologically normal epithelium: 14 bronchial biopsies from non-cancer patients of which all but one were negative and 32 bronchial margins from resected carcinomas, of which 17 showed infrequent solitary cells with p53-positive nuclei in predominantly basal locations scattered throughout the epithelium. These results for resection margins were confirmed by use of a second antibody, DO-1. Sixty-nine per cent of the corresponding carcinomas were p53 positive, but in 15 cases the p53 reactivity differed from resection margins. No correlation between p53 expression and any of the clinicopathological characteristics of these tumours was found. This study supports the observation that abnormal p53 expression may be an early but not obligatory event in malignant transformation in lung.     

p53 mutation and allelic loss of chromosome 3p, 9p of preneoplastic lesions in patients with nonsmall cell lung carcinoma

BACKGROUND: An accumulation of mutations can result in carcinogenesis. Comparing genetic alterations in preneoplastic lesions with those seen in cancer in the same patient may be helpful in the early diagnosis of lung carcinoma or preneoplastic lesions. METHODS: To identify genetic alterations that may play a role in the development of nonsmall cell lung carcinoma (NSCLC), the authors examined the p53 gene and microsatellite markers on chromosome 3p (D3S643, D3S1317), 9p (D9S171, IFNA) in 35 bronchial metaplastic lesions and 28 alveolar hyperplastic lesions from 61 patients. RESULTS: A total of 8 metaplastic lesions (1 squamous metaplasia and 7 dysplasias) and 3 alveolar hyperplastic lesions (with atypia) showed genetic alterations, including loss of heterozygosity (LOH) of 3p, 9p and mutations of the p53 gene. In an analysis of microsatellite markers, 5 of 35 cases of squamous cell carcinoma (SCC) and 3 of 26 cases of adenocarcinoma (Ad) showed LOH in both preneoplastic lesions and synchronous cancers. Nine patients (25.7%) with SCC and 6 patients (23.1%) with Ad were shown to have mutations of the p53 gene by single-strand conformation polymorphism. In 2 of these 9 patients with SCC, the same mutation was observed in both dysplasia and SCC. CONCLUSIONS: These findings suggest that several genetic alterations may occur in preneoplastic lesions or the early stage of SCC of the lung, whereas the genetic alterations examined appeared to occur relatively late in the pathogenesis of pulmonary adenocarcinoma.     

Topological analysis of p21WAF1/CIP1 expression in esophageal squamous dysplasia.

In the normal stratified squamous epithelium of the esophagus, only the third to the fifth layers of cells express the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21). Using immunohistochemical staining, we examined the topological distribution of cells expressing p21, p53, Ki67, and cytokeratin 10 (CK10), a differentiation marker of esophageal squamous cell carcinoma (SCC), in 25 superficial SCCs and 72 dysplastic lesions of the esophagus. Image analysis of p21, p53, and Ki67 expression was also performed in 48 dysplastic lesions. In superficial SCCs, although Ki67- and p53-expressing cells were mainly distributed in the deep layers of tumors despite tumor differentiation, the distribution of p21 correlated with tumor differentiation. In dysplastic lesions, p53- and Ki67-coexpressing cells tended to locate in the same layers and expand in the lower layers of epithelium with the progression of dysplasia. p21-expressing cells shifted to the upper layers of the epithelium with the progression of dysplasia. However, this change was heterogeneous; in some lesions, p21-expressing cells were confined to the superficial layers of atypical cells (confined type), whereas in others, p21-overexpressing cells were scattered among atypical cells (scattered type). CK10 expression was observed in 25% of dysplastic lesions, and the frequency of CK10 expression was significantly higher in the scattered than in the confined type. Our results suggest that esophageal squamous dysplasia represents the earliest pathological process in esophageal squamous carcinogenesis. Our results also suggest that differentiation of esophageal SCC is determined at the stage of dysplasia, and that p21 plays a critical role in the differentiation process.     

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.     

Genetic progression and heterogeneity associated with the development of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma is a common fatal cancer, and Shanxi province, a region in north-central China, has some of the highest esophageal cancer rates in the world. Chromosomal regions with frequent allelic loss may point to major susceptibility genes that will assist us in understanding the molecular events involved in esophageal carcinogenesis and may serve as the basis for the development of markers for genetic susceptibility and screening for early detection of this cancer. This study was designed to identify events in the molecular progression of precursor and invasive lesions of squamous esophageal cancer. Twelve marker loci identified during our previous studies as having some of the highest rates of loss of heterozygosity (LOH) in invasive esophageal cancer were evaluated in laser-microdissected DNA obtained from low- and high-grade dysplastic lesions and invasive tumor foci from 10 fully embedded esophageal resection specimens. Each resection specimen contained a spectrum of disease, from epithelium that appeared histologically normal to invasive cancer, including a single dominant tumor surrounded by a region of precursor lesions (low- and high-grade dysplasia) and occasional "remote," nonadjacent precancerous foci. Using the 12 polymorphic markers, LOH was found in all of the three stages of disease. The frequency of LOH for all of the markers together increased with increasing disease severity. Among the informative low-grade dysplasia samples, LOH was detected with markers D3S1766 (3p), D4S2632 (4p), D9S910 (9q), and D13S1493 (13q), suggesting that LOH at these loci may be associated with early stages of tumor initiation and/or progression. LOH was detected among the informative high-grade (but not low-grade) dysplasia samples for the other eight markers tested, suggesting that LOH at these loci may occur later in the neoplastic process. In addition to the association between disease progression and these genetic changes, considerable genetic heterogeneity was found in each fully embedded resection specimen both between and within geographically separate neoplastic lesions.     

Expression of p53R2, newly p53 target in oral normal epithelium,epithelial dysplasia and squamous cell carcinoma

Recently, the p53R2 gene has been isolated and shown to play a crucial role in DNA repair after DNA damage. The p53R2 gene encodes the p53 inducible ribonucleotide reductase small subunit 2 homologue, which is part of the p53 pathway. However, the function of p53R2 in human cancer is still unclear. We investigated p53R2 mRNA expression in human oral normal epithelium, epithelial dysplasias and squamous cell carcinomas (SCCs). Surgical or biopsy-proven specimens of 10 normal epithelium, 48 epithelial dysplasias and 63 SCCs were collected in our department. Then, p53R2 was identified by in situ hybridization to visualize and localize the expression of specific mRNAs. The authors examined the p53 gene mutation by polymerase chain reaction-single strand conformation polymorphism analysis. p53, mdm2, p21^WAF1/CIP1 and Ki-67 expression was detected by immunohistochemistry. p53R2 expression was detected in none of ten normal epithelium (0%), ten of 48 dysplasias (20.8%) and 33 of 63 SCCs (52.4%). In oral SCC, the expression of p53R2 was significantly associated with tumor size, lymph node metastasis and histological differentiation (P=0.014, 0.046 and 0.022, respectively). p53R2 expression was significantly associated with p53 abnormality in epithelial dysplasia and SCC (P=0.034 and 0.009, respectively). Of 63 patients, 37 received preoperative radiochemotherapy. p53R2 mRNA expression was significantly associated with the pathologic response to radiochemotherapy (P=0.031). This study suggested that p53R2 expression could be associated with oral carcinogenesis. The presence of p53R2 mRNA expression would be a predictive factor for tumor development, tumor cell differentiation and the sensitivity to radiochemotherapy in oral SCC.     

The p53 status of cultured human premalignant oral keratinocytes.

Around 60% of oral squamous cell carcinomas (SCCs) have been shown to harbour p53 mutations, and other studies have demonstrated mutant p53 genes in normal and dysplastic squamous epithelium adjacent to these SCCs. In line with these earlier studies we show here that DOK, a keratinocyte cell line derived from a dysplasia, displays elevated levels of p53 protein and harbours a 12 bp in-frame deletion of the p53 gene spanning codons 188-191. In contrast, the coding region of the p53 gene was normal in a series of six benign recurrent laryngeal papillomas and a series of four premalignant oral erythroplakia biopsies and their cell cultures. All but one of these lesions were free of malignancy at the time of biopsy, in contrast to the premalignant lesions studied by previous investigators, but keratinocytes cultured from these lesions all displayed a partially transformed phenotype that was less pronounced than that of DOK. Since three out of four of the erythroplakia patients developed SCC within 1 year of biopsy, these lesions were by definition premalignant. The availability of strains of partially transformed keratinocytes from premalignant erythroplakias which possess normal p53 genes should enable us to test the role of mutant p53 in the progression of erythroplakia to SCC. The premalignant tissues and cultures were also tested for the presence of human papillomavirus (HPV), which is known to inactivate p53 function in some cases. Only the benign papillomas were shown to contain high levels of either HPV 6 or HPV 11 E6 DNA, but not both, and none of the samples contained detectable levels of HPV 16, HPV 18 or HPV 33 E6 DNA or L1 DNA of several other HPV types. There was therefore no evidence to suggest that p53 was being inactivated by a highly oncogenic HPV in these samples.     

The value of epidermal growth-factor receptor (egfr) as a tumor-marker for human oral premalignant and malignant lesions

A previous study demonstrated increased epidermal growth factor receptor (EGFR) in oral dysplasia while another showed decreased EGFR in oral dysplasia. The present study examined immunohistochemical expression of EGFR in 33 dysplastic oral lesions as well as in 9 normal oral mucosa specimens, 12 hyperplastic oral lesions and 10 oral squamous cell carcinomas (SCCs). There were no significant differences in EGFR staining either in intensity or in the epithelial layers stained among the normal oral epithelium, hyperplastic and dysplastic lesions. In addition, no significant difference was noted between keratinized and non-keratinized specimens and among lesions from different sites. Oral SCCs demonstrated significantly stronger staining than the normal oral mucosa, hyperplastic and dysplastic lesions (p=0.0011). At this time, the conflicting data on the EGFR expression in oral dysplastic lesions indicate that this receptor is not a good marker for oral dysplasia. Because most of the available data (including our results) show that the majority of oral SCC overexpress EGFR, this receptor may be useful in the diagnosis and treatment of some oral cancers.     

Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression

Although an important risk factor for oral cancer is the presence of epithelial dysplasia, most of these lesions will not progress to malignancy. Presently, for the individual patient with dysplasia, there are few reliable markers that may indicate the likelihood of progression to oral cancer. Cathepsin L is a lysosomal protease that degrades extracellular matrix material. Because cathepsin L is frequently overexpressed in oral squamous cell carcinoma (SCC) we hypothesized that it is also overexpressed in oral premalignancy and that premalignant lesions that progressed to oral cancer expressed higher levels of cathepsin L than those premalignant lesions that did not. In this retrospective pilot study we examined changes in cathepsin L expression at the mRNA level using quantitative TaqMan RT-PCR and at the protein level by immunohistochemistry in 33 routinely processed oral dysplastic lesions and 14 SCCs obtained from 33 patients. Sixteen of the dysplastic lesions progressed to oral SCC and 17 did not after several years of follow-up. Cathepsin L mRNA was overexpressed in 16/33 (48%) dysplastic lesions and in 9/14 (64%) oral SCC. Cathepsin L protein was also overexpressed in a large proportion of dysplasias and cancers. Overexpression was independent of dysplasia grade and identified in both those patients who progressed to oral SCC and in those who did not. Levels of cathepsin L mRNA and protein did not differ significantly in the progressing versus non-progressing dysplasias (P=0.27). However, cathepsin L mRNA and protein were significantly lower in the non-progressing dysplasias when compared to the oral cancers (P=0.03) but not in the progressing dysplasias suggesting a trend for dysplasias with overexpressed cathepsin L to be more likely to progress to oral cancer.     

Expression of the BCL-2 protein in normal and dysplastic bronchial epithelium and in lung carcinomas.

Although expression of the bcl-2 protein has been investigated in a number of non-haematological malignancies, little is known of its distribution in premalignant lesions. Expression of bcl-2 was investigated immunohistochemically in archival biopsies of normal (n = 8) and dysplastic bronchial epithelium (n = 56) and in 31 bronchial resection margins and their corresponding carcinomas. All dysplasias had lost the prominent basal staining pattern seen in histologically normal epithelium. Two were negative and six had occasional basal positive cells. In 37 cases up to 66% of the epithelial cells throughout the full epithelial thickness were bcl-2 positive with weak to moderate staining intensity. In 11 cases, all severe dysplasias, strong expression was observed in > 90% of the epithelial cells. Four patterns of bcl-2 expression in dysplasias were identified and an increasingly aberrant pattern of bcl-2 expression correlated with an increasing grade of dysplasia (Spearman''s rank correlation, P < or = 0.0001). Sixty-five per cent of the carcinomas contained bcl-2-positive cells. Patients with non-small-cell lung carcinomas (n = 27) in which > 50% of the tumour cells were bcl-2 positive showed a survival advantage compared with those with 0-25% bcl-2-positive cells (P = 0.02). No correlation was found between p53 expression (Walker et al., 1994) and bcl-2 expression in dysplasias or carcinomas.     

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.     

p53 overexpression in normal and dysplastic tissues adjacent to p53 negative squamous cell carcinomas of the head and neck

p53 overexpression was present in the normal or dysplastic epithelium, but absent in the adjacent invasive cancers of five patients with head and neck squamous cell carcinomas (HNSCC), when p53 immunostaining (IHC) was performed. In three of the five p53 immunoreactive dysplasias and adjacent p53 negative invasive cancers single stranded conformation polymorphism (SSCP) results from exon 7 and 8 were also obtained. Bandshifts in exon 7 were detected in two dysplasias, and bandshifts in exon 8 were found in a third. Sequencing of exon 7 in the first dysplasia with bandshift indicated a deletion of codon 241-242 (loss of CT) resulting in a frame shift. In the second dysplasia with bandshift a mutation was observed in codon 244 resulting in a Gly-->Arg substitution in the protein sequence. In the adjacent IHC p53 negative invasive cancer lesions, no bandshifts could be observed by SSCP, and sequencing did not reveal any mutated p53. WAF1/p21 (IHC) expression was assayed to study p53 function. Image cytometry (ICM) DNA analysis, estimating genetic instability, showed progress in DNA aberration for invasive cancer lesions as compared with the dysplasias. Human papillomavirus (HPV DNA) was not detected by a polymerase chain reaction (PCR) in any of the five cancers thus excluding possible p53 degradation caused by HPV protein. In conclusion, the finding of p53 mutations in mild, moderate, and severe dysplasia indicates that p53 mutation, not only p53 immunoreactivity, can be an early event in HNSCC carcinogenesis. The lack of p53 immunoreactivity in the invasive cancers adjacent to p53 positive dysplasias could possibly be attributed to loss of the mutant allele, or clonal heterogeneity.     

Cyclo-oxygenase-1 and -2 expression in human oral mucosa, dysplasias and squamous cell carcinomas and their pathological significance

Cyclo-oxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostanoids. The expression of its isoforms, COX-1 and -2 is found in many human malignancies. This study analyzed the correlation between COX expression and the pathobiological nature of human oral mucosa, dysplasias and squamous cell carcinomas (SCCs). We examined 9 specimens of normal oral epithelia, 65 lesions with dysplasias and 50 SCCs. Labeling indices (LIs) for COX-1, COX-2, Ki-67 and P53, microvessel density (MVD) and apoptotic index (AI) were evaluated using immunohistochemistry and TUNEL methods. Western blot analysis of COX-1 and -2 was performed on four human oral SCC cell lines, all of which showed expression. The LIs for COX-1 and -2 were higher for the dysplasias than the SCCs. LIs of COX-2 but not COX-1 correlated with the histological grade of dysplasia, being highest for the severe dysplasias (p < 0.05). In contrast, the COX-2 LIs as well as COX-1 were significantly (p < 0.05) inversely correlated with the histological differentiation of the SCCs. COX-2 expression was significantly correlated with LIs of COX-1 for dysplasia (p < 0.05), but not for the SCCs. In addition no significant relationship was noted between COX-2 expression and the Lis of Ki-67, P53, AI as well as MVD for the dysplasias and SCCs. The expression of COX-1 and -2 is correlated with early stage tumorigenesis and cellular differentiation of SCCs in the oral dysplasia-carcinoma sequence.     

Genetic progression and divergence in superficial esophageal squamous cell carcinoma by loss of heterozygosity analysis

Esophageal squamous cell carcinoma (SCC) is one of the most common fatal carcinomas worldwide and has some of the most malignant characteristics among gastrointestinal tumors. Although a high frequency of loss of heterozygosity (LOH) for various genes has been observed in esophageal SCCs, these findings do not provide any information regarding the genetic pathways that may underlie the development and progression of this type of tumor. To clarify the temporal and topographic pathways in the genetic evolution of esophageal SCC, we microdissected multiple foci from superficial mucosal invasive foci of tumors. We then carried out LOH analyses of the microdissected neoplastic foci. Sixteen superficial esophageal SCCs were examined. Three to six carcinoma foci from each superficial esophageal SCC were individually microdissected. We used 12 oligonucleotide primer pairs specific for the microsatellite markers for which frequent LOH in esophageal SCC has been reported. All tumors exhibited LOH of at least three microsatellite loci. A frequent homogeneous LOH pattern was detected for TP53 (60%), D16S518 (43%) and D3S1234 (29%), suggesting that the loss of these alleles is an early event in the development of esophageal SCC. A heterogeneous LOH pattern was detected for D13S325 (87%), D10S559 (73%), D3S1568 (58%), D3S1234 (57%) and D3S1621 (56%), suggesting that the loss of these alleles is a late event in the development of esophageal SCC. All tumors showed the LOH pattern of single clonal neoplasms with genetic progression and divergence. In conclusion, by extensive sampling of SCC lesions with microdissection and LOH analysis of multiple chromosomal loci, we successfully demonstrated dynamic and successive accumulation of genetic alterations in early SCC.     

p53 and Bcl-2 expression in pneumoconiosis-related pre-cancerous lesions and lung cancers: Frequent and preferential p53 expression in pneumoconiotic bronchiolar dysplasias

To explore the mechanism by which lung cancers excessively arise from pneumoconiosis, we determined the altered expression of p53 and Bcl-2 by immunohistochemistry (IHC) in lung cancers, dysplasias and non-cancerous pulmonary epithelia in pneumoconiotics in comparison with those from non-pneumoconiotic patients. We examined p53 expression in squamous cell carcinomas (SCCs) and dysplasias separately in the central and peripheral zones of bronchial trees, based on observations that SCCs from pneumoconiotic patients occurr more frequently in peripheral epithelia than those from non-pneumoconiotic patients (55 of 72 SCCs with pneumoconiosis vs. 33 of 72 SCCs without pneumoconiosis). Forty-one of 72 patients with pneumoconiosis-related lung cancers had altered p53 expression, which was comparable to the positivity of p53 expression in lung cancers without pneumoconiosis. p53 expression was observed significantly more frequently in bronchiolar dysplasias with pneumoconiosis than in those from non-pneumoconiotic patients (13 of 23 vs. 4 of 22), while p53 expression was found in bronchial dysplasias with pneumoconiosis as frequently as those without pneumoconiosis. Moreover, in patients with pneumoconiosis, bronchiolar dysplasias exhibited p53 expression more frequently than bronchial dysplasias (13 of 23 vs. 4 of 19). When comparison was restricted to bronchiolar dysplasias from patients without lung cancer, p53 expression had a strikingly higher frequency in the dysplasias with pneumoconiosis than in those from non-pneumoconiotic patients (8 of 15 vs. 0 of 14). Bcl-2 occasionally was expressed in squamous metaplasias and basal cell hyperplasias, in contrast to p53, for which immunostaining was negative in these lesions. Altogether, our results show that pre-cancerous and/or cancerous targets in pneumoconiosis may be distributed over a more peripheral zone than those in patients without pneumoconiosis. Int. J. Cancer 75:504–511, 1998.© 1998 Wiley-Liss, Inc.     

Frequent loss of heterozygosity on chromosome 10p14-p15 in esophageal dysplasia and squamous cell carcinoma

High frequencies of loss of heterozygosity (LOH) on chromosome 10p14-p15 have been reported in various tumors, including glioma, pulmonary carcinoid and cervical, hepatic and prostatic carcinomas. These findings suggest the presence of a tumor suppressor gene at the loci. However, analysis of LOH on chromosome 10p14-p15 in esophageal tumors has not been reported. Therefore, we examined LOH on chromosome 10p14-p15 in 88 esophageal squamous cell carcinomas (SCC) (35 superficial- and 53 advanced-types) and 44 dysplasias by microsatellite assay. Five oligonucleotide primer sets for microsatellite loci D10S191, D10S501, D10S559, D10S558 and D10S249 were used. In dysplasias, frequent LOH was detected with markers D10S191 (26%) and D10S249 (33%). In superficial esophageal SCCs, frequent LOH was detected with markers D10S191 (26%), D10S559 (50%), D10S558 (29%) and D10S249 (33%). In advanced esophageal SCCs, we found frequent LOH was detected with markers D10S191 (38%), D10S501 (25%) and D10S559 (30%). There were no significant correlations between LOH on chromosome 10p14-p15 and clinicopathologic features, including patient age, sex, tumor location, depth of invasion and lymph node metastasis. These data suggest that a putative tumor suppressor gene for esophageal carcinogenesis may be located on chromosome 10p14-p15 and that malfunction of this gene may be involved in the development but not progression of esophageal tumors.