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.     

Telomerase RNA expression during progression of gastric cancer

Telomerase, an enzyme associated with cellular immortality and malignancy, is stringently repressed in most normal somatic cells but is reactivated in malignant tumor cells and immortal cell lines, indicating that activation of telomerase may play an important role in tumorigenesis and immortalization. The pattern of human telomerase RNA (hTR) expression during progression of gastric cancer was investigated by a radioactive in situ hybridization (ISH) assay. Paraffin-embedded sections of 85 archival samples from Korean patients with benign and various malignant stages of gastric carcinomas as well as normal and regenerative tissues were studied. In normal gastric mucosae and regenerative lesions such as chronic peptic ulcer and hyperplastic polyps, only a weak degree of hTR expression was noted, and the expression was limited to basal cells of the gastric glands. Also, a moderate degree of hTR expression was present in the germinal centers of lymphoid follicles present in the submucosa. In tubular adenomas, the degree of hTR expression was also generally weak, but, unlike normal gastric mucosa, the expression was rather diffuse and occasionally focal in distribution. However, moderate to intense and usually diffuse hTR expression was present in all cancerous tissues at different stages. Although some heterogeneity of hTR expression was noted, there was a tendency for intensity of hTR expression to increase gradually as the cancer progressed to a more advanced stage. Our results indicate that upregulation of telomerase expression is associated with gastric cancer development or plays some role in gastric carcinogenesis. Upregulation of hTR expression detected by ISH assay may be a useful marker or tool for the early detection of gastric cancer.     

Telomerase in (pre)neoplastic cervical disease

This study was performed to determine upregulation of the human telomerase RNA component (hTR) and mRNA of the catalytic subunit of telomerase (hTERT) in (pre)malignant cervical lesions, to analyze possible intralesional heterogeneity of hTR expression, and to relate hTR and hTERT mRNA levels to telomerase activity levels and human papillomavirus (HPV) typing. hTR expression was determined by in situ hybridization (ISH) on paraffin-embedded sections, obtained from patients with cervical intraepithelial neoplasia (CIN) I-III or cervical cancer and from normal controls. hTR and hTERT mRNA expression were determined by semiquantitative rt-PCR on frozen samples from the same lesions. Data on telomerase activity and HPV were obtained from a previous study. hTR expression as determined by ISH was observed in 0 of 8 normal cervices, 1 of 14 CIN I, 15 of 28 CIN II, 21 of 30 CIN III, and 16 of 18 cervical cancer specimens. In general, hybridization patterns for hTR expression were homogeneous throughout the lesion. Frequency of hTR expression was related to grade of CIN/cervical cancer (P<.001). hTR expression, as determined by rt-PCR, was detected in 8 of 8 normal cervices, 2 of 2 CIN I, 12 of 14 CIN II, 23 of 23 CIN III, and 16 of 17 cervical cancer specimens. hTERT mRNA was detected in 1 of 8 normal cervices, 1 of 2 CIN I, 5 of 14 CIN II, 14 of 23 CIN III, and 11 of 17 cervical cancer specimens. hTR as determined by rt-PCR was not related to grade of CIN/cervical cancer, whereas hTERT mRNA expression was related to grade of CIN/cervical cancer (P<.01). hTR expression, as determined by ISH and hTERT mRNA expression by rt-PCR, were related to telomerase activity levels (P<.001, P<.05, respectively) and presence of oncogenic types of HPV (both P<.05). Our data show frequent upregulation of hTR and hTERT mRNA expression in CIN lesions, which appear to occur earlier than induction of telomerase activity. The fact that semiquantitative hTERT mRNA as well as hTR levels are related to telomerase activity levels illustrates that in (pre)malignant cervical lesions upregulation of both telomerase components may be important for functional telomerase.     

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.     

Suprabasal 40 kd keratin (K19) expression as an immunohistologic marker of premalignancy in oral epithelium.

The authors have studied the expression of keratin 19 in normal oral mucosa and in oral lesions exhibiting a range of histopathologic changes that are thought to precede squamous cell carcinoma. Formalin-fixed, paraffin-embedded sections were pretreated with pronase and stained with a K19-specific antibody by the avidin-biotin immunoperoxidase method. In nonkeratinized mucosa, whether normal or benign hyperplastic, K19 was detectable in the basal cell layer. In keratinized mucosa, whether normal or benign hyperplastic, there was no detectable K19. All lesions from any oral site that exhibited atypia diagnosed from hematoxylin and eosin stained sections as moderate-to-severe dysplasia or carcinoma in situ, whether hyperkeratotic or not, stained strongly for K19 in the basal and suprabasal cell layers. The number of cell layers that were K19-positive correlated with the level in the epithelium to which dysplasia persisted. Suprabasal K19 staining tended to occur in regions of the epithelium in which expression of the terminal differentiation protein involucrin was delayed or absent. Thus, K19 expression may be linked to the retention of stem cell character or a state otherwise uncommitted to terminal squamous differentiation. Suprabasal K19 staining is clearly correlated with premalignant change in oral epithelium and therefore promises to be a useful tool in oral histopathologic diagnosis.     

Patterns of keratin 19 expression in normal, metaplastic, condylomatous, atrophic, dysplastic, and malignant cervical squamous epithelium.

Keratin 19 (K-19) expression has been strongly correlated with dysplasia in oral epithelium. Expression of K-19 was evaluated by immunoperoxidase staining in formalin-fixed normal ectocervical tissue, normal endocervical tissue, cervical dysplasia, squamous metaplasia, atrophic epithelium, cervical condylomas, and invasive carcinoma to determine if a correlation of K-19 expression with dysplasia was present in the cervical epithelium. Uniform expression of K-19 was seen in endocervical epithelium and in the basal layer of normal ectocervical epithelium in all areas where these epithelia were present. Cervical dysplasia without associated condylomatous changes showed increased expression of K-19 in suprabasal epithelium, corresponding to the level of immature cells. Squamous metaplasia was characterized by scattered cells with increased staining (patch-quilt pattern). There was considerable overlap in the patterns of K-19 expression in dysplastic and metaplastic epithelium. Thus K-19 staining pattern could not be used as a distinctive marker for dysplasia in the cervical epithelium. Atrophic epithelium showed a characteristic uniform but low-level expression of K-19 in suprabasal areas. This pattern may be of diagnostic use in differentiating atrophic lesions from dysplasia. Condylomas showed focal loss of K-19 in the basal layer, suggesting induction of premature differentiation in the basal layer by human papillomavirus infection. Invasive carcinomas showed variable patterns. K-19 is a marker of immature cervical squamous epithelium, with generally distinctive but sometimes overlapping patterns of expression in various diagnostic categories.     

Markers for dysplasia of the upper aerodigestive tract. Suprabasal expression of PCNA, p53, and CK19 in alcohol-fixed, embedded tissue.

Recognition of premalignant lesions in the oral epithelium has the potential to increase survival rates for squamous cell carcinoma of the oral cavity. It has previously been reported that cytokeratin 19 (CK19), a 40-kd epithelial cytoskeletal protein within the suprabasal squamous epithelium, is a specific marker of moderate-to-severe dysplasia and carcinoma in situ in oral cavity squamous epithelium. In contrast, normal epithelium and hyperplastic lesions reportedly express CK19 only in the basal layer if at all. The authors chose to test and extend this hypothesis by studying suprabasal CK19 expression and dysplasia of the oral cavity and upper aerodigestive tract in paraffin-embedded specimens that had been fixed in alcohol, a superior fixative for the preservation of cytokeratins. The authors examined 56 alcohol-fixed, paraffin-embedded specimens including 37 from the oral cavity, using two antibodies specific for CK19 (Ks19.1 and 4.62), an antibody to the nuclear proliferation marker, proliferating cell nuclear antigen (PCNA) (19A2), and an antibody to the putative tumor suppressor gene, p53 (pAb1801). The lesions were classified as normal, hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia/carcinoma in situ, or invasive squamous cell carcinoma, following standard histologic criteria. Immunocytochemically stained sections were scored for the presence or absence of suprabasal CK19, suprabasal PCNA, and p53 positivity, regardless of location. The immunostaining patterns of the two anti-CK19 antibodies were essentially equivalent. Except for one laryngeal specimen, normal epithelium, when positive, showed CK19 expression only in scattered cells throughout the basal layer. Proliferating cell nuclear antigen-positive nuclei were found exclusively in the basal layer. In areas of hyperplasia, CK19 immunostaining was absent or confined to the basal layer in 20 of 38 specimens and was expressed in suprabasal cells in 18 of 38 hyperplastic specimens. Proliferating cell nuclear antigen immunostaining in all cases of hyperplasia was limited to the basal layer. Severe dysplasia and carcinoma in situ showed suprabasal CK19 staining in six of nine specimens and no CK19 staining in three of nine specimens. In contrast, suprabasal PCNA immunostaining was found in all dysplasia and carcinoma in situ cases. p53 expression was detected in three of nine severe dysplasia/CIS specimens and was immunocytochemically undetectable in all normal, hyperplasia, and mild to moderate dysplasia specimens. The authors conclude that suprabasal CK19 expression is neither a sensitive nor a specific marker of premalignancy in oral epithelium and cannot be used to distinguish hyperplasia from dysplasia. In contrast, a strong correlation between suprabasal expression of PCNA, a marker for proliferating cells, and dysplasia/carcinoma in situ was evident.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involucrin in intraepithelial and invasive squamous cell carcinomas of the cervix: an immunohistochemical study

Immunohistochemical staining for involucrin, a cytoplasmic protein synthesized during squamous maturation, was assessed in histologic sections from hysterectomy and cone biopsy specimens from patients with cervical neoplasia. In normal and condylomatous squamous epithelium, diffuse cytoplasmic staining was seen in the suprabasal layers, with no staining of the basal cells. Staining was absent in two cases of cervical intraepithelial neoplasia (CIN), grade III, in which the lesions were composed entirely of undifferentiated cells and markedly decreased in cases involving large numbers of basal cells. In 19 of 23 cases (83 per cent) of CIN, however, focal staining for involucrin was seen in large differentiated cells in the more superficial layers, and in two cases of keratinized CIN diffuse suprabasal staining was observed. Similarly, strong staining for involucrin was present in differentiated areas in one case of microinvasive squamous cell carcinoma and in 93 per cent of cases of infiltrating squamous cell carcinoma. These findings suggest that involucrin is a marker for maturation in cervical squamous epithelial neoplasms. Patterns of immunohistochemical staining for involucrin in keratinized dysplasia and differentiated squamous carcinomas should be taken into consideration if loss of involucrin staining is used as a criterion for neoplastic transformation of cervical epithelium, as has been proposed.     

Assessment of cell proliferation in benign, premalignant and malignant skin lesions.

A deeper understanding of the variance of epidermal cell proliferation may eventually increase the reproducibility of diagnostic classification. A prospective study of 46 consecutive, unselected biopsies from benign (keratoacanthoma n=14), premalignant (actinic keratosis n=15 and Bowen disease n=10) and malignant (squamous cell carcinoma n=7) skin lesions was studied to assess the presence and extent of differences in expression of the proliferation marker Ki-67 using a monoclonal antibody directed against a c-DNA defined subsegment (MIB-1) and a noncross-linking, proprietary fixative BoonFix. MIB-1 was expressed in the adjacent, non-affected skin in a scattered to confluent linear pattern in the basal/suprabasal cell layer. In actinic keratosis, MIB-1 expression, in addition to basal/suprabasal layers, extended to mid-zones of the epidermis. An interesting feature in actinic keratosis as well as in Bowen disease was the expression of MIB-1 in the epithelium lining the hair follicles. In Bowen disease, MIB-1 was observed throughout the full thickness of the epidermis, unequivocally separating this entity from others under study. In invasive squamous cell carcinoma, MIB-1 expression was not consistent between and within cases. MIB-1 positivity was variably found in all layers of the epidermis, but showed a chaotic and haphazard pattern with total loss of polarity. Keratoacanthoma cases showed highly variable MIB-1 expression, ranging from no expression to expression in both basal/suprabasal and mid-zone layers of the epidermis. These results warrant further study of modulation of cell proliferation in actinic keratosis.     

Development and characterization of a canine oral mucosa equivalent in a serum-free environment

The objectives of this study were to develop a serum-free system for culturing canine oral keratinocytes, the construction and characterization of a canine ex vivo produced oral mucosa equivalent (EVPOME), and transduction green fluorescent protein (GFP) into keratinocytes as a post-grafting tracking marker. Dissociated canine buccal mucosa keratinocytes were cultured in a chemically defined serum-free medium, Epilife trade mark. First-passage keratinocytes were transfected with the GFP gene using a lentiviral vector, sorted by flow cytometer and seeded onto a dermal equivalent, AlloDerm(R) to form EVPOMEs. The EVPOME was characterized by histology and immunohistochemistry, for p63, Ki-67, and involucrin. Laser confocal microscopy was used to locate GFP-transfected keratinocytes within the EVPOME. Cultured canine oral keratinocytes grew rapidly over the first three passages and then the proliferative rate decreased. The canine EVPOME formed a well-stratified epithelial layer. The majority of p63 and Ki-67 immunopositive cells were located in the basal layer whereas cytoplasmic involucrin expression was seen in the suprabasal layers, similar to native canine buccal mucosa. Under laser confocal microscopy, significant green fluorescence was observed throughout the EVPOME. In conclusion, canine EVPOMEs were successfully fabricated in a defined serum-free system with similar characteristics to native buccal mucosa. GFP-transfected canine oral keratinocytes could be identified within the EVPOME.     

Overexpression of human telomerase RNA is an early event in oesophageal carcinogenesis

 Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumour progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human oesophageal dysplasia and cancer by using in situ mRNA hybridization (ISH) with avidin–biotin staining. Ki-67 immunoreactivity was also examined. We analysed 51 squamous cell carcinomas, 9 dysplasias and 60 normal mucosae. The integrity of the mRNA in each sample was verified by using a poly d(T)₂₀ probe. Seventy-six samples (63%) showed no mRNA degradation; these included 30 carcinomas, 7 dysplasias and 39 normal mucosae. At the single-cell level, high levels of hTR expression were found in the cytoplasm and especially in the nucleus. Most (>90%) cancer cells demonstrated high levels of hTR expression in 29 (97%) of the 30 tumours. Most dysplastic cells also showed high levels of hTR in all 7 dysplastic cases. In all 39 normal mucosae, most basal cells indicated high levels of hTR expression, which were also seen in infiltrating lymphocytes. The distribution of hTR-expressing cells was similar to that of Ki-67-positive cells. These data suggest that overexpression of hTR may be correlated with the proliferative activity that defined by Ki-67 immunoreactivity and is an early event in carcinogenesis of the oesophagus. Received: 14 December 1998 / Accepted: 20 January 1999     

端粒酶hTR和hTRT在肿瘤中的表达及意义

目的 :探讨端粒酶hTR、hTRT基因表达在人肿瘤中的意义 ,并与 p5 3蛋白改变相比较 ,评估其对肿瘤临床诊断的价值。方法 :用原位杂交及免疫组化技术检测了 115例恶性肿瘤、6 0例良性及癌前病变中hTR和hTRT的表达 ,并与 p5 3基因蛋白改变相比较。结果 :hTR、hTRT在人恶性肿瘤中的平均检出率分别为 81%、83%。在转移性癌中两者均为 10 0 %的阳性率 ,而在慢性肝炎伴肝硬化中hTR、hTRT分别可见 5 0 %、6 0 %的弱阳性表达 ,其他良性病变及癌前病变均为阴性。p5 3蛋白在人恶性肿瘤中的平均阳性率为 4 9 6 % :在良性病变及癌前病变均为阴性。结论 :恶性肿瘤中hTR和hTRT明显高于 p5 3的检出率 ,且在良恶性病变中的表达差异有显著性 (P <0 0 5 ) ,并提示端粒酶hTR、hTRT与癌的生物学行为有密切关系 ,端粒酶基因hTR、hTRT的原位杂交检测可以作为一个比 p5 3更好的肿瘤诊断的新标记物。     

Immunohistochemical expression of p16 protein in oral squamous cell carcinoma and lichen planus

Epithelial carcinogenesis is a multistep process. Specific genetic events lead to malignant transformation of oral epithelium. Oral squamous cell carcinoma (OSCC) may be preceded by potentially malignant lesions such as oral lichen planus (OLP). The p16 protein functions as a negative regulator of the cell cycle progression. Altered pattern of p16 serves as a biomarker for oral mucosal dysplasia and malignant growth. The purpose of this study was to evaluate p16 expression in OSCC and OLP to determine whether it can be a useful marker for early detection of carcinogenesis. We examined p16 expression in 45 OSCCs (15 grade I, 15 grade II, and 15 grade III), 15 OLPs without dysplasia, and 8 normal mucosal specimens with immunohistochemistry. p16 was interpreted as positive if more than 70% of tumor cells showed brown nuclear and cytoplasmic staining. All of the OSCC and control group samples showed negative immunoreactivity, whereas 26.7% of OLP samples were positive for p16. Our findings suggest that p16 expression could not be used as a helpful marker for detection of development toward malignancy in OLP samples.     

Detection of telomerase in hepatocellular carcinomas using a PCR ELISA assay: comparison with hTR expression

BACKGROUND: While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected in many immortal cell lines and various cancers. AIM: To investigate telomerase expression in hepatocellular carcinoma, and to assess the expression of the RNA component of telomerase, hTR. METHODS: 39 hepatocellular carcinomas were studied using a telomerase polymerase chain reaction (PCR) enzyme linked immunosorbent assay, which does not require radioactive PCR amplification and yields a semiquantitative measurement. Expression of hTR was also assessed by a non-radioactive in situ hybridisation procedure. The correlations between these two markers and the clinicopathological data were analysed. RESULTS: Telomerase activity was detected in 23 of 39 hepatocellular carcinoma specimens (59%). Comparison of hepatocellular carcinoma with and without telomerase expression, or with high and low telomerase (10 cases v 13 cases), showed no differences in the principal clinicopathological data. Although median survival was lower in the group with detectable telomerase activity than in that with undetectable activity (510 v 720 days) the difference was not significant (log-rank test, p = 0.08). hTR expression was detected in 11 of 14 cases of hepatocellular carcinoma tested (78%) and in four of 12 samples of adjacent non-cancerous tissue (33%). Five tumours and four non-cancerous tissues were positive for hTR, whereas no telomerase activity was detected in these. CONCLUSIONS: The presence of telomerase activity in hepatocellular carcinomas is confirmed. No correlation was observed between clinicopathological data and telomerase expression in hepatocellular carcinoma, but survival seemed better in the absence of telomerase expression. hTR seems to be more widely expressed than telomerase.     

Keratin expression in the normal anal canal

The pattern of epithelial keratin expression in the normal anal canal has not been extensively defined and is a necessary prerequisite to the interpretation of alterations in these intermediate filaments in pathological anal epithelial lesions. Thirty-five frozen tissue specimens of resected haemorrhoids were investigated immunohistolo-gically for expression of 14 individual keratins (K) using a panel of 17 monoclonal antibodies. Perianal skin showed basal expression of karatinocyte Ks 5, 14 and 17, and suprabasal expression of keratinocyte Ks 14, 10, 1 and 16. Anal squamous epithelium showed persistent basal K5 and 17, basal and suprabasal K4, 13 and 16 positivity, with sporadic expression of K1 and 10. The expression of simple epithelial keratins in squamous epithelium adjacent to the anal transitional zone varied with basal expression of K7, K8, K18 and K19 and sporadic suprabasal expression of K7 and K19. The anal transitional zone (ATZ) expressed K19, as found in transitional epithelia elsewhere. The full thickness of epithelium was positive for the simple epithelial Ks 7, 8 18 and 19. Marked heterogeneity of keratinocyte keratin expression was seen. Basal layers expressed Ks 4, 13, 14 and 17 and variably K16, while suprabasal layers expressed Ks4 and 13, 14 and 17 and variably K16, while suprabasal layers expressed Ks4 and 13 and variably K14, 16 and 17. This anomalous expression of keratinocyte K4 and 13 has also been documented in transitional epithelium of the bladder. The anal glands and ducts showed a keratin distribution similar to the transition zone. Rectal columnar epithelium expressed simple keratins 7, 8 18 and 19. In addition, low levels of keratinocyte keratins were found as indicated by heterogeneous staining for K4, 13, 14 and 16. The overall pattern, particularly in the region of the anal transitional zone and immediately adjacent squamous and columnar epithelia, is of a flexible epithelial cell population able to express a range of keratins unrestricted by a particular morphological phenotype. In the light of these results, analysis of changes in keratin distribution within anal carcinomas may assist classification by providing information on the state of differentiation and histogenesis of these tumours.     

Expression of Ep-CAM in cervical squamous epithelia correlates with an increased proliferation and the disappearance of markers for terminal differentiation.

Ep-CAM, an epithelial adhesion molecule, is absent in normal squamous epithelia but can be detected in some squamous carcinomas. Using a panel of monoclonal antibodies to keratinocyte differentiation and proliferation markers, we investigated the association of EP-CAM expression with differentiation-related and/or neoplastic changes in cervical epithelium. Normal endocervical glandular epithelium (Both columnar and reserve cells) appeared strongly positive for EP-CAM, whereas ectocervical squamous epithelial cells did not express this molecule. Expression of Ep-CAM (in basal cells) was sometimes observed in morphologically normal ectocervical tissue but only in areas bordering cervical intraepithelial neoplasia (CIN) lesions. At the early stages of neoplasia the expression of Ep-CAM was regularly present in squamous epithelium, in general consistent with the areas of atypical, undifferentiated cells. Thus, in CIN grades I and II, the basal/suprabasal layers of the epithelia were positive, whereas in CIN grade III lesions, up to 100% of the cells over the whole thickness of the epithelium sometimes excluding the very upper layers, expressed Ep-CAM. A clear increase, not only in number of positive cells but also in levels of Ep-CAM expression (intensity) was observed during progression from CIN I to CIN III. Expression of Ep-CAM in ectocervical lesions did not coincide with a reappearance of the simple epithelium cytokeratins (CK8 and CK18). On the other hand, expression of Ep-CAM in atypical cells of CIN lesions correlated with the disappearance of CK13, which normally marks cells undergoing squamous differentiation. As was shown with Ki-67, a marker for proliferating cell populations, the areas of Ep-CAM expression were also the areas of enhanced proliferation. Cells expressing Ep-CAM did not express involucrin, a marker for cells committed to terminal differentiation. In the majority of both squamous and adenocarcinomas of the cervix a strong expression of Ep-CAM was observed, although some decrease in the expression (both the intensity and the number of positive cells), as compared with CIN III lesions, was observed in the areas of squamous differentiation. This study demonstrates that the expression of Ep-CAM in cervical squamous epithelium is associated with abnormal proliferation of cell populations that are not committed to terminal differentiation.     

Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.