Telomerase catalytic subunit in laryngeal carcinogenesis--an immunohistochemical study.

We recently demonstrated that (1) telomerase catalytic subunit messenger RNA (mRNA) relative quantities increase progressively with the degree of laryngeal epithelial abnormalities and that (2) telomerase catalytic subunit gene re-expression represents an early event in laryngeal carcinogenesis. The aim of the study was to determine whether telomerase catalytic protein immunohistochemisty reflects telomerase catalytic subunit gene expression in different grades of laryngeal epithelial abnormalities and squamous cell carcinomas of the larynx. Telomerase catalytic protein was analysed immunohistochemically in 106 laryngeal epithelial tissue samples: 10 normal epithelia, 15 squamous cell hyperplasias, 14 basal/parabasal cell hyperplasias, 10 atypical hyperplasias, eight intraepithelial carcinomas and 49 squamous cell carcinomas. At least 200 nuclei of each lesion were quantified per slide and the number of positive signals per nucleus was expressed as a telomerase catalytic protein index. The mean telomerase catalytic protein index increased progressively with the degree of laryngeal epithelial abnormalities: from 0.17 in normal epithelia, 0.44 in squamous cell hyperplasia, 0.54 in basal/parabasal cell hyperplasia, 0.91 in atypical hyperplasia, 1.05 in intraepithelial carcinoma to 0.96 in squamous cell carcinomas. Statistical analysis revealed three different groups of laryngeal epithelial changes according to the number of telomerase catalytic protein signals per nucleus: (1) normal epithelium, (2) regenerative epithelium (squamous cell hyperplasia, basal/parabasal cell hyperplasia), and (3) atypical hyperplasia, intraepithelial carcinoma and squamous cell carcinoma (P<0.0033). Telomerase catalytic protein immunohistochemistry parallels well with telomerase catalytic subunit mRNA relative quantities in laryngeal carcinogenesis. In normal and regenerative laryngeal epithelia, telomerase catalytic protein is present in occasional basal/parabasal nuclei, becomes undetectable with maturation or differentiation of epithelial cells, and reflects the regenerative capacity of squamous epithelium. Nevertheless, several telomerase catalytic protein signals in the majority of nuclei in precancerous lesions, intraepithelial carcinomas and squamous cell carcinomas, are consistent with telomerase catalytic subunit gene re-expression, an early event in laryngeal carcinogenesis.     

Human telomerase catalytic subunit gene re-expression is an early event in oral carcinogenesis

AIMS: Detection of telomerase catalytic subunit (hTERT) mRNA has been used as a surrogate marker for estimation of telomerase activity. The exact role and timing of telomerase re-activation, a key enzyme implicated in cellular immortalization and transformation, in the multistep process of oral carcinogenesis is still unknown. The aim was to test the hypothesis that (i) quantitative rather than qualitative differences exist in the level of hTERT mRNA expression between normal oral mucosa, different grades of oral epithelial abnormalities and squamous cell carcinomas of the oral cavity, and that (ii) hTERT gene re-expression is an important, probably early event in oral carcinogenesis. METHODS AND RESULTS: The relative quantity of hTERT mRNA was analysed in 45 frozen oral epithelia representing different morphological stages of oral carcinogenesis classified according to the Ljubljana classification and in 37 oral squamous cell carcinomas, using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. hTERT mRNA was not detected in normal or reactive hyperplastic oral epithelia, but was present in 43% of atypical hyperplasias (premalignant lesions), 60% of intraepithelial carcinomas and 68% of oral squamous cell carcinomas. Statistical analysis revealed two groups of oral epithelial changes, with significant differences in the levels of hTERT mRNA expression: 1, normal and reactive hyperplastic oral epithelium, and 2, atypical hyperplasia, intraepithelial carcinomas and squamous cell carcinomas. CONCLUSION: These data suggest that hTERT gene re-expression represents an early event in the multistep process of oral carcinogenesis, already detectable at the stage of precancerous oral epithelial changes. Nevertheless, other genetic aberrations appear to be necessary for progression of oral epithelial abnormalities towards invasive squamous cell carcinoma.     

Expression of human telomerase catalytic protein in gallbladder carcinogenesis

BACKGROUND: Telomerase catalytic subunit (hTERT) gene re-expression is a rate limiting step for the activity of telomerase, a key enzyme implicated in cellular immortalisation and transformation. AIMS: To determine the potential role of hTERT protein in gallbladder carcinogenesis. MATERIAl/METHODS: hTERT protein was analysed by means of immunohistochemistry in 89 gallbladder tissue samples: 16 normal epithelia, 14 reactive hyperplasias, 15 low grade dysplasias, 16 high grade dysplasias, and 28 adenocarcinomas. At least 200 nuclei were assessed for each slide and the mean number of positive signals for each nucleus was expressed as the hTERT index. RESULTS: The mean hTERT index increased progressively with the degree of gallbladder epithelial abnormalities: from 0.03 in normal epithelia, 0.04 in hyperplastic epithelia, 0.25 in low grade dysplasia, 0.82 in high grade dysplasia, to 0.93 in adenocarcinoma. Statistical analysis revealed that three different groups of gallbladder epithelial changes can be distinguished according to the number of hTERT signals for each nucleus: (1) normal and regenerative gallbladder epithelium, (2) low grade dysplasia, and (3) high grade dysplasia and adenocarcinoma (p < 0.001). CONCLUSIONS: The occasional presence of hTERT protein in normal and regenerative gallbladder mucosa reflects their regenerative capacity. Nevertheless, significantly higher hTERT indices in low and high grade dysplastic epithelia and in gallbladder adenocarcinomas are probably a consequence of hTERT re-expression--an early event in the multistep process of gallbladder carcinogenesis.     

Epithelial abnormalities and precancerous lesions of anterior urethra in patients with penile carcinoma: a report of 89 cases.

Urethral and penile tissues and their neoplasms are considered anatomically and pathogenetically different. Since we observed urethral dysplastic lesions and some similarities between noninvasive and invasive lesions of the anterior urethra and glans, we designed this study to document epithelial urethral abnormalities in patients with penile squamous cell carcinoma. We examined urethral epithelia from 170 penectomies with invasive squamous cell carcinoma finding a variety of primary epithelial abnormalities in 89 cases (52%) and secondary invasion of penile carcinoma to urethra in 42 cases (25%). Patients' average age was 68 years. Primary tumors measured 4 cm in average diameter and the majority were squamous cell carcinoma of the usual (67%) or verrucous type (15%). Primary epithelial abnormalities found were squamous intraepithelial lesions, metaplasias and microglandular hyperplasias. Urethral squamous intraepithelial lesions of high grade was found in six patients and of low grade in eight cases. Squamous metaplasia, seen in 69 cases, was the most frequent finding. Metaplasias were classified as nonkeratinizing and keratinizing. Nonkeratinizing metaplasias (57 cases) were variegated in morphology: simplex (26 cases), hyperplastic (12 cases), clear cell (11 cases) and spindle (8 cases). Keratinizing metaplasias (12 cases) showed hyperkeratosis and were more frequently associated with verrucous than nonverrucous penile squamous cell carcinoma. Microglandular hyperplasia was present in eight cases. Lichen sclerosus was associated with simplex squamous metaplasia in four cases. Despite the large size of the primary tumors, direct urethral invasion by penile carcinoma was present in only 25% of the cases. The presence of precancerous lesions in urethra of patients with penile carcinoma indicates urethral participation in the pathogenesis of penile cancer. Simplex squamous metaplasia is a common finding probably related to chronic inflammation. Keratinizing and hyperplastic squamous metaplasias may be important in the pathogenesis of special types of penile carcinomas such as verrucous carcinoma.     

Downregulation of transforming growth factor beta type II receptor in laryngeal carcinogenesis

AIMS: To investigate whether anomalies of transforming growth factor beta type II receptor (TGF-beta RII) expression occur in the early stages of laryngeal carcinogenesis and to assess their importance in the development of laryngeal squamous cell carcinoma. TGF-beta RII status was examined in laryngeal premalignant lesions coupled with malignant evolution and compared with a control group of similar lesions without progression to cancer. METHODS: Immunohistochemical staining for TGF-beta RII was performed on 15 paraffin wax embedded biopsies from patients with precancerous laryngeal lesions who subsequently developed invasive squamous cell carcinoma of the larynx, and on 30 control biopsies from patients who did not develop cancer in a comparable follow up period. In addition, DNA extracted from 18 preneoplastic lesions and eight squamous cell carcinomas was amplified by the polymerase chain reaction at the poly A and the poly GT regions of the TGF-beta RII gene. RESULTS: In the group of lesions with progression to carcinoma, 11 of 15 cases showed loss (< 20% of epithelial cells) of TGF-beta RII immunoreactivity, whereas among non-evolved lesions only five of 30 had similar altered expression of the receptor (p < 0.001, two tailed Fisher's exact test). All squamous cell carcinomas showed a degree of receptor expression comparable with that of the corresponding preneoplastic lesion, with the exception of one case, in which loss of the receptor was evident only in invasive cancer. Mutation of the poly A sequence of the TGF-beta RII gene was identified in only one precancerous lesion and in the subsequent squamous cell carcinoma. CONCLUSIONS: These findings indicate that the downregulation of TGF-beta RII is an early event in laryngeal carcinogenesis, which may result in the loss of TGF-beta mediated growth inhibition, thereby facilitating the progression of laryngeal precancerous lesions to squamous cell carcinoma.     

Localization of telomerase hTERT protein and hTR in benign mucosa, dysplasia, and squamous cell carcinoma of the cervix

Telomerase has been detected by telomerase repeat amplification protocol (TRAP) assay in cervical dysplasia and squamous cell carcinoma but not in most normal cervical tissues. In the present study, the cellular localization of the protein catalytic subunit of telomerase (hTERT) and the RNA component (hTR) were investigated by a sensitive immunohistochemical technique and by in situ hybridization, respectively. hTERT protein was detected in all diagnostic categories of cervical specimens. hTERT was localized predominantly to the lower suprabasal levels of normal squamous mucosa but was detected throughout virtually all levels of the lesional epithelium in low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma (SCC). Telomerase expression correlated with hTERT detection in SCC and HSIL but was not detected by TRAP assay in most samples of normal mucosa or LSIL. The distribution of hTR correlated with the localization of hTERT in HSIL and SCC but was restricted to the basal and suprabasal cell layers in normal mucosa and LSIL.     

Expression of the RNA component of human telomerase (hTR) in prostate cancer, prostatic intraepithelial neoplasia, and normal prostate tissue.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)-based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non-radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin-embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate.     

Clonal analysis of esophageal squamous cell carcinoma with intraepithelial components.

OBJECTIVE: In tumors of the upper aerodigestive tract, field carcinogenesis is a prevailing concept which suggests that such tumors are commonly of multiclonal origin. METHODS: To test this possibility, we applied a PCR-based clonality assay utilizing the polymorphic locus of the human androgen receptor gene (HUMARA) in female patients with esophageal squamous cell carcinomas (SCCs). DNA was extracted from small pieces of tissues microdissected from multiple points of intraepithelial and invasive parts of each tumor and the adjacent epithelia in 12 cases. The HUMARA locus was PCR amplified with or without prior digestion with Hpa II. PCR products were analyzed by a genetic analyzer and polyacrylamide gel electrophoresis followed by silver staining. RESULTS: In each of 8 informative cases, the pattern of X chromosome inactivation of the major cell population in each sample was common among the samples from the invasive part and among those samples, if any, from the intraepithelial part, and was concordant between the intraepithelial and invasive parts in 5 cases and discordant in 1 case. Out of the samples of adjacent epithelia, a monoclonal pattern was demonstrated in 8 basal cell hyperplasias and 3 dysplasias, of which 2 and 1, respectively, showed inactivation patterns discordant with those of the concomitant cancers. CONCLUSION: Esophageal SCCs may often be preceded or accompanied by multiclonal precancerous lesions, and may develop through the outgrowth of single or less commonly multiple dominant clones.     

食管鳞癌中hTERT表达、DNA含量与细胞增殖周期的关系

目的探讨食管鳞癌中hTERT表达与细胞DNA含量、细胞增殖周期的关系。方法采用免疫组化检测hTERT表达,流式细胞术检测hTERT和DNA含量及增殖指数。结果(1)癌组hTERT阳性率和荧光指数(FI)显著高于癌旁组(P<0.01);(2)癌组DNA指数(DI)和异倍体率均高于癌旁组(P<0.05),癌组增殖指数(PI)显著高于癌旁组(P<0.01)。结论hTERT过量表达、DNA含量增加和增殖指数升高与食管鳞癌的发生、发展密切相关。     

Epithelial lesions associated with invasive penile squamous cell carcinoma: a pathologic study of 288 cases

A heterogeneous spectrum of epithelial alterations and atypical lesions affect the squamous epithelium of penile mucosal anatomical compartments. Analogous to other genital sites, the terminology utilized to define the lesions is variable. The few pathologic studies of penile precancerous lesions are mostly related to carcinoma in situ and human papilloma virus (HPV), and the information on low-grade atypical lesions is limited. The objective of this study was to comprehensively describe the morphologic features of all epithelial alterations, benign and atypical, low grade and high grade, associated with invasive squamous cell carcinoma of the penis and to investigate their relation with each other and with subtypes of invasive carcinoma. We also propose herein a simple and reproducible nomenclature for penile precancerous abnormalities until more biological, molecular, or epidemiologic information on the lesions is available. Two hundred and eighty-eight penectomy and circumcision specimens with invasive squamous cell carcinoma were pathologically evaluated. Carcinomas were classified as usual, verrucous, papillary not otherwise specified, warty (condylomatous), basaloid, and mixed. Associated lesions were classified as squamous hyperplasia and squamous intraepithelial lesions of low and high grade (LGSIL and HGSIL). In LGSIL, atypia was confined to the lower third, and in HGSIL, atypical cells affected at least two thirds of the squamous epithelium. Subtypes of SIL were squamous, warty, basaloid, warty-basaloid, and papillary. Squamous hyperplasia, the most common lesion, was found in 83% of the cases, followed by LGSIL (59%) and HGSIL (44%). In 62% of the cases more than 1 associated lesion was present per specimen. A sequence from squamous hyperplasia to low-grade to high-grade SIL was seen frequently. Squamous hyperplasia was more commonly associated with usual squamous, papillary, and verrucous than with warty and basaloid invasive carcinomas. LGSIL was associated with all types of squamous cell carcinoma but was rarely present adjacent to basaloid or verrucous tumors. HGSIL was present in two thirds of invasive warty, basaloid, and mixed warty-basaloid tumors, in about half of usual squamous cell carcinomas, and was absent in papillary and verrucous carcinomas. Correlation of special types of invasive carcinomas with subtypes of SIL revealed morphologic correspondence of invasive tumor and the associated intraepithelial lesion. Squamous LGSIL was preferentially associated with verrucous, papillary, and usual squamous cell carcinomas; warty LGSIL, with invasive warty and mixed warty-basaloid carcinomas. High-grade SIL of the squamous type was frequently found in squamous cell carcinoma of usual type but was rarely present with warty or basaloid carcinomas. Basaloid HGSIL was associated with basaloid carcinoma, and HGSIL of warty type, with either warty or mixed warty-basaloid carcinomas. The high frequency of squamous hyperplasia and LGSIL and preferential association with usual, verrucous, and papillary carcinomas plus the subtle morphologic differences of the 2 lesions suggest that, despite its benign appearance, squamous hyperplasia is a precursor of the aforementioned carcinomas. The association and histologic similarities between high-grade SIL of the basaloid, warty, or mixed forms with their invasive counterparts indicate these lesions are their likely precursors.